REGULATION OF TRANSCRIPTION THROUGH CTCF LOOP ANCHORS
Disclosed are methods of altering expression of a gene with a promoter region CTCF binding site. Also disclosed are compositions and methods useful for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site. Further disclosed are cells and non-human animals with modified a promoter region CTCF binding site, as well as methods for screening for compounds that can modify the expression of a gene with a promoter region CTCF binding site.
This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2017/065918, filed Dec. 12, 2017, which claims the benefit of U.S. Provisional Application Ser. No. 62/433,234, filed Dec. 12, 2016, the contents of which are hereby incorporated by reference in their entirety. International Application No. PCT/2017/065918 was published under PCT Article 21(2) in English.
GOVERNMENT SUPPORTThis invention was made with government support under Grant Nos. HG002668 and CA109901 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTIONThe ability to activate transcription of specific genes is fundamental to the establishment of gene expression programs that define cell identity. To accomplish this, transcription factors (TFs) bind enhancer elements and regulate transcription from the promoters of nearby or distant genes through physical contacts that involve looping of DNA between enhancers and promoters (Bonev and Cavalli, 2016; Buecker and Wysocka, 2012; Bulger and Groudine, 2011; Fraser et al., 2015; Heard and Bickmore, 2007; de Laat and Duboule, 2013; Müller et al., 1989; Pombo and Dillon, 2015; Spitz, 2016; Tolhuis et al., 2002). However, the mechanisms that ensure that specific enhancers interact with specific promoters are not well understood. Most study of eukaryotic enhancer-promoter interactions has focused on cofactors that lack DNA binding capabilities and bridge enhancer-bound transcription factors and the promoter-bound transcription apparatus (Allen and Taatjes, 2015; Deng et al., 2012; Jeronimo et al., 2016; Kagey et al., 2010; Malik and Roeder, 2010, 2016; Petrenko et al., 2016; Phillips-Cremins et al., 2013). Some enhancer-promoter interactions are likely determined by the nature of transcription factors bound at the two sites (Muerdter and Stark, 2016), but there is little understanding of the rules that govern these interactions.
Recent studies have revealed that specific chromosome structures play important roles in gene control. Enhancer-promoter interactions generally occur within larger chromosomal loop structures formed by the interaction of CTCF proteins bound to each of the loop anchors (Dekker and Mirny, 2016; Fraser et al., 2015; Gibcus and Dekker, 2013; Gorkin et al., 2014; Hnisz et al., 2016; Merkenschlager and Nora, 2016). These loop structures, variously called TADs, loop domains, CTCF contact domains and insulated neighborhoods, tend to insulate enhancers and genes within the CTCF-CTCF loops from elements outside those loops (Dixon et al., 2012, 2016; Dowen et al., 2014; Franke et al., 2016; Hnisz et al., 2016; Ji et al., 2016; Lupiáñez et al., 2015; Narendra et al., 2015; Nora et al., 2012; Phillips-Cremins et al., 2013; Rao et al., 2014; Tang et al., 2015). Constraining DNA interactions within CTCF-CTCF loop structures in this manner may facilitate proper enhancer-promoter contacts.
SUMMARY OF THE INVENTIONCTCF does not generally occupy enhancer and promoter elements (Cuddapah et al., 2009; Dixon et al., 2012; Handoko et al., 2011; Ji et al., 2016; Kim et al., 2007; Parelho et al., 2008; Phillips-Cremins et al., 2013; Rao et al., 2014; Rubio et al., 2008; Tang et al., 2015; Wendt et al., 2008), but where CTCF does bind these elements, it may engender enhancer-promoter interactions (Guo et al., 2015; Lee et al., 2017; Splinter et al., 2006; de Wit et al., 2015). This consideration led us to further investigate the class of human genes that contain CTCF-bound sites at promoters to learn whether these are utilized to facilitate contacts with enhancers via CTCF-CTCF interactions. We report here that 2000 human genes have highly conserved promoter-proximal sites that are bound by CTCF regardless of the cell type examined and that these sites can form contacts with diverse cell-type specific enhancers. These genes appear to have evolved CTCF enhancer-docking sites in order to facilitate contacts with the diverse CTCF-bound enhancers formed by cell-type specific transcription factors during development, and thus experience activation in a broad range of cell types. Interestingly, this set of genes with CTCF-bound enhancer-docking sites includes many important cancer-associated genes, and the enhancer-docking site of one of these, MYC, was studied in detail.
Elevated expression of the c-MYC transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome (Berns et al., 2013; Grotzer et al, 2001; Nesbit et al., 1999; Rao et al., 1998; Dang, 2012). There has been considerable interest in understanding the mechanisms responsible for aberrant transcriptional regulation of MYC in tumor cells. Promoter-proximal regulatory sequences were identified in early studies, but these were not sufficient to recapitulate endogenous patterns of MYC expression (Lavenu et al., 1994; Wierstra, 2008). Subsequent reports noted putative regulatory elements that occur over 1 megabase (Mb) away from MYC, suggesting that distal elements might be involved in MYC regulation (Ahmadiyeh et al., 2010; Hallikas et al., 2006; Pomerantz et al., 2009; Sotelo et al., 2010; Tuupanen et al., 2009; Wright et al., 2010; Yochum et al., 2008). Recent studies have described large tumor-specific super-enhancers in the 3 Mb region surrounding the MYC gene (Chapuy et al, 2013; Herranz et al., 2014; Hnisz et al., 2013; Lin et al., 2016; Wang et al., 2015; Xiang et al., 2014; Zhang et al, 2015). It is not clear how these large enhancer clusters, which differ in size, composition and distance from MYC, all accomplish the same task of stimulating MYC overexpression in a broad spectrum of tumors. Molecular features common to these regulatory elements might prove to be valuable for therapeutic targeting in cancer.
Genetic and epigenetic perturbation of the MYC enhancer-docking site reduces CTCF binding, super-enhancer interaction, MYC gene expression and tumor cell proliferation. These observations reveal a mechanism for enhancer-promoter interaction that is employed during development to allow genes to have cell-specific contacts with diverse enhancers and is exploited by cancer cells to facilitate oncogenic expression of genes driven by diverse super-enhancers.
We show here that diverse tumor-specific super-enhancers acquired throughout the 3 Mb MYC insulated neighborhood functionally interact with a single conserved site containing densely clustered CTCF motifs in the MYC promoter. CRISPR-mediated deletion analysis shows that this common CTCF site is required for super-enhancer looping to the MYC promoter, high MYC expression and rapid cell proliferation in multiple cancers. Targeted methylation of the MYC enhancer anchor by dCAS9-DNMT3A-3L fusion proteins abrogate CTCF binding with consequent loss of MYC expression, suggesting a common vulnerability and a novel approach for therapeutic targeting. Disruption of CTCF protein binding with the MYC promoter CTCF binding site by compounds including targeted nucleic acid derivatives are predicted to have a similar effect.
Disclosed herein are methods of altering expression of a gene with a promoter region (i.e., within or proximal (e.g., within 2.5 kilobases of the transcription start site) to the promoter) CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by altering the interaction or binding between CTCF protein and CTCF binding site(s) to a promoter region of the gene. In some aspects, the gene is MYC. Generally said alteration will comprise inhibiting or reducing the binding of CTCF protein and CTCF binding site in the promoter region of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), thereby reducing or eliminating expression of the gene. However said alteration may alternatively comprise enhancing the binding of CTCF protein to CTCF binding site in the promoter region of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), thereby enhancing expression. Methods of therapy corresponding to alteration of expression of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) are also encompassed.
Disclosed herein are compositions useful for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site and/or a portion within 200-300 nucleotides of either side of the promoter region CTCF binding site. In some aspects, the disease or condition is cancer.
Also disclosed herein are compositions useful for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition comprises a mixture of proteins (e.g., a fusion protein comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity) and nucleic acid sequences (e.g., one or more guide RNAs, one or more sequences encoding guide RNA). In some embodiments, the composition comprises one or more guide sequences and one or more nucleic acids encoding a fusion protein comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity. In some embodiments, the composition comprises one or more nucleic acids encoding a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity and one or more nucleic acids encoding guide sequences. In some embodiments, the guide sequences are homologous or complementary to at least a portion of the MYC promoter region CTCF binding site and/or a portion within 200-300 nucleotides upstream or downstream of the MYC promoter region CTCF binding site. In some embodiments, the guide sequences are homologous or complementary to at least a portion of a promoter region CTCF binding site listed in Table S1 and/or a portion within 200-300 nucleotides upstream or downstream of the promoter region CTCF binding site listed in Table S1. In some embodiments, the guide sequences are homologous or complementary to at least a portion of a promoter region CTCF binding site for TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, or CSNK1A1 and/or a portion within 200-300 nucleotides upstream or downstream of the promoter region CTCF binding site for TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, or CSNK1A1. In some aspects, the disease or condition is cancer.
In some aspects, the effector domain comprises DNMT3A-3L or DNMT3A-3L lacking the 5′ nuclear localization signal (NLS) domain. In some aspects, the catalytically inactive site specific nuclease is a catalytically inactive Cas (e.g., Cas9); in other aspects the catalytically inactive site specific nuclease is a catalytically inactive Cpf1. In some aspects, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DNMT3A-3L or dCas9-DNMT3A-3L lacking the 5′ NLS.
Also disclosed herein are methods for modulating methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a cell comprising introducing into the cell a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby methylating or demethylating the promoter region CTCF binding site. In some aspects, methylation of the promoter region CTCF binding site of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by about 2-fold, 2.5-fold, 2.7-fold, 3.0 fold, or 4.0 fold or more. In some aspects, methylation of the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. In some aspects, methylation of CpG in the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more.
Also disclosed herein are methods of modulating the expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a subject in need thereof comprising introducing into the subject a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby modulating the expression of mRNA of the gene in cells of the subject. In some aspects, expression of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some aspects, protein expression is increased or decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
In some aspects, the effector domain comprises DNMT3A-3L. In some aspects, the catalytically inactive site specific nuclease is a catalytically inactive Cas (e.g., Cas9). In some aspects, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DNMT3A-3L or dCas9-DNMT3A-3L without the 5′ NLS.
In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 genomic sequences (e.g., regions of the MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S 1promoter region CTCF binding site) are modified in the cell. In some embodiments, the genomic sequences to be modified are CTCF binding motifs. The cell may be a stem cell, a neuron, a post-mitotic cell, or a fibroblast. In some aspects, the cell is a human cell or a mouse cell. In some aspects, the cell is a cancer cell.
In certain embodiments, the methods further comprise introducing the cell into a non-human mammal. The non-human mammal may be a mouse.
Also disclosed are isolated modified cells produced by the methods described herein.
Also disclosed herein are methods of treating a subject in need thereof, comprising administering to the subject a composition that suppresses, reduces or eliminates the binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some aspects the subject is human. In some aspects, the subject has cancer. In some aspects the cancer is colorectal cancer, leukemia or breast cancer.
Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising contacting a cell with a test agent; and measuring methylation in the promoter region CTCF binding site, wherein the test agent is identified as a compound that modulates expression if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent differs from the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent.
Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising contacting a cell with a test agent; and measuring binding between CTCF protein and CTCF binding site(s) within the promoter region, wherein the test agent is identified as a compound that modulates expression of the gene if the level of binding between CTCF protein and CTCF binding site(s) within the promoter region in the cell contacted with the test agent differs from the level of binding in a control cell not contacted with the test agent.
In some aspects, the test agent comprises a small molecule. In some aspects, the test agent comprises a nucleic acid. In some aspects, the compound is identified as an anti-cancer agent.
The above discussed, and many other features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange; 10th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, ncbi.nlm nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at omia.angis.org.au/contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.
In some aspects, the invention is directed to compositions for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51), comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site. In one aspect, the invention is directed to compositions for treating a disease or condition involving over-expression of MYC, comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the MYC promoter CTCF binding site.
In some aspects, the invention is directed to compositions for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), comprising one or more nucleic acids encoding a catalytically inactive site specific nuclease fused to an effector domain having methylation activity and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site. In some aspects, the invention is directed to compositions for treating a disease or condition involving over-expression of MYC comprising one or more nucleic acids encoding a catalytically inactive site specific nuclease fused to an effector domain having methylation activity and one or more guide sequences homologous or complementary to at least a portion of the MYC promoter CTCF binding site.
In some aspects, the composition is capable of modulating the expression (e.g., increasing or decreasing the expression) of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by changing the degree of methylation of the promoter region CTCF binding site. In some embodiments, the composition is capable of increasing methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition is capable of decreasing methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition is capable of modulating expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by modulating the binding of CTCF to the promoter region CTCF binding site. In some embodiments, the composition is capable of decreasing binding of CTCF to the promoter region CTCF binding site. In some embodiments, the composition is capable of increasing binding of CTCF to the promoter region CTCF binding site.
In some aspects, the composition is capable of modulating the expression of MYC by changing the degree of methylation of the MYC promoter CTCF binding site. In some aspects, the composition is capable of modulating the expression of MYC by changing the degree of methylation of the MYC promoter CTCF binding site. In some embodiments, the composition is capable of increasing methylation of the MYC promoter CTCF binding site. In some embodiments, the composition is capable of decreasing methylation of the MYC promoter CTCF binding site. In some embodiments, the composition is capable of modulating MYC expression by modulating the binding of CTCF to the MYC promoter CTCF binding site. In some embodiments, the composition is capable of decreasing binding of CTCF to the MYC promoter CTCF binding site. In some embodiments, the composition is capable of increasing binding of CTCF to the MYC promoter CTCF binding site.
Without being limited to theory, it is believed based on the results shown herein that CTCF associates with the MYC promoter region and homodimerizes with a CTCF associated with a MYC enhancer region. The homodimerization forms a promoter-enhancer DNA loop, bringing enhancers into physical proximity with the MYC promoter region and increasing expression of MYC. The degree of methylation of the MYC promoter region modulates CTCF binding and modulates MYC expression.
Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating promoter region CTCF binding site methylation of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51). Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating expression of a gene having a promoter region CTCF binding site. Some aspects of the invention are directed towards a method of treating a patient with a disease or condition involving over-expression or under-expression of a gene having a promoter region CTCF binding site by administering a composition described herein and modulating expression of the gene. In some embodiments, the disease or condition is cancer.
In some embodiments, a CTCF binding site as used herein refers to a domain comprising one or more (e.g., 2, 3, 4, 5, 6 or more) CTCF binding motifs. In some embodiments, the CTCF binding site comprises a nucleotide sequence proximate to (e.g, within about 50 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides) either or both ends of a CTCF binding motif. In some embodiments, the CTCF binding site comprises a domain with one or more (e.g., 2, 3, 4, 5, 6 or more) CTCF binding motifs and a nucleotide sequence proximate to (e.g, within about 50 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides) either or both ends of the one or more CTCF binding motifs. It is understood by a person of skill in the art that not every nucleotide in a promoter region CTCF binding site may interact with CTCF.
Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating MYC promoter CTCF binding site methylation. Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating MYC expression. Some aspects of the invention are directed towards a method of treating a patient with a disease or condition involving MYC over-expression or under-expression by administering a composition described herein and modulating MYC expression. In some embodiments, the disease or condition is cancer and administration of the composition reduces MYC expression.
The MYC promoter CTCF binding site as used herein refers to a domain of the MYC promoter comprising multiple (e.g., 2, 3, 4, 5, 6 or more) CTCF binding motifs. It is understood by a person of skill in the art that not every nucleotide in the MYC promoter CTCF binding site may interact with CTCF.
The terms “disease,” “disorder” or “condition” are used interchangeably and may refer to any alteration from a state of health and/or normal functioning of an organism, e.g., an abnormality of the body or mind that causes pain, discomfort, dysfunction, distress, degeneration, or death to the individual afflicted. Diseases include any disease known to those of ordinary skill in the art. In some embodiments a disease is a chronic disease, e.g., it typically lasts or has lasted for at least 3-6 months, or more, e.g., 1, 2, 3, 5, 10 or more years, or indefinitely. Disease may have a characteristic set of symptoms and/or signs that occur commonly in individuals suffering from the disease. Diseases and methods of diagnosis and treatment thereof are described in standard medical textbooks such as Longo, D., et al. (eds.), Harrison's Principles of Internal Medicine, 18th Edition; McGraw-Hill Professional, 2011 and/or Goldman's Cecil Medicine, Saunders; 24 edition (Aug. 5, 2011). In certain embodiments a disease is a multigenic disorder (also referred to as complex, multifactorial, or polygenic disorder). Such diseases may be associated with the effects of multiple genes, sometimes in combination with environmental factors (e.g., exposure to particular physical or chemical agents or biological agents such as viruses, lifestyle factors such as diet, smoking, etc.). A multigenic disorder may be any disease for which it is known or suspected that multiple genes (e.g., particular alleles of such genes, particular polymorphisms in such genes) may contribute to risk of developing the disease and/or may contribute to the way the disease manifests (e.g., its severity, age of onset, rate of progression, etc.) In some embodiments a multigenic disease is a disease that has a genetic component as shown by familial aggregation (occurs more commonly in certain families than in the general population) but does not follow Mendelian laws of inheritance, e.g., the disease does not clearly follow a dominant, recessive, X-linked, or Y-linked inheritance pattern. In some embodiments a multigenic disease is one that is not typically controlled by variants of large effect in a single gene (as is the case with Mendelian disorders). In some embodiments a multigenic disease may occur in familial form and sporadically. Examples include, e.g., Parkinson's disease, Alzheimer's disease, and various types of cancer. Examples of multigenic diseases include many common diseases such as hypertension, diabetes mellitus (e.g., type II diabetes mellitus), cardiovascular disease, cancer, and stroke (ischemic, hemorrhagic). In some embodiments a disease, e.g., a multigenic disease is a psychiatric, neurological, neurodevelopmental disease, neurodegenerative disease, cardiovascular disease, autoimmune disease, cancer, metabolic disease, or respiratory disease. In some embodiments the disease or condition involves overexpression of MYC. In some embodiments the disease or condition involves aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1.
In some embodiments, the disease or condition involving over-expression of MYC, or aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1, is cancer which term is generally used interchangeably to refer to a disease characterized by one or more tumors, e.g., one or more malignant or potentially malignant tumors. The term “tumor” as used herein encompasses abnormal growths comprising aberrantly proliferating cells. As known in the art, tumors are typically characterized by excessive cell proliferation that is not appropriately regulated (e.g., that does not respond normally to physiological influences and signals that would ordinarily constrain proliferation) and may exhibit one or more of the following properties: dysplasia (e.g., lack of normal cell differentiation, resulting in an increased number or proportion of immature cells); anaplasia (e.g., greater loss of differentiation, more loss of structural organization, cellular pleomorphism, abnormalities such as large, hyperchromatic nuclei, high nuclear to cytoplasmic ratio, atypical mitoses, etc.); invasion of adjacent tissues (e.g., breaching a basement membrane); and/or metastasis. Malignant tumors have a tendency for sustained growth and an ability to spread, e.g., to invade locally and/or metastasize regionally and/or to distant locations, whereas benign tumors often remain localized at the site of origin and are often self-limiting in terms of growth. The term “tumor” includes malignant solid tumors, e.g., carcinomas (cancers arising from epithelial cells), sarcomas (cancers arising from cells of mesenchymal origin), and malignant growths in which there may be no detectable solid tumor mass (e.g., certain hematologic malignancies). Cancer includes, but is not limited to: breast cancer; biliary tract cancer; bladder cancer; brain cancer (e.g., glioblastomas, medulloblastomas); cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic leukemia and acute myelogenous leukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma; adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastoma; melanoma, oral cancer including squamous cell carcinoma; ovarian cancer including ovarian cancer arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; neuroblastoma, pancreatic cancer; prostate cancer; rectal cancer; sarcomas including angiosarcoma, gastrointestinal stromal tumors, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; renal cancer including renal cell carcinoma and Wilms tumor; skin cancer including basal cell carcinoma and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullary carcinoma. It will be appreciated that a variety of different tumor types can arise in certain organs, which may differ with regard to, e.g., clinical and/or pathological features and/or molecular markers. Tumors arising in a variety of different organs are discussed, e.g., the WHO Classification of Tumours series, 4th ed, or 3rd ed (Pathology and Genetics of Tumours series), by the International Agency for Research on Cancer (IARC), WHO Press, Geneva, Switzerland, all volumes of which are incorporated herein by reference. In some embodiments, the cancer is lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, (e.g., a Non-Hodgkin lymphoma, e.g., diffuse large B-cell lymphoma, Burkitts lymphoma) ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, or uterine cancer. The type of cancer is not limited as long as over-expression of MYC, or aberrant expression of aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1, is exhibited. In some embodiments, the cancer is colorectal cancer, leukemia (e.g., acute T-cell leukemia, Chronic Myeloid Leukemia), or breast cancer. In some embodiments the cancer is neuroblastoma and administering the composition increases methylation of the CTCF binding site within the N-MYC promoter, thereby decreasing expression of N-MYC. In some embodiments the cancer is lung cancer and administering the composition increases methylation of the CTCF binding site within the L-MYC promoter, thereby decreasing expression of L-MYC.
In some embodiments, a method of treating a subject in need of treatment for cancer comprises measuring expression and/or activity of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 in a cancer (e.g., in a sample obtained from a cancer (e.g., a biopsy sample, circulating cancer cells, etc.)); determining that the cancer comprises cells with aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 relative to a reference value; and administering a composition disclosed herein that modulates expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 to the subject. Measuring expression may comprise measuring mRNA or protein. Measuring activity may comprise measuring expression of one or more target genes of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1. In some embodiments a reference value may be a value for a normal, non-cancer cell, e.g., of the same cell type as the cancer cell. In some aspects, a method of treating cancer may further comprise administering a second ant-cancer agent (e.g., a conventional chemotherapy agent, a molecularly targeted therapy, a cancer immunotherapy agent, radiotherapy, or a combination thereof).
In some embodiments a method of treating a subject in need of treatment for cancer comprises measuring expression and/or activity of MYC in a cancer (e.g., in a sample obtained from a cancer (e.g., a biopsy sample, circulating cancer cells, etc.)); determining that the cancer comprises cells that over-express MYC relative to a reference value; and administering a composition disclosed herein that reduces MYC expression to the subject. Measuring expression may comprise measuring MYC mRNA or protein. Measuring MYC activity may comprise measuring expression of one or more MYC target genes. In some embodiments a reference value may be a value for a normal, non-cancer cell, e.g., of the same cell type as the cancer cell. In some embodiments a method comprises determining that the cancer comprises cells that harbor three or more copies of MYC (MYC amplification) or a MYC translocation; and administering a composition disclosed herein that reduces MYC expression to the subject. In some aspects, a method of treating cancer may further comprise administering a second ant-cancer agent (e.g., a conventional chemotherapy agent, a molecularly targeted therapy, a cancer immunotherapy agent, radiotherapy, or a combination thereof).
In some embodiments, the disease or condition involving over-expression of the MYC gene is a proliferative disease such as restenosis or polycystic kidney disease.
By “MYC” as used herein refers to nucleic acid sequences encoding any MYC protein, peptide, or polypeptide having MYC activity. The term “MYC” is also meant to include other MYC encoding sequence, such as MYC isoforms (e.g., N-MYC, L-MYC, etc.), mutant MYC genes, splice variants of MYC genes, and MYC gene polymorphisms. In some embodiments, MYC is NCBI Gene ID 4609
In some embodiments, the MYC promoter CTCF binding site is located 2 kb upstream of the major transcript start site (E. M. Klenova et al., ref (32), incorporated by reference in its entirety). In some embodiments, the MYC promoter CTCF binding site is located at Chr8:128746041-128746751 (Genome build GR37/HG19). In some embodiments, the MYC is N-MYC. In some embodiments, the N-MYC promoter CTCF binding site is located at Chr2:16079556-16080469 (Genome build GR37/HG19). In some embodiments, the MYC is L-MYC. In some embodiments, the L-MYC promoter binding site is located at chr1:40367702-40368974 (Genome build GR37/HG19).
As used herein, the terms “site specific nuclease” and “a targetable nuclease” are used interchangeably. Site specific nucleases and targetable nucleases are known in the art. See U.S. Pat. Pub. Nos. 20140068797, 20140186919, 20140170753 and WO/2014/172470, incorporated herein by reference in their entireties. In some embodiments, a site specific nuclease is a targetable nuclease. In some embodiments, a targetable nuclease is a site specific nuclease. In some embodiments, the site-specific nuclease is a Cas protein. In some embodiments, the site-specific nuclease is catalytically inactive. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein. A variety of CRISPR associated (Cas) genes or proteins which are known in the art can be used in the compositions and methods of the invention and the choice of Cas protein will depend upon the particular situation (e.g., www.ncbi.nlm.nih.gov/gene/?term=cas9). Specific examples of Cas proteins include Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 and Cas10. In a particular aspect, the Cas nucleic acid or protein used in the compositions is Cas9. In some embodiments a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a particular Cas protein, e.g., a particular Cas9 protein, may be selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram positive bacteria or a gram negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus, (e.g., a S. pyogenes, a S. thermophilus) a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter. In some embodiments nucleic acids encoding two or more different Cas proteins, or two or more Cas proteins, may be present in the composition, e.g., to allow for recognition and modification of sites comprising the same, similar or different PAM motifs.
In some embodiments, the Cas protein is Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is Cpf1 from any bacterial species or functional portion thereof. In certain embodiments, a Cpf1 protein is a Francisella novicida U112 protein or a functional portion thereof, a Acidaminococcus sp. BV3L6 protein or a functional portion thereof, or a Lachnospiraceae bacterium ND2006 protein or a functional portion thereof. Cpf1 protein is a member of the type V CRISPR systems. Cpf1 protein is a polypeptide comprising about 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Catalytically inactive Cpf1 is known in the art. See US Pat. Pub. No. 20160208243, incorporated by reference in its entirety.
In some embodiments, the Cas protein is a variant polypeptide with at least about 50%, 60%. 70%, 80%, 90%, 95%, or 99% sequence identity to a naturally occurring Cas protein.
In some embodiments a Cas9 nickase may be generated by inactivating one or more of the Cas9 nuclease domains. In some embodiments, an amino acid substitution at residue 10 in the RuvC I domain of Cas9 converts the nuclease into a DNA nickase. For example, the aspartate at amino acid residue 10 can be substituted for alanine (Cong et al, Science, 339:819-823). Other amino acids mutations that create a catalytically inactive Cas9 protein include mutating at residue 10 and/or residue 840. Mutations at both residue 10 and residue 840 can create a catalytically inactive Cas9 protein, sometimes referred herein as dCas9. In some embodiments, dCas9 is a D10A and a H840A Cas9 mutant that is catalytically inactive.
In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein.
As used herein an “effector domain” is a molecule (e.g., protein) that modulates the expression and/or activation of a genomic sequence (e.g., gene). The effector domain may have methylation activity (e.g., DNA methylation activity). In some aspects, the effector domain targets one or both alleles of a gene. The effector domain can be introduced as a nucleic acid sequence and/or as a protein. In some aspects, the effector domain can be a constitutive or an inducible effector domain. In some aspects, a Cas (e.g., dCas) nucleic acid sequence or variant thereof and an effector domain nucleic acid sequence are introduced into the cell as a chimeric sequence. In some aspects, the effector domain is fused to a molecule that associates with (e.g., binds to) Cas protein (e.g., the effector molecule is fused to an antibody or antigen binding fragment thereof that binds to Cas protein). In some aspects, a Cas (e.g., dCas) protein or variant thereof and an effector domain are fused or tethered creating a chimeric protein and are introduced into the cell as the chimeric protein. In some aspects, the Cas (e.g., dCas) protein and effector domain bind as a protein-protein interaction. In some aspects, the Cas (e.g., dCas) protein and effector domain are covalently linked. In some aspects, the effector domain associates non-covalently with the Cas (e.g., dCas) protein. In some aspects, a Cas (e.g., dCas) nucleic acid sequence and an effector domain nucleic acid sequence are introduced as separate sequences and/or proteins. In some aspects, the Cas (e.g., dCas) protein and effector domain are not fused or tethered.
As shown herein, fusions of a catalytically inactive (D10A; H840A) Cas9 protein (dCas9) tethered with all or a portion of (e.g., biologically active portion of) an (one or more) effector domain create chimeric proteins that can be guided to specific DNA sites by one or more RNA sequences (sgRNA) to modulate activity and/or expression of MYC. In specific aspects, fusions of a dCas9 tethered with all or a portion of an effector domain create chimeric proteins that can be guided to specific DNA sites by one or more RNA sequences to modulate or modify methylation of MYC. As used herein, a “biologically active portion of an effector domain” is a portion that maintains the function (e.g. completely, partially, minimally) of an effector domain (e.g., a “minimal” or “core” domain). The fusion of the Cas9 (e.g., dCas9) with all or a portion of one or more effector domains created a chimeric protein.
Examples of effector domains include a transcription(al) activating domain, a coactivator domain, a transcription factor, a transcriptional pause release factor domain, a negative regulator of transcriptional elongation domain, a transcriptional repressor domain, a chromatin organizer domain, a remodeler domain, a histone modifier domain, a DNA modification domain, a RNA binding domain, a protein interaction input device domain (Grunberg and Serrano, Nucleic Acids Research, 38 (8): 2663-2675 (2010), and a protein interaction output device domain (Grunberg and Serrano, Nucleic Acids Research, 38 (8): 2663-2675 (2010). As used herein a “protein interaction input device” and a “protein interaction output device” refers to a protein-protein interaction (PPI). In some aspect, binding partners are targeted to different sites in the genome using the catalytically inactive Cas protein. The binding partners interact, thereby bringing the targeted loci into proximity.
In some aspects, the effector domain is a DNA modifier. Specific examples of DNA modifiers include 5hmc conversion from 5mC such as Tet1 (Tet1CD); DNA demethylation by Tet1, ACID A, MBD4, Apobec1, Apobec2, Apobec3, Tdg, Gadd45a, Gadd45b, ROS1; DNA methylation by Dnmtl, DNMT3A, Dnmt3b, CpG Methyltransferase M.SssI, and/or M.EcoHK31I. In specific aspects, an effector domain is DNMT3A. In some aspects, the effector domain is the C-terminal domain of DNMT3A (i.e., DNMT3A-C). In some aspects, the DNMT3A-C effector domain is complexed with the C-terminal portion of DNMT3L (DNMT3L-C). In some aspects, a chimeric protein comprising DNMT3A-C and DNMT3L-C (sometimes referred to herein as DNMT3A-3L) is used for the effector domain. In some aspects, DNMT3A-3L is a single chain fusion protein as provided in Siddique, et al. (2013) incorporated herein by reference in its entirety. In some embodiments, the effector domain is DNMT3A-3L without the 5′ NLS. In some embodiments, dCas9 is fused to DNMT3A-3L or DNMT3A-3L without the 5′ NLS.
DNA methylation is established by two de novo DNA methyltransferases (DNMT3A/B), and is maintained by DNMT1 (Smith and Meissner, (2013). DNA methylation: roles in mammalian development. Nature reviews Genetics 14, 204-220). Gene activation during development is associated with demethylation of promoter and enhancer sequences. In addition, demethylation can be achieved through oxidation of the methyl group by TET (ten-eleven translocation) dioxygenases to form 5-hydroxymethylcytosine (5-hmC), and then restoration into unmodified cytosines by either DNA replication-dependent dilution or DNA glycosylase-initiated base excision repair (BER), a process termed as active demethylation and proposed to operate during specific developmental stages such as preimplantation embryos or in post-mitotic neurons.
In one aspect of the invention, fusion of the dCas9 to an effector domain can be to that of a single copy or multiple/tandem copies of full-length or partial-length effectors. Other fusions can be with split (functionally complementary) versions of the effector domains. In some embodiments, the effector domain can include full-length or partial-length effectors from more than one effector (e.g., DNMT3A and DNMT3L). Effector domains for use in the methods include any one of the following classes of proteins: proteins that mediate drug inducible looping of DNA and/or contacts of genomic loci, proteins that aid in the three-dimensional proximity of genomic loci bound by dCas9 with different sgRNA.
Other examples of effector domains are described in PCT Application No. PCT/US2014/034387 and U.S. application Ser. No. 14/785,031, which are incorporated herein by reference in their entirety.
In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having DNA methylation activity is dCas9-DMNT3A-3L or dCas9-DNMT3A-3L without the 5′ NLS.
In some aspects the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a nucleic acid sequence that encodes a fusion protein (chimeric protein) comprising all or a portion of a Cas (e.g., dCas) protein fused to all or a portion of one or more effector domains. In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a fusion protein comprising all or a portion of a Cas (e.g., dCas) protein fused to all or a portion of one or more effector domains. In some aspects all or a portion of the Cas (e.g., dCas) protein targets but does not cleave a nucleic acid sequence. In some aspects, the Cas (e.g., dCas) protein can be fused to the N-terminus or C-terminus of one or more effector domains. In some aspects, the portion of the effector domain modulates the methylation of the genomic sequence (e.g., demethylates or methylates the genomic sequence). In some aspects, the effector domain comprises the c-terminal portions of DNMT3A and DNMT3L. In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a fusion protein comprising dCas9-DNMT3A-3L or dCas9-DNMT3A-3L without the 5′ NLS.
In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9), an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more guide sequences. In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) nucleic acids encoding a catalytically inactive nuclease (e.g., dCas9), an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more guide sequences.
In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a mixture of nucleic acids and polypeptides. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9), an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more nucleic acids encoding one or more guide sequences. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9) and effector (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) fusion protein and one or more nucleic acids encoding one or more guide sequences. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) one or more nucleic acids encoding a catalytically inactive nuclease (e.g., dCas9) and an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more nucleic acids encoding one or more guide sequences. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9) and effector (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) fusion protein and one or more nucleic acids encoding one or more guide sequences. Every combination of encoding nucleic acids (e.g., encoding a catalytically inactive site specific nuclease, effector, catalytically inactive nuclease-effector fusion protein, and/or guide sequence) with or without non-encoded components (e.g., a catalytically inactive site specific nuclease, effector, catalytically inactive nuclease-effector fusion protein, and/or guide sequence) having the capability to modulate MYC expression are contemplated herein.
In some aspects, the nucleic acid sequence encoding the fusion protein and/or the one or more guide sequences are isolated. An “isolated,” “substantially pure,” or “substantially pure and isolated” nucleic acid sequence, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA or cDNA library). For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. An “isolated,” “substantially pure,” or “substantially pure and isolated” protein (e.g., chimeric protein; fusion protein), as used herein, is one that is separated from or substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system, or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example, as determined by agarose gel electrophoresis or column chromatography such as HPLC. Preferably, an isolated nucleic acid molecule comprises at least about 50%, 80%, 90%, 95%, 98% or 99% (on a molar basis) of all macromolecular species present.
In one aspect, fusion of catalytically inactive site specific nuclease (e.g, a catalytically inactive Cas protein) with all or a portion of one or more effector domains comprise one or more linkers. As used herein, a “linker” is something that connects or fuses two or more moieties (e.g see Hermanson, Bioconjugate Techniques, 2nd Edition, which is hereby incorporated by reference in its entirety). As will be appreciated by one of ordinary skill in the art, a variety of linkers can be used. In one aspect, a linker comprises one or more amino acids. In some aspects, a linker comprises two or more amino acids. In one aspect, a linker comprises the amino acid sequence GS. In some aspects, fusion of Cas9 (e.g., dCas9) with two or more effector domains comprises one or more interspersed linkers (e.g., GS linkers) between the domains. In some aspects, one or more nuclear localization sequences may be located between the catalytically inactive nuclease (e.g., dCas9) and the effector domain. For example, a fusion protein may include dCas9-NLS-DNMT3A or dCas9-NLS-DNMT3A-3L. In some embodiments, the one or more nuclear localization sequences may be located anywhere in the fusion protein. In some embodiments, the fusion protein does not comprise a NLS, or does not comprise an NLS located between the catalytically inactive nuclease (e.g., dCas9) and the effector domain.
In some aspects, one or more guide sequences include sequences that recognize DNA in a site-specific manner. For example, guide sequences can include guide ribonucleic acid (RNA) sequences utilized by a CRISPR system or sequences within a TALEN or zinc finger system that recognize DNA in a site-specific manner. In some embodiments, the guide sequences comprise a portion that is complementary or homologous to a portion of each of the one or more genomic sequences and comprise a binding site for the catalytically inactive site specific nuclease. In some embodiments, one or more guide sequences do not comprise a binding site for the catalytically inactive site specific nuclease. In some embodiments, one or more guide sequences comprise a moity that blocks (e.g., sterically blocks) CTCF binding when the one or more guide sequences is bound to genomic sequences. In some embodiments, the guide sequence is referred to as guide RNA (gRNA) or single guide RNA (sgRNA).
In some aspects, a single guide sequence can be complementary or homologous to one or more (e.g., all) of the genomic sequences that are being modulated or modified. In one aspect, a single guide is complementary or homologous to a single target genomic sequence. In a particular aspect in which two or more target genomic sequences are to be modulated or modified, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) guide sequences are introduced wherein each guide sequence is complementary or homologous to (specific for) one target genomic sequence. In some aspects, two or more, three or more, four or more, five or more, or six or more guide sequences are complementary or homologous to (specific for) different parts of the same target sequence. In one aspect, two or more guide sequences bind to different sequences of the same region of DNA. In some aspects, a single guide sequence is complementary or homologous to at least two target or more (e.g., all) of the genomic sequences. It will also be apparent to those of skill in the art that the portion of the guide sequence that is complementary or homologous to one or more of the genomic sequences and the portion of the guide sequence that binds to the catalytically inactive site specific nuclease can be introduced as a single sequence or as 2 (or more) separate sequences into a cell. In some embodiments the sequence that binds to the catalytically inactive site specific nuclease comprises a stem-loop.
In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a portion of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a nucleotide sequence of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) and/or a sequence within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the promoter region CTCF binding site. The portion of the guide sequence homologous or complementary to the promoter region CTCF binding site or adjacent sequence (e.g., within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the promoter region CTCF binding site) can comprise 15 nt or more, 18 nt or more, or 20 nt or more.
In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a portion of the MYC promoter CTCF binding site. In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a nucleotide sequence of the MYC promoter CTCF binding site and/or a sequence within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the MYC promoter CTCF binding site. The portion of the guide sequence homologous or complementary to the MYC promoter CTCF binding site or adjacent sequence (e.g., within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the MYC promoter CTCF binding site) can comprise 15 nt or more, 18 nt or more, or 20 nt or more.
In some embodiments, guide sequence used to modify gene expression (e.g., MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 gene expression) is a naturally occurring RNA sequence, a modified RNA sequence (e.g., a RNA sequence comprising one or more modified bases), a synthetic RNA sequence, or a combination thereof. As used herein a “modified RNA” is an RNA comprising one or more modifications (e.g., RNA comprising one or more non-standard and/or non-naturally occurring bases and/or modifications to the backbone, internucleoside linkage(s) and/or sugar). Methods of modifying bases of RNA are well known in the art. Examples of such modified bases include those contained in the nucleosides 5-methylcytidine (5mC), pseudouridine (Ψ), 5-methyluridine, 2′O-methyluridine, 2-thiouridine, N-6 methyladenosine, hypoxanthine, dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G). It should be noted that any number of bases, sugars, or backbone linkages in a RNA sequence can be modified in various embodiments. It should further be understood that combinations of different modifications may be used. In some embodiments an RNA comprises one or more modifications selected from: phosphorothioate, 2′-OMe, 2′-F, 2′-constrained ethyl (2′-cEt), 2′-OMe 3′ phosphorothioate (MS), and 2′-OMe 3-thioPACE (MSP) modifications. In some embodiments a modification may stabilize the RNA and/or increase its binding affinity to a complementary sequence.
In some embodiments, the one or more guide sequences comprise at least one locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units, such as from about 3-7 or 4-8 LNA units, or 3, 4, 5, 6 or 7 LNA units. In some embodiments, all the nucleotides of the one or more guide sequences are LNA. In some embodiments, the one or more guide sequences may comprise both beta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof. In some embodiments all LNA cytosine units are 5′methyl-cytosine.
In some aspects, the RNA sequence is a morpholino. Morpholinos are typically synthetic molecules, of about 25 bases in length and bind to complementary sequences of RNA by standard nucleic acid base-pairing. Morpholinos have standard nucleic acid bases, but those bases are bound to morpholine rings instead of deoxyribose rings and are linked through phosphorodiamidate groups instead of phosphates. Morpholinos do not degrade their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates, siRNA). Instead, morpholinos act by steric blocking and bind to a target sequence within a RNA and block molecules that might otherwise interact with the RNA.
In some embodiments, an RNA sequence can vary in length from about 8 base pairs (bp) to about 200 bp. In some embodiments, each of the one or more guide sequences can be about 9 to about 190 bp; about 10 to about 150 bp; about 15 to about 120 bp; about 20 to about 100 bp; about 30 to about 90 bp; about 40 to about 80 bp; about 50 to about 70 bp in length.
Chemical modifications and methods of synthesizing guide RNAs (guide sequences) are known in the art. See WO/2016/164356, herein incorporated by reference in its entirety.
The portion of each genomic sequence (e.g., MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 promoter region CTCT binding sequence) to which each guide sequence is complementary or homologous to can also vary in size. In particular aspects, the portion of each genomic sequence to which the guide sequence is complementary or homologous to can be about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38 39, 40, 41, 42, 43, 44, 45, 46 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 81, 82, 83, 84, 85, 86, 87 88, 89, 90, 81, 92, 93, 94, 95, 96, 97, 98, or 100 nucleotides (contiguous nucleotides) in length. In some embodiments, each guide sequence can be at least about 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc. identical, complementary or similar to the portion of each genomic sequence. In some embodiments, each guide sequence is completely or partially identical, complementary or similar to each genomic sequence. For example, each guide sequence can differ from perfect complementarity or homology to the portion of the genomic sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. nucleotides. In some embodiments, one or more guide sequences are perfectly complementary or homologous (100%) across at least about 10 to about 25 (e.g., about 20) nucleotides of the genomic sequence.
As will be apparent to those of ordinary skill in the art, the one or more RNA sequences can further comprise one or more expression control elements. For example, in some embodiments the RNA sequences comprises a promoter, suitable to direct expression in cells, wherein the portion of the RNA sequence is operably linked to the expression control element(s). The promoter can be a viral promoter (e.g., a CMV promoter) or a mammalian promoter (e.g., a PGK promoter). The RNA sequence can comprise other genetic elements, e.g., to enhance expression or stability of a transcript. In some embodiments the additional coding region encodes a selectable marker (e.g., a reporter gene such as green fluorescent protein (GFP)).
As described herein, the one or more guide sequences also comprise a (one or more) binding site for a (one or more) catalytically inactive site specific nuclease. The catalytically inactive site specific nuclease may be a catalytically inactive CRISPR associated (Cas) protein. In a particular aspect, upon hybridization of the one or more guide sequences to the one or more genomic sequences, the catalytically inactive site specific nuclease binds to the one or more guide sequences.
In some aspects, the guide sequences are ribonucleic acid guide sequences. In some aspects, each guide sequence is from about 10 base pairs to about 150 base pairs in length. In some aspects, the composition comprises at least two guide sequences. In some aspects, the compositions and methods disclosed herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more guide sequences. In some embodiments, the compositions and methods disclosed herein can comprise 1, 2 or 5 guide sequences.
In some aspects, the one or more guide sequences comprise a sequence homologous to a sequence selected from the group consisting of SEQ ID NOS. 1-8.
There are various ways that a polypeptide comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity can be delivered to a cell or subject, e.g., by administering a nucleic acid that encodes the polypeptide, which nucleic acid may be, e.g., a viral vector or may be a translatable nucleic acid (e.g, synthetic modified mRNA. In some embodiments a nucleic acid sequence encoding a polypeptide is codon optimized for expression in mammalian cells, e.g., human cells. Examples of modified mRNA are described in Warren et al. (Cell Stem Cell 7(5):618-30, 2010, Mandal P K, Rossi D J. Nat Protoc. 2013 8(3):568-82, US Pat. Pub. No. 20120046346 and/or PCT/US2011/032679 (WO/2011/130624). Additional examples are found in numerous PCT and US applications and issued patents to Moderna Therapeutics, e.g., PCT/US2011/046861; PCT/US2011/054636, PCT/US2011/054617, U.S. Ser. No. 14/390,100 (and additional patents and patent applications mentioned in these.) In some embodiments the guide sequence can be delivered as a nucleic acid that encodes the guide sequence. In some embodiments a nucleic acid comprises a first portion that encodes a polypeptide comprising a catalytically inactive site-specific nuclease fused to an effector domain and a second portion that encodes a guide RNA. One of ordinary skill in the art will appreciate that a nucleic acid that encodes a polypeptide or RNA may be operably linked to a promoter capable of directing expression in a cell or subject, e.g., a mammalian cell or subject.
Administration may be by any route (e.g., oral, intravenous, intraperitoneal, gavage, topical, transdermal, intramuscular, enteral, subcutaneous), may be systemic or local, may include any dose (e.g., from about 0.01 mg/kg to about 500 mg/kg), may involve a single dose or multiple doses. In some embodiments administration may be performed by direct administration to a tissue or organ (e.g., skin, heart, liver, lung, kidney, brain, eye, muscle, bone, nerve) or tumor. The nucleic acid(s) or protein(s) may be physically associated with, e.g., encapsulated, e.g., in lipid-containing particles, e.g., solid lipid nanoparticles, liposomes, polymeric particles (e.g., PLGA particles). In some embodiments one or more nucleic acids may be administered using a vector (e.g., a viral vector such as an adenoviral vector, lentiviral vector, or adeno-associated virus vector). In some embodiments one or more nucleic acids, proteins, and/or vectors may be combined with a pharmaceutically acceptable carrier to produce a pharmaceutical composition, which may be administered to a subject.
In some embodiments a nucleic acid, polypeptide, or particle may be targeted to cells of a particular type, e.g., cancer cells of a particular type or expressing a particular cell surface marker. For example, a nucleic acid, protein, or a particle comprising a nucleic acid or vector may comprise or be conjugated to a targeting moiety that binds to a marker expressed at the surface of a target cell (e.g., binds to a tumor antigen or a receptor expressed by the target cell). A targeting moiety may comprise, e.g., an antibody or antigen-binding portion thereof, an engineered protein capable of specific binding, a nucleic acid aptamer, a ligand, etc.
In some embodiments, nucleic acids encoding one or more components (e.g., catalytically inactive site specific nuclease, effector domain, catalytically inactive site specific nuclease-effector domain fusion protein, one or more guide sequences) are delivered by one or more viral vectors e.g., a retroviral vector such as a lentiviral vector or gamma retroviral vector, or an adenoviral or AAV vector. In some embodiments, the nucleic acids encoding a catalytically inactive site specific nuclease, effector domain, and/or catalytically inactive site specific nuclease-effector domain fusion protein are codon-optimized for expression in a subject (e.g., human).
In some aspects, the invention is directed towards a composition that inhibits binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition comprises a small molecule or a nucleic acid derivative. In some embodiments, the composition binds to CTCF. In some embodiments, the composition binds to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1).
Also disclosed herein are methods for methylating a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a cell comprising introducing into the cell a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby methylating the promoter region CTCF binding site.
In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein as described herein. In some embodiments, the effector domain having methylation activity is DMNT3A-3L or DMNT3A-3L without a 5′ NLS as described herein. In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS as described herein. In some aspects, expression of a gene with a promoter region CTCF binding site is modulated. In some aspects, expression of a gene with a promoter region CTCF binding site is decreased. In some aspects, expression of a gene with a promoter region CTCF binding site is increased.
In some aspects, the invention is directed towards a composition that inhibits binding of CTCF to the MYC promoter CTCF binding site. In some embodiments, the composition comprises a small molecule or a nucleic acid derivative. In some embodiments, the composition binds to CTCF. In some embodiments, the composition binds to the MYC promoter CTCF binding site.
Also disclosed herein are methods for methylating a MYC promoter CTCF binding site in a cell comprising introducing into the cell a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the MYC promoter CTCF binding site, thereby methylating the MYC promoter CTCF binding site.
In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein as described herein. In some embodiments, the effector domain having methylation activity is DMNT3A-3L as described herein. In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L as described herein. In some aspects, MYC expression is modulated. In some aspects, MYC expression is decreased. In some aspects, MYC expression is increased.
In some embodiments, the guide sequences are ribonucleic acid guide sequences as described herein. In some embodiments, the guide sequence is from about 10 base pairs to about 150 base pairs in length. In some embodiments, the one or more guide sequences comprise two or more guide sequences. In some embodiments, the one or more guide sequences comprise a sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS. 1-8.
The methods described herein can be used to modify or modulate one or more genomic sequences (e.g., MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site) in a variety of cells, which includes somatic cells, stem cells, mitotic or post-mitotic cells, neurons, fibroblasts, or zygotes. A cell, zygote, embryo, or post-natal mammal can be of vertebrate (e.g., mammalian) origin. In some aspects, the vertebrates are mammals or avians. Particular examples include primate (e.g., human), rodent (e.g., mouse, rat), canine, feline, bovine, equine, caprine, porcine, or avian (e.g., chickens, ducks, geese, turkeys) cells, zygotes, embryos, or post-natal mammals. In some embodiments, the cell, zygote, embryo, or post-natal mammal is isolated (e.g., an isolated cell; an isolated zygote; an isolated embryo). In some embodiments, a mouse cell, mouse zygote, mouse embryo, or mouse post-natal mammal is used. In some embodiments, a rat cell, rat zygote, rat embryo, or rat post-natal mammal is used. In some embodiments, a human cell, human zygote or human embryo is used. The methods described herein can be used to modify or modulate one or more genomic sequences (e.g., methylate or demethylate a promoter region CTCF binding site) in a mammal (e.g., a mouse, a human) in vivo.
Stem cells may include totipotent, pluripotent, multipotent, oligipotent and unipotent stem cells. Specific examples of stem cells include embryonic stem cells, fetal stem cells, adult stem cells, and induced pluripotent stem cells (iPSCs) (e.g., see U.S. Published Application Nos. 2010/0144031, 2011/0076678, 2011/0088107, 2012/0028821 all of which are incorporated herein by reference).
Somatic cells may be primary cells (non-immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line capable of prolonged proliferation in culture (e.g., for longer than 3 months) or indefinite proliferation (immortalized cells). Adult somatic cells may be obtained from individuals, e.g., human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art. Somatic cells of use in aspects of the invention include mammalian cells, such as, for example, human cells, non-human primate cells, or rodent (e.g., mouse, rat) cells. They may be obtained by well-known methods from various organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, breast, reproductive organs, muscle, blood, bladder, kidney, urethra and other urinary organs, etc., generally from any organ or tissue containing live somatic cells. Mammalian somatic cells useful in various embodiments include, for example, fibroblasts, Sertoli cells, granulosa cells, neurons, pancreatic cells, epidermal cells, epithelial cells, endothelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), macrophages, monocytes, mononuclear cells, cardiac muscle cells, skeletal muscle cells, etc.
In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a mouse cell. In some embodiments, the cell is a cancer cell as disclosed herein.
In some aspects, methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) is increased by about 2-fold, 2.5-fold, 2.7-fold, 3.0 fold, 3.5-fold, 4.0 fold or more. In some aspects, methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some aspects, methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, methylation of the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. In some aspects, methylation of CpGs in the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. Reporters of genomic methylation are described in U.S. application Ser. No. 15/078,851, which is incorporated herein by reference in its entirety. Any method known in the art may be used to measure genomic methylation and is not limited.
In some aspects, expression of a gene having a promoter region CTCF binding site is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, expression of a gene having a promoter region CTCF binding site is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more. Methods of measuring gene expression are known in the art. Any method known in the art may be used to measure gene expression and is not limited.
In some aspects, methylation of the MYC promoter CTCF binding site is increased by about 2-fold, 2.5-fold, 2.7-fold, 3.0 fold, 3.5-fold, 4.0 fold or more. In some aspects, methylation of the MYC promoter CTCF binding site is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some aspects, methylation of the MYC promoter CTCF binding site is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, methylation of the MYC promoter region CTCF binding site is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. In some aspects, methylation of CpGs in the MYC promoter region CTCF binding site is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more.
In some aspects, MYC expression is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, MYC expression is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more. Methods of measuring MYC expression are known in the art. Any method known in the art may be used to measure MYC expression and is not limited.
In some aspects, the invention is directed to a method of producing a nonhuman mammal carrying modifications in a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising introducing into a zygote or an embryo a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity, and one or more guide sequences. The zygote or the embryo is maintained under conditions in which the guide sequence hybridizes to a portion of each of the one or more genomic sequences, and the catalytically inactive site specific nuclease fused to an effector domain either methylates or demethylates the genomic sequence, thereby producing an embryo having one or more modified genomic sequences. The embryo having one or more modified genomic sequences may be transferred into a foster nonhuman mammalian mother. The foster nonhuman mammalian mother is maintained under conditions in which one or more offspring carrying the one or more modified genomic sequences are produced, thereby producing a nonhuman mammal carrying modifications in one or more genomic sequences.
In some aspects, the invention is directed to a method of producing a nonhuman mammal carrying modifications in a MYC promoter CTCF binding site comprising introducing into a zygote or an embryo a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity, and one or more guide sequences. The zygote or the embryo is maintained under conditions in which the guide sequence hybridizes to a portion of each of the one or more genomic sequences (e.g., MYC promoter CTCF binding site), and the catalytically inactive site specific nuclease fused to an effector domain either methylates or demethylates the genomic sequence (e.g., MYC promoter CTCF binding site), thereby producing an embryo having one or more modified genomic sequences. The embryo having one or more modified genomic sequences (e.g., MYC promoter CTCF binding site) may be transferred into a foster nonhuman mammalian mother. The foster nonhuman mammalian mother is maintained under conditions in which one or more offspring carrying the one or more modified genomic sequences (e.g., MYC promoter CTCF binding site) are produced, thereby producing a nonhuman mammal carrying modifications in one or more genomic sequences (e.g., MYC promoter CTCF binding site).
As will be apparent to those of skill in the art, the nonhuman mammals can also be produced using methods described herein and/or with conventional methods, see for example, U.S. Published Application No. 2011/0302665. A method of producing a non-human mammalian embryo can comprise injecting non-human mammalian ES cells (e.g., iPSCs) into non-human tetraploid blastocysts and maintaining said resulting tetraploid blastocysts under conditions that result in formation of embryos, thereby producing a non-human mammalian embryo. In some embodiments, said non-human mammalian cells are mouse cells and said non-human mammalian embryo is a mouse. In some embodiments, said mouse cells are mutant mouse cells and are injected into said non-human tetraploid blastocysts by microinjection. In some embodiments laser-assisted micromanipulation or piezo injection is used. In some embodiments, a non-human mammalian embryo comprises a mouse embryo.
Another example of such conventional techniques is two step cloning which involves introducing embryonic stem (ES) and/or induced pluripotent stem (iPS) cells into a blastocyst (e.g., a tetraploid blastocyst) and maintaining the blastocyst under conditions that result in development of an embryo. The embryo is then transferred into an appropriate foster mother, such as a pseudopregnant female (e.g., of the same species as the embryo). The foster mother is then maintained under conditions that result in development of live offspring.
Another example is the use of the tetraploid complementation assay in which cells of two mammalian embryos are combined to form a new embryo (Tarn and Rossant, Develop, 750:6156-6163 (2003)). The assay involves producing a tetraploid cell in which every chromosome exists fourfold. This is done by taking an embryo at the two-cell stage and fusing the two cells by applying an electrical current. The resulting tetraploid cell continues to divide, and all daughter cells will also be tetraploid. Such a tetraploid embryo develops normally to the blastocyst stage and will implant in the wall of the uterus. In the tetraploid complementation assay, a tetraploid embryo (either at the morula or blastocyst stage) is combined with normal diploid embryonic stem cells (ES) from a different organism. The embryo develops normally; the fetus is exclusively derived from the ES cell, while the extraembryonic tissues are exclusively derived from the tetraploid cells.
Another conventional method used to produce nonhuman mammals includes pronuclear microinjection. DNA is introduced directly into the male pronucleus of a nonhuman mammal egg just after fertilization. Similar to the two-step cloning described above, the egg is implanted into a pseudopregnant female. Offspring are screened for the integrated transgene. Heterozygous offspring can be subsequently mated to generate homozygous animals.
A variety of nonhuman mammals can be used in the methods described herein. For example, the nonhuman mammal can be a rodent (e.g., mouse, rat, guinea pig, hamster), a nonhuman primate, a canine, a feline, a bovine, an equine, a porcine or a caprine.
In some aspects, various mouse strains and mouse models of human disease are used in conjunction with the methods of producing a nonhuman mammal carrying mutations or other modifications (e.g., altered methylation) in one or more target nucleic acid sequences described herein (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site). One of ordinary skill in the art appreciates the thousands of commercially and non-commercially available strains of laboratory mice for modeling human disease. Mice models exist for diseases such as cancer, cardiovascular disease, autoimmune diseases and disorders, inflammatory diseases, diabetes (type 1 and 2), neurological diseases, and other diseases. Examples of commercially available research strains include, and is not limited to, 11BHSD2 Mouse, GSK3B Mouse, 129-E Mouse HSD1 1B1 Mouse, AK Mouse Immortomouse®, Athymic Nude Mouse, LCAT Mouse, B6 Albino Mouse, Lox-1 Mouse, B6C3F1 Mouse, Ly5 Mouse, B6D2F1 (BDF1) Mouse, MMP9 Mouse, BALB/c Mouse, NIH-III Nude Mouse, BALB/c Nude Mouse, NOD Mouse, NOD SCID Mouse, Black Swiss Mouse, NSE-p25 Mouse, C3H Mouse, NU/NU Nude Mouse, C57BL/6-E Mouse, PCSK9 Mouse, C57BL/6N Mouse, PGP Mouse (P-glycoprotein Deficient), CB6F1 Mouse, repTOP™ ERE-Luc Mouse, CD-I® Mouse, repTOP™ mitolRE Mouse, CD-I® Nude Mouse, repTOP™ PPRE-Luc Mouse, CD1-E Mouse, Rip-HAT Mouse, CD2F1 (CDF1) Mouse, SCID Hairless Congenic (SHC™) Mouse, CF-1TM Mouse, SCID Hairless Outbred (SHO™) Mouse, DBA/2 Mouse, SJL-E Mouse, Fox Chase CB17™ Mouse, SKH1-E Mouse, Fox Chase SCID® Beige Mouse, Swiss Webster (CFW®) Mouse, Fox Chase SCID® Mouse, TARGATT™ Mouse, FVB Mouse, THE POUND MOUSE™, and GLUT 4 Mouse. Other mouse strains include BALB/c, C57BL/6, C57BL/10, C3H, ICR, CBA, A/J, NOD, DBA/1, DBA/2, MOLD, 129, HRS, MRL, NZB, NIH, AKR, SJL, NZW, CAST, KK, SENCAR, C57L, SAMR1, SAMP1, C57BR, and NZO.
In some aspects, the method of producing a nonhuman mammal carrying modifications in one or more genomic sequences (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site) further comprises mating one or more commercially and/or non-commercially available nonhuman mammal with the nonhuman mammal carrying modifications in one or more genomic sequences (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site) produced by the methods described herein. The invention is also directed to nonhuman mammals produced by the methods described herein.
As will be apparent to those of skill in the art, a variety of methods can be used to introduce nucleic acid and/or protein into a cell, zygote, embryo, and or mammal. Suitable methods include calcium phosphate or lipid-mediated transfection, electroporation, injection, and transduction or infection using a vector (e.g., a viral vector such as an adenoviral vector, lentiviral vector, or adeno-associated viral vector). In some aspects, the nucleic acid and/or protein is complexed with a vehicle, e.g., a cationic vehicle, that facilitates uptake of the nucleic acid and/or protein, e.g., via endocytosis.
The method described herein can further comprise isolating the cell or zygote produced by the methods. Thus, in some aspects, the invention is directed to a cell or zygote (an isolated cell or zygote) produced by the methods described herein. In some aspects, the disclosure provides a clonal population of cells harboring the modification(s), replicating cultures comprising cells harboring the modification(s) and cells isolated from the generated animals.
The methods described herein can further comprise crossing the generated animals with other animals harboring genetic modifications (optionally in same strain background) and/or having one or more phenotypes of interest (e.g., disease susceptibility—such as NOD mice). In addition, the methods may comprise modifying a cell, zygote, and/or animal from a strain that harbors one or more genetic modifications and/or has one or more phenotypes of interest (e.g., disease susceptibility). In some aspects, the genetic modifications are epigenetic modifications.
The methods described herein can further comprise assessing whether the one or more target nucleic acids have been modified and/or modulated using a variety of known methods.
In some embodiments methods described herein are used to produce multiple genetic modifications in a cell, zygote, embryo, or animal, wherein at least one of the genetic modifications methylates or demethylates a CTCF region binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) promoter region CTCF binding site, and at least one of the genetic modifications is in a different gene or genomic location. In some embodiments, a genetic modification includes epigenetic modifications. The resulting cell, zygote, embryo, or animal, or a cell, zygote, embryo, or animal generated therefrom, is analyzed. In some embodiments at least one of the genetic modifications may be conditional (e.g., the effect of the modification, such as gene methylation or demethylation, only becomes manifest under certain conditions, which are typically under control of the artisan). In some embodiments animals are permitted to develop at least to post-natal stage, e.g., to adult stage. The appropriate conditions for the modification to produce an effect (sometimes termed “inducing conditions”) are imposed, and the phenotype of the animal is subsequently analyzed. A phenotype may be compared to that of an unmodified animal or to the phenotype prior to the imposition of the inducing conditions.
Analysis may comprise any type of phenotypic analysis known in the art, e.g., examination of the structure, size, development, weight, or function, of any tissue, organ, or organ system (or the entire organism), analysis of behavior, activity of any biological pathway or process, level of any particular substance or gene product, etc. In some embodiments analysis comprises gene expression analysis, e.g., at the level of mRNA or protein. In some embodiments such analysis may comprise, e.g., use of microarrays (e.g., oligonucleotide microarrays, sometimes termed “chips”), high throughput sequencing (e.g., RNASeq), ChIP on Chip analysis, ChlPSeq analysis, etc. In some embodiments high content screening may be used, in which elements of high throughput screening may be applied to the analysis of individual cells through the use of automated microscopy and image analysis (see, e.g., Zanella et al, (2010). High content screening: seeing is believing. Trends Biotechnol. 28:237-245). In some embodiments analysis comprises quantitative analyses of components of cells such as spatio-temporal distributions of individual proteins, cytoskeletal structures, vesicles, and organelles, e.g., when contacted with test agents, e.g., chemical compounds. In some embodiments activation or inhibition of individual proteins and protein-protein interactions and/or changes in biological processes and cell functions may be assessed. A range of fluorescent probes for biological processes, functions, and cell components are available and may be used, e.g., with fluorescence microscopy. In some embodiments cells or animals generated according to methods herein may comprise a reporter, e.g., a fluorescent reporter or enzyme (e.g., a luciferase such as Gaussia, Renilla, or firefly luciferase) that, for example, reports on the expression or activity of particular genes. Such reporter may be fused to a protein, so that the protein or its activity is rendered detectable, optionally using a non-invasive detection means, e.g., an imaging or detection means such as PET imaging, MRI, fluorescence detection. Multiplexed genome editing according to the invention may allow installation of reporters for detection of multiple proteins, e.g., 2-20 different proteins, e.g., in a cell, tissue, organ, or animal, e.g., in a living animal.
Multiplexed genome editing or modification according to the present invention may be useful to determine or examine the biological role(s) and/or roles in disease of genes of unknown function. For example, discovery of synthetic effects caused by modifications in first and second genes (e.g., wherein one of the modifications comprises altered methylation of a CTCF region binding site of a gene) may pinpoint a genetic or biochemical pathway in which such gene(s) or encoded gene product(s) is involved.
In some embodiments it is contemplated to use, in methods described herein, cells or zygotes generated in or derived from animals produced in projects such as the International Knockout Mouse Consortium (IKMC), the website of which is http://www.knockoutmouse.org). In some embodiments it is contemplated to cross animals generated as described herein with animals generated by or available through the IKMC. For example, in some embodiments a mouse gene to be modified according to methods described herein is any gene from the Mouse Genome Informatics (MGI) database for which sequences and genome coordinates are available, e.g., any gene predicted by the NCBI, Ensembl, and Vega (Vertebrate Genome Annotation) pipelines for mouse Genome Build 37 (NCBI) or Genome Reference Consortium GRCm38.
In some embodiments, a gene or genomic location to be modified is included in a genome of a species for which a fully sequenced genome exists. Genome sequences may be obtained, e.g., from the UCSC Genome Browser (http://genome.ncsc.edu/index.html). For example, in some embodiments a human gene or sequence to be modified according to methods described herein may be found in Human Genome Build hg19 (Genome Reference Consortium). In some embodiments a gene is any gene for which a Gene ID has been assigned in the Gene Database of the NCBI (http://www.ncbi.nlm.nih.gov/gene). In some embodiments a gene is any gene for which a genomic, cDNA, mRNA, or encoded gene product (e.g., protein) sequence is available in a database such as any of those available at the National Center for Biotechnology Information (www.nchi.nih.gov) or Universal Protein Resource (www.uniprot.org). Databases include, e.g., GenBank, RefSeq, Gene, UniProtKB/SwissProt, UniProtKB/Tremb1, and the like.
In some embodiments animals generated according to methods described herein may be useful in the identification of candidate agents for treatment of disease and/or for testing agents for potential toxicity or side effects. In some embodiments any method described herein may comprise contacting an animal generated according to methods described herein, e.g., any genetically modified animal generated as described herein, with a test agent (e.g., a small molecule, nucleic acid, polypeptide, lipid, etc.). In some embodiments contacting comprises administering the test agent. Administration may be by any route (e.g., oral, intravenous, intraperitoneal, gavage, topical, transdermal, intramuscular, enteral, subcutaneous), may be systemic or local, may include any dose (e.g., from about 0.01 mg/kg to about 500 mg/kg), may involve a single dose or multiple doses. In some embodiments a method may further comprise analyzing the animal. Such analysis may, for example assess the effect of the test agent in an animal having a genetic modification(s) introduced according to the methods. In some embodiments a test agent that reduces or enhances an effect of one or more genetic modification(s) may be identified. In some embodiments if a test agent reduces or inhibits development of a disease associated with or produced by the genetic modification(s), (or reduces or inhibits one or more symptoms or signs of such a disease) the test agent may be identified as a candidate agent for treatment of a disease associated with or produced by the genetic modification(s) or associated with or produced by naturally occurring mutations in a gene or genomic location harboring the genetic modification.
The term “small molecule” refers to an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than abou t 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups. In some embodiments a small molecule is an artificial (non-naturally occurring) molecule. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, the term “small molecule” excludes molecules that are ingredients found in standard tissue culture medium.
In some embodiments a cell may be a diseased cell or may originate from a subject suffering from a disease, e.g., a disease affecting the cell or organ from which the cell was obtained. In some embodiments a mutation is introduced into a genomic region of the cell that is associated with a disease (e.g., any disease of interest, such as diseases mentioned herein). For example, in some embodiments it is of interest to methylate or demethylate a gene or genomic location (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site) that is known or suspected to be involved in disease pathogenesis and/or known or suspected to be associated with increased or decreased risk of developing a disease or particular manifestation(s) of a disease. In some embodiments it is of interest to methylate or demethylate a gene or genomic location (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site) and determine whether such modification alters the risk of developing a disease or one or more manifestations of a disease, alters progression of the disease, or alters the response of a subject to therapy or candidate therapy for a disease. In some embodiments it is of interest to modify an abnormal or disease-associated nucleotide or sequence (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site) to one that is normal or not associated with disease. In some embodiments this may allow production of genetically matched cells or cell lines (e.g., iPS cells or cell lines) that differ only at one or more selected sites of genetic modification (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site). Multiplexed genome editing as described herein may allow for production of cells or cell lines that are isogenic except with regard to, e.g., between 2 and 20 selected sites of genetic alterations (e.g., within a Promoter region CTCF binding site of a gene, within a MYC Promoter CTCF binding site). This may allow for the study of the combined effect of multiple modifications that are suspected of or known to play a role in disease risk, development or progression.
Also disclosed herein are methods of modulating the expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a subject in need thereof comprising introducing into the subject a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby modulating the expression of the gene in cells of the subject. In some embodiments, the effector domain has methylation activity and the expression of a gene with a promoter region CTCF binding site is decreased. In some embodiments, the effector domain has methylation activity and the expression of a gene with a promoter region CTCF binding site is increased. In some embodiments, the effector domain has demethylation activity and the expression of a gene with a promoter region CTCF binding site is increased. In some embodiments, the effector domain has methylation activity and the expression of a gene with a promoter region CTCF binding site is decreased. In some embodiments, the effector domain has demethylation activity and the expression of a gene with a promoter region CTCF binding site is decreased. In some embodiments, the effector domain has methylation activity and MYC expression is decreased. In some embodiments, the effector domain has demethylation activity and MYC expression is increased.
In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein as described herein. In some embodiments, the effector domain having methylation activity is DMNT3A-3L as described herein.
In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS as described herein.
In some embodiments, the guide sequences are ribonucleic acid guide sequences as described herein. In some embodiments, the guide sequence is from about 10 base pairs to about 150 base pairs in length. In some embodiments, the one or more guide sequences comprise two or more guide sequences.
In some embodiments, the one or more guide sequences comprise a sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS. 1-8.
In some aspects, expression of a gene having a promoter region CTCF binding site is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, expression of a gene having a promoter region CTCF binding site is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more. In some aspects, MYC expression is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, MYC expression is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more.
In some aspects, the effector domain comprises DNMT3A-3L. In some aspects, the catalytically inactive site specific nuclease is a catalytically inactive Cas (e.g., Cas9). In some aspects, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DNMT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS.
Some aspects of the disclosure are related to methods of treating a subject in need thereof, comprising administering to the subject a composition that enhances, suppresses, reduces or eliminates the binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51). Also disclosed herein are methods of treating a disease or condition involving aberrant MYC expression in a subject, comprising administering to the subject a composition that modulates the binding of CTCF to a MYC promoter CTCF binding site. In some aspects, aberrant MYC expression is over-expression. In some aspects, aberrant MYC expression is under-expression. In some aspects, the subject has cancer.
In some aspects, the composition is a composition described herein. In some aspects, the composition comprises a nucleic acid sequence, protein, organic molecule, inorganic molecule, or small molecule. In some aspects, the composition reduces binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TGIF1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, composition increases binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some aspects the subject is human. The disease or condition involving aberrant gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) expression in a subject is any disease or condition described herein. In some aspects the disease or condition is cancer as described herein. In some aspects, the cancer is colorectal cancer, leukemia or breast cancer.
Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising, contacting a cell with a test agent; and measuring methylation in the promoter region CTCF binding site, wherein the test agent is identified as a compound that modulates expression of the gene if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent differs from the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent. In some aspects, the test agent is identified as an anti-cancer compound if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent is higher than the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent.
Also disclosed herein are methods of screening for a compound that modulates MYC expression comprising, contacting a cell with a test agent; and measuring methylation in a MYC promoter CTCF binding site, wherein the test agent is identified as a compound that modulates MYC expression if the level of methylation of the MYC promoter CTCF binding site in the cell contacted with the test agent differs from the level of methylation of said MYC promoter CTCF binding site in a control cell not contacted with the test agent. In some aspects, the test agent is identified as an anti-cancer compound if the level of methylation of the MYC promoter CTCF binding site in the cell contacted with the test agent is higher than the level of methylation of said MYC promoter CTCF binding site in a control cell not contacted with the test agent.
Methods of measuring methylation are known in the art and are not limited. In some embodiments the cells used in the method comprise cancer cells.
Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising contacting a cell with a test agent; and measuring binding between CTCF protein and CTCF binding site(s) within the promoter region, wherein the test agent is identified as a compound that modulates expression of the gene if the level of binding between CTCF protein and CTCF binding site(s) within the promoter region in the cell contacted with the test agent differs from the level of binding in a control cell not contacted with the test agent.
Also disclosed herein are methods of screening for a compound that modulates MYC expression comprising contacting a cell with a test agent; and measuring binding between CTCF protein and CTCF binding site(s) within the MYC promoter, wherein the test agent is identified as a compound that modulates MYC expression if the level of binding between CTCF protein and CTCF binding site(s) within the MYC promoter in the cell contacted with the test agent differs from the level of binding in a control cell not contacted with the test agent.
Also disclosed herein are methods of screening for a compound that modulates MYC expression comprising, contacting a cell with a test agent; and measuring binding of CTCF to the MYC promoter CTCF binding site, wherein the test agent is identified as a compound that modulates MYC expression if the level of binding of CTCF to the MYC promoter CTCF binding site in the cell contacted with the test agent differs from the level of binding of CTCF to the MYC promoter CTCF binding site in a control cell not contacted with the test agent. In some aspects, the test agent is identified as an anti-cancer compound if the level of binding of CTCF to the MYC promoter CTCF binding site in the cell contacted with the test agent is lower than the level of binding of CTCF to the MYC promoter CTCF binding site in a control cell not contacted with the test agent. Methods of measuring binding of CTCF to a DNA site of interest are known in the art and are not limited. For example, one could use ChIP-Seq. In some embodiments the cells used in the method comprise cancer cells.
One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more nucleic acids, polypeptides, cells, species or types of organism, disorders, subjects, or combinations thereof, can be excluded.
Where the claims or description relate to a composition of matter, e.g., a nucleic acid, polypeptide, cell, or non-human transgenic animal, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”. “Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered “isolated.”
Specific examples of these methods are set forth below in the Examples.
EXAMPLESProper regulation of gene expression is dependent on specific interactions between enhancers and promoters, but the mechanisms responsible for this specificity are not well-understood. We have identified a class of human genes that utilize CTCF-CTCF interactions to connect different cell-type specific enhancers with a single promoter-proximal element that functions as a docking site for those enhancers. At these genes, the enhancers are often bound by CTCF in a cell-type specific fashion whereas the promoter-proximal enhancer-docking sites are constitutively bound by CTCF. The proto-oncogene MYC, which is controlled by different cell-type specific enhancers during development, is a prominent example of a gene regulated in this fashion. We find that many human cancer cells acquire super-enhancers at the MYC locus and exploit this CTCF-mediated enhancer-docking mechanism to express MYC at oncogenic levels. Genetic and epigenetic perturbation of the MYC enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression and cell proliferation. Additional genes with roles in cancer employ a CTCF-bound enhancer-docking site to engender interactions with tumor specific CTCF-bound enhancers. Thus, a CTCF-dependent enhancer-docking mechanism, which facilitates interaction with cell-specific enhancers during development, is exploited by cancer cells to dysregulate expression of prominent oncogenes. Oncogene enhancer-docking sites can be repressed by dCas9-DNMT—mediated DNA methylation and may thus represent a common vulnerability in multiple human cancers.
CTCF does not generally occupy enhancer and promoter elements (Cuddapah et al., 2009; Dixon et al., 2012; Handoko et al., 2011; Ji et al., 2016; Kim et al., 2007; Parelho et al., 2008; Phillips-Cremins et al., 2013; Rao et al., 2014; Rubio et al., 2008; Tang et al., 2015; Wendt et al., 2008), but where CTCF does bind these elements, it may engender enhancer-promoter interactions (Guo et al., 2015; Lee et al., 2017; Splinter et al., 2006; de Wit et al., 2015). This consideration led us to further investigate the class of human genes that contain CTCF-bound sites at promoters to learn whether these are utilized to facilitate contacts with enhancers via CTCF-CTCF interactions. We report here that 2000 human genes have highly conserved promoter-proximal sites that are bound by CTCF regardless of the cell type examined and that these sites can form contacts with diverse cell-type specific enhancers. These genes appear to have evolved CTCF enhancer-docking sites in order to facilitate contacts with the diverse CTCF-bound enhancers formed by cell-type specific transcription factors during development, and thus experience activation in a broad range of cell types. Interestingly, this set of genes with CTCF-bound enhancer-docking sites includes many important cancer-associated genes, and the enhancer-docking site of one of these, MYC, was studied in detail. Genetic and epigenetic perturbation of the MYC enhancer-docking site reduces CTCF binding, super-enhancer interaction, MYC gene expression and tumor cell proliferation. These observations reveal a mechanism for enhancer-promoter interaction that is employed during development to allow genes to have cell-specific contacts with diverse enhancers and is exploited by cancer cells to facilitate oncogenic expression of genes driven by diverse super-enhancers.
Example 1—Promoter-Proximal CTCF-Bound Sites are Putative Enhancer-Docking SitesTo gain insights into the interactions between genes and their regulatory elements that may be mediated by CTCF, we focused our study on genes that have CTCF-bound sites at their promoters and are expressed in multiple cell types through the activity of different cell-specific enhancers (
Previous studies have established that tumor cells acquire tumor-specific super-enhancers at various sites throughout the MYC locus (
While all cells examined appear to share the TAD-spanning 2.8 Mb loop encompassing MYC, the loop structures within the neighborhood were found to be markedly different among the tumor types. The internal loops were dominated by interactions between a MYC promoter-proximal CTCF site and diverse cell specific super-enhancers (
Further examination of the MYC promoter-proximal region revealed three constitutive CTCF binding sites (
To determine whether the putative enhancer-docking site plays a functional role in MYC expression through DNA loop formation, we used the CRISPR/Cas9 system to delete a 210 bp segment centered on this site in the Chronic Myeloid Leukemia (CML) cell line K562 (
If the putative MYC enhancer-docking site contributes to looping interactions with distal enhancers, then the loss of this site in AK562 cells should cause a decrease in DNA interactions between the MYC promoter and the distal super-enhancers located nearly 2 Mb away in the downstream portion of the insulated neighborhood. We used chromosome conformation capture combined with high-throughput sequencing (4C-seq) to compare the interactions in K562 and AK562 cells (
If the MYC enhancer-docking site functions similarly across a variety of tumor cells, then deletion of the site in various tumor cells should consistently cause reduced MYC expression. Indeed, when the CRISPR/Cas9 system was used to delete the 210 bp segment in colorectal cancer cells (HCT-116), acute T-cell leukemia cells (Jurkat) and breast cancer cells (MCF7), we consistently observed a reduction in MYC transcripts (
Importance of CTCF Motif Sequence in Enhancer-Docking Site
The enhancer-docking site contains multiple putative CTCF motifs, of which one stood out in terms of conservation and JASPAR score (
Loss of MYC Expression Upon Methylation of Enhancer-Docking Site
CTCF binding is abrogated when its sequence motif is methylated (Bell and Felsenfeld, 2000; Maurano et al., 2015), and the MYC enhancer-docking site occurs within a CpG island that is consistently hypomethylated in different tumor types (
MYC Enhancer-Docking Site in Normal Development and Differentiation
It seems unlikely that the MYC enhancer-docking site would have evolved to facilitate the development of diverse cancers, so we explored the possibility that this site might typically be employed during normal development and differentiation, when MYC expression contributes to normal cellular proliferation and different cell-type specific transcription factors establish the different cell states. Examination of enhancer and promoter-capture Hi-C data in a variety of normal cell types that express MYC (Javierre et al., 2016) revealed that cell-type specific enhancers do indeed loop to the MYC enhancer-docking site (
Enhancer-Docking Sites at Additional Genes with Prominent Roles in Cancer
Our initial analysis of putative enhancer-docking sites suggested that additional cancer-associated genes may be regulated in a manner similar to MYC (
We describe here a class of human genes that utilize CTCF-CTCF interactions to connect different cell-type specific enhancers with a single promoter-proximal element that functions as a docking site for those enhancers. These CTCF-mediated enhancer-promoter interactions are generally nested within larger CTCF-mediated loops that function as insulated neighborhoods. At these genes with CTCF-mediated enhancer docking, the enhancers are often bound by CTCF in a cell-type specific fashion whereas the promoter-proximal enhancer-docking sites tend to be constitutively bound by CTCF.
The proto-oncogene MYC, which is controlled by different cell-type specific enhancers during development, is a prominent oncogene and an example of a gene regulated in this fashion. Many different human cancer cells acquire super-enhancers within the ˜3 MB MYC TAD/insulated neighborhood and we show here that these exploit a CTCF-mediated enhancer-docking mechanism to express MYC at oncogenic levels. Because tumor super-enhancers can encompass genomic regions as large as 200 kb, and CTCF occupies sites that occur on average every 10 kb, there is considerable opportunity for super-enhancers to adventitiously contain a CTCF-bound site, which in turn could serve to interact with an enhancer-docking site.
Additional genes with roles in cancer employ this CTCF-mediated enhancer-docking mechanism to engender interactions with tumor-specific enhancers. For example, at CSNK1A1, a drug target in AML tumor cells ({umlaut over (J)}r{dot over (a)}s et al., 2014), VEGFA, which is upregulated in many cancers (Goel and Mercurio, 2013), and RUNX1, a well-defined oncogene in AML (Deltcheva and Nimmo, 2017; Ito et al., 2015), the evidence indicates that super-enhancers in these cancer cells use a CTCF enhancer-docking site to interact with the oncogene. Thus, a CTCF-dependent enhancer-docking mechanism, which presumably facilitates interaction with different cell-specific enhancers during development, is exploited by cancer cells to dysregulate expression of prominent oncogenes.
MYC dysregulation is a hallmark of cancer (Bradner et al., 2017). The c-Myc TF is an attractive target for cancer therapy because of the role that excessive c-Myc levels play in a broad spectrum of aggressive cancers (Felsher and Bishop, 1999; Jain et al., 2002; Soucek et al., 2008, 2013), but direct pharmacologic inhibition of MYC remains an elusive challenge in drug discovery (Bradner et al., 2017). The MYC enhancer-docking site, and presumably those of other oncogenes, can be repressed by dCas9-DNMT-mediated DNA methylation. Oncogene enhancer-docking sites may thus represent a common vulnerability in multiple human cancers.
Materials and Methods:
Star Methods
Experimental Model and Subject Details
Cell Lines
K562 (female), MCF7 (female), HCT-116 (male), Jurkat (male) and HEK293T cells were purchased from ATCC (CCL-243, HTB-22, CCL-247, TIB-152, CRL-3216) and propagated according to ATCC guidelines in RPMI-1640 with GlutaMax (Life Technologies 61870-127) or DMEM, high glucose, pyruvate (Life Technologies 11995-073), supplemented with 10% fetal bovine serum (Sigma). Cells were maintained at 37° C. and 5% CO2.
Method Details
CRISPR/Cas9 Genome Editing
Genome editing was performed using CRISPR/Cas9 essentially as described (Ran et al., 2013; Shalem et al., 2014). The genomic sequences complementary to all guide RNAs are listed in Table S2
For generation of Myc-cover clones, target-specific oligonucleotides were cloned into the pX330 plasmid which carries a U6 promoter, chimeric guide RNA, and a codon-optimized version of Cas9. pX330 was a kind gift of F. Zhang (Cong et al., 2013) (Addgene: 42230). For the generation of Myc-cover line clones, 2 million cells were transfected with 10 ug of DNA with 50 uL of 1 mg/ml PEI and sorted for presence of GFP after 2 days. Individual cells were then propagated in to clonal lines.
For the 210-Δ experiments (
Virus Production and Generation of Cell Lines
For virus production, HEK293T cells grown to 50-75% confluency on a 15 cm dish and then transfected with 15 μg plasmid of interest, 11.25 μg psPAX (Addgene 12260), and 3.75 μg pMD2.G (Addgene 12259). psPAX and pMD2.G were kind gifts of Didier Trono. After 12 hours, media was replaced. Viral supernatant was collected 24 hours after media replacement (36 hrs post transfection) and fresh media was added. Viral supernatant was collected again 48 hours after the media replacement (60 hours post transfection). Viral supernatant was cleared of cells by either centrifugation at 500×g for 10 minutes or filtration through a 0.45-micron filter. The virus was concentrated with Lenti-X concentrator (Clonetech 631231) per manufacturers' instruction. Concentrated virus was resuspended in either DMEM or RPMI (depending on the cell line being infected) and added to 5 million cells in the presence of polybrene (Millipore TR-1003) at 8 ug/mL. After 24 hours, viral media was removed and fresh media containing drug was added. Drug concentrations are as follows: Puromycin (Thermo A1113802) (2 ug/mL), Geneticin (Thermo 10131027) (800 ug/mL), Blasticidin (Invivogen ant-b1-1) (10 ug/mL). Cells were selected until all cells on non-transduced plates died. The viral plasmid containing pGK-MYC-tdTomato was deposited on Addgene (Plasmid #85675).
RNA Isolation and Quantitative RT-PCR
RNA was isolated using the RNeasy, RNeasy plus or AllPrep kit (QIAGEN 74004, 80204) and reverse transcribed using oligo-dT primers (Promega C1101) and SuperScript III reverse transcriptase (Thermo 18080093) according to the manufacturers' instructions. Quantitative real-time PCR was performed on a 7000 AB Detection System using Taqman probes for MYC (Hs00153408_m1) and GAPDH (Hs02758991_g1) in conjunction with Taqman 2× master mix (Thermo 4304437). For detection of endogenous MYC only in experiments utilizing the MYC cover, primers specific to the endogenous copy of MYC (Table S2) were designed against a MYC 3′ UTR region not present in the cover construct and qPCR was conducted with SYBR green PCR master mix (Thermo 4309155).
ChIP-seq
ChIP was performed as described in (Lee et al., 2006) with a few adaptations. ˜30 million K562 cells were crosslinked for 10 min at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution (11% formaldehyde, 50 mM HEPES pH 7.3, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) to the growth media followed by 5 min quenching with 125 mM glycine. Cells were washed twice with PBS, then the supernatant was aspirated and the cell pellet was flash frozen at −80 C. 100 μl of Protein G Dynabeads (Thermo 10003D) were blocked with 0.5% BSA (w/v) in PBS. Magnetic beads were bound with 40 μl of anti-CTCF antibody (Millipore 07-729). Nuclei were isolated as previously described (Lee et al., 2006), and sonicated in lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH8.0, 0.1% SDS, and 1% Triton X-100) on a Misonix 3000 sonicator for 5 cycles at 30s each on ice (18-21 W) with 60 s on ice between cycles. Sonicated lysates were cleared once by centrifugation and incubated overnight at 4° C. with magnetic beads bound with antibody to enrich for DNA fragments bound by the indicated factor. Beads were washed with wash buffer A (50 mM HEPES-KOH pH7.9, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.1% Na-Deoxycholate, 1% Triton X-100, 0.1% SDS), B (50 mM HEPES-KOH pH7.9, 500 mM NaCl, 1 mM EDTA pH 8.0, 0.1% Na-Deoxycholate, 1% Triton X-100, 0.1% SDS), C (20 mM Tris-HCl pH8.0, 250 mMLiCl, 1 mM EDTA pH 8.0, 0.5% Na-Deoxycholate, 0.5% IGEPAL C-630 0.1% SDS) and D (TE with 50 mM NaCl) sequentially. DNA was eluted in elution buffer (50 mM Tris-HCL pH 8.0, 10 mM EDTA, 1% SDS). Cross-links were reversed overnight at 65° C. RNA and protein were digested using RNase A and Proteinase K, respectively and DNA was purified with phenol chloroform extraction and ethanol precipitation. Purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. Libraries for Illumina sequencing were prepared following the Illumina TruSeq DNA Sample Preparation v2 kit. Amplified libraries were size-selected using a 2% gel cassette in the Pippin Prep system from Sage Science set to capture fragments between 200 and 400 bp. Libraries were quantified by qPCR using the KAPA Biosystems Illumina Library Quantification kit according to kit protocols. Libraries were sequenced on the Illumina HiSeq 2500 for 40 bases in single read mode.
4C-seq
A modified version of 4C-seq (van de Werken et al., 2012a, 2012b) was developed. The major change was the ligation is performed in intact nuclei (in situ). This change was incorporated because previous work has noted that in situ ligation dramatically decreases the rate of chimeric ligations and background interactions (Nagano et al., 2015; Rao et al., 2014).
Approximately 5 million K562 cells were resuspended in 5 mL 10% FBS/PBS. 5 mL of 4% formaldehyde in 10% FBS/PBS was added and cells were crosslinked for 10 minutes while rotating at room temperature. Glycine was added to a final concentration of 0.125 M and cells were centrifuged at 300×g for 5 minutes. Cells were washed twice with PBS, transferred to an eppendorf tube, snap frozen and stored at −80. Pellets were gently resuspended in Hi-C lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal) with 1× protease inhibitors (Roche, 11697498001). Cells were incubated on ice for 30 minutes then washed once with 500 uL of ice-cold Hi-C lysis buffer with no protease inhibitors. Pellets were resuspended in 50 uL of 0.5% SDS and incubated at 62° C. for 7 minutes. 145 uL of water and 25 uL of 10% Triton X-100 were added and tubes incubated at 37° C. for 15 minutes. 25 uL of 10× New England Biolabs CutSmart buffer and 200 units of NlaIII (NEB R0125L) enzyme were added and the chromatin was digested for four hours at 37 degrees in a thermomixer at 500 RPM. 200 additional units of NlaIII was spiked in and digest continued for 12 hours. Then, 200 additional units of NlaIII was spiked in and digest continued for four more hours. Restriction enzyme was inactivated by heating to 62° C. for 20 minutes while shaking at 500 rpm. Proximity ligation was performed in a total of 1200 uL with 2000 units of T4 DNA ligase (NEB M0202M) for six hours at room temperature. After ligation samples were spun down for 5 minutes at 2500 g and resuspended in 300 uL 10 mM Tris-HCl, 1% SDS and 0.5 mM NaCl with 1000 units of Proteinase K. Samples were reversed cross-linked overnight at 68° C.
Samples were then phenol-chloroform extracted and ethanol precipitated and the second digestion was performed overnight in 450 uL with 50 units of CviQI (NEB R0639L). Samples were phenol-chloroform extracted and ethanol precipitated and the second ligation was performed in 14 mL total with 6700 units of T4 DNA ligase (NEB 0202M) at 16° C. overnight. Samples were ethanol precipitated, resuspended in 500 uL Qiagen EB buffer, and purified with a Qiagen PCR kit.
The concentration was measured with a Nanodrop and PCR amplification was performed with 16 50 uL PCR reactions using Roche Expand Long Template polymerase (Roche 11759060001). Reaction conditions are as follows: 11.2 uL Roche Expand Long Template Polymerase, 80 uL of 10×Roche Buffer 1, 16 uL of 10 mM dNTPs (Promega PAU1515), 112 uL of 10 uM forward primer, 112 uL of 10 uM reverse primer (Table S2), 200 ng template, and milli-q water till 800 uL total. Reactions were mixed and then distributed into 16 50 uL reactions for amplification. Cycling conditions were a “Touchdown PCR” based on reports that this decreases non-specific amplification of 4 C libraries (Ghavi-Helm et al., 2014). The conditions are: 2′ 94° C., 10″ 94° C., 1′ 63° C., 3′ 68° C., repeat steps 2-4 but decrease annealing temperature by one degree, until 53° C. is reached at which point reaction is cycled an additional 15 times at 53° C., after 25 total cycles are performed the reaction is held for 5′ at 68° C. and then 4° C. Libraries were cleaned-up using a Roche PCR purification kit (Roche 11732676001) using 4 columns per library. Reactions were then further purified with Ampure XP beads (Agencourt A63882) with a 1:1 ratio of bead solution to library following the manufactures instructions. Samples were then quantified with Qubit and the KAPA Biosystems Illumina Library Quantification kit according to kit protocols. Libraries were sequenced on the Illumina HiSeq 2500 for 40 bases in single read mode.
HiChIP
HiChIP was performed essentially as described (Mumbach et al., 2016). 10 million HCT116 cells were crosslinked for 10 min at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution (11% formaldehyde, 50 mM HEPES pH 7.3, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) to the growth media followed by 5 min quenching with 125 mM glycine. Cells were washed twice with PBS, then the supernatant was aspirated and the cell pellet was flash frozen in liquid nitrogen. Frozen crosslinked cells were stored at −80° C.
The crosslinked pellets were thawed on ice, resuspended in 500 μL of ice-cold Hi-C Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40 with protease inhibitors), and rotated at 4° C. for 30 minutes. Nuclei were spun down at 2500 rcf for 5 minutes at 4° C., and washed once with 500 μL of ice-cold Hi-C Lysis Buffer. Supernatant was removed, and the pellet was resuspended in 100 μL of 0.5% SDS. Nuclei were incubated at 62° C. for 7 minutes, and SDS was quenched by addition of 285 μL of H2O and 50 μL of 10% Triton X-100 for 15 minutes at 37° C. After the addition of 50 μL of 10×NEB Buffer 2 and 400 U of MboI restriction enzyme (NEB, R0147), chromatin was digested overnight at 37° C. The following day, the MboI enzyme was inactivated by incubating the nuclei at 62° C. for 20 minutes.
To fill in the restriction fragment overhangs and mark the DNA ends with biotin, the following was added: 37.5 μL 0.4 mM biotin-ATP (19524-016, Invitrogen) 1.5 μL of 10 mM dCTP (N0441S, NEB), 1.5 μL of 10 mM dTTP (N0443S, NEB), 1.5 μL of 10 mM dGTP (N0442S, NEB), 10 μL of 5 U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210), and the tubes were incubated at 37° C. for 1 hour with rotation. Next, the following mix was added for the proximity ligation step: 150 μL of 10×NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202), 125 μL 10% Triton X-100, 3 μL 50 mg/mL BSA, 10 μL 400 U/μL T4 DNA Ligase (NEB, M0202), 660 μL H2O, and the nuclei suspension was incubated at room temperature for 6 hours with rotation. Nuclei were pelleted at 2500 rcf for 5 minutes and supernatant was removed.
Pellets were resuspended in 880 μL in Nuclear Lysis Buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS with protease inhibitors), and the lysates were sonicated on a Covaris S220 instrument using the following parameters: Fill Level 10, Duty Cycle 5, PIP 140, Cycles/Burst 200, for 4 minutes. Sonicated lysates were spun down at 16100 rcf for 15 minutes at 4° C., and the supernatant was transferred to a fresh tube. The supernatant was split into two Eppendorf tubes (about 400 μL of lysate in each), and 800 μL of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 7.5, 167 mM NaCl) was added to each tube. 60 μL of Protein G beads (Life Technologies) were washed in ChIP dilution buffer, resuspended in 100 μL ChIP dilution buffer and 50 μL were added to each of the two tubes of lysates. Tubes were then rotated for 1 hour at 4° C. to preclear the lysates. Dynabeads were separated on a magnetic stand, and the supernatant was moved to a fresh tube. 3.5 μg of SMC1A antibody (Bethyl A300-OSSA) were added to each tube, and tubes were incubated at 4° C. overnight with rotation. The next day, 60 μL of Protein G beads were washed ChIP Dilution Buffer, resuspended in 100 μL ChIP Dilution Buffer, and 50 μL was added to each sample tube. Samples were then incubated for 2 hours at 4° C. with rotation. Beads were washed twice with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 7.5, 150 mM NaCl), twice with High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 7.5, 500 mM NaCl), twice with LiCl buffer (10 mM Tris pH 7.5, 250 mM LiCl, 1% NP-40, 1% Sodium-deoxycholate, 1 mM EDTA). Beads were then resuspended in 100 μL of DNA Elution Buffer (50 mM NaHCO3, 1% SDS), incubated for 10 minutes at room temperature with rotation and 3 minutes at 37° C. with shaking. Beads were separated on a magnetic stand, and supernatant was transferred to a fresh tube. Beads were then mixed with another 100 μL of DNA Elution Buffer, incubated for 10 minutes at room temperature with rotation and 3 minutes at 37° C. with shaking. Beads were separated on a magnetic stand, and supernatant was combined with the previous round of supernatant. 10 μL of Proteinase K (20 mg/ml) was added to each sample and samples were incubated at 55° C. for 45 minutes with shaking. Temperature was then increased to 67° C., and samples were incubated for 1.5 hours with shaking. Samples were purified on a Zymo column (Zymo Research).
Fragmentation of the ChIP DNA was performed using the Tn5 transposase (Illumina). First, 5 μL of Streptavidin M-280 magnetic beads were washed with Tween Wash Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), resuspended in 10 μL of Binding Buffer (10 mM, Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl), and added to the samples. Samples were then rotated for 15 minutes at room temperature. Beads were separated on a magnet, and supernatant was discarded. Beads were washed twice with 500 μL of Tween Wash Buffer and incubated at 55° C. for 2 minutes shaking. Beads were then washed with 100 μL of 1× TD Buffer (Nextera DNA sample preparation kit, FC-121-1030, Illumina), and tagmented using the Nextera DNA sample preparation kit (FC-121-1030, Illumina) 0.5 μL TDE1 enzyme was used to tagment 10 ng of ChIP DNA (quantified after the previous Zymo column purification). Tagmentation was performed for 10 minutes at 55° C. with shaking. Beads were then separated on a magnet, and supernatant was discarded. Beads were washed with 50 mM EDTA at 50° C. for 30 minutes, and twice with 50 mM EDTA at 50° C. for 3 minutes. Beads were then washed twice in Tween Wash Buffer at 55° C. for 2 minutes, and once with 10 mM Tris for 1 minute at room temperature. The tagmented library still bound to the beads was amplified by 12 cycles of PCR using the Nextera DNA sample preparation kit. The library was then purified on a Zymo column, size-selected (300-700 bp) using AMPure beads (Agencourt) per manufacturers' instructions, and sequenced 100×100 on an Illumina Hi-Seq 2500.
Targeted Methylation and Bisulfite Sequencing.
To perform targeted methylation, HCT-116 cells or HEK293T were transfected with a dCas9-DNMT3A-3L construct with or without guides. To generate the dCas9-DNMT3A-3L construct, dCas9 was isolated from pSQL1658 (Addgene: 51023) by PCR. Cas9 was removed from pX330-Cas9 (Addgene: 42230) by Agel and EcoRI restriction digest. dCas9 was inserted into pX330 to create pX330-dCas9. DNMT3A-3L carrier plasmid was a generous gift from the Jeltsch lab (Siddique et al., 2013). DNMT3A-3L was cloned into pX330-dCas9 using Pmel and Ascl sites to create pX330-dCas9-DNMT3A-3L (no guides). Guide RNAs were added to pX330-dCas9-DNMT3A-3L by digesting pX330-dCas9-DNMT3A-3L with Bbsl followed by ligation of annealed oligos (Table S2) to create pX330-dCas9-DNMT3A-3L-guide with three different guides. An additional plasmid containing two guides, lentiGuide-Puro-double_guide, was also generated. Double guide containing gBlock (individual guide sequences Table S2) was cloned into lentiGuide-Puro (Addgene: 52963) as described (Vidigal and Ventura, 2015) to create lentiGuide-Puro-double_guide. Two hundred fifty thousand HCT-116 or HEK293T cells were transfected with either 750 ng of pX330-dCas9-DNMT3A-3L (no guides) (Addgene: 85701), 250 ng lentiGuide-Puro, and 5 uL of lmg/ml PEI, or 250 ng of pX330-dCas9-DNMT3A-3L-guide1, 250 ng of pX330-dCas9-DNMT3A-3L-guide2, 250 ng of pX330-dCas9-DNMT3A-3L-guide3, 250 ng lentiGuide-Puro-double_guide, and 5 uL of 1 mg/ml PEI, and harvested after two days. HCT-116 cells were harvested and sorted for GFP presence after two days, HEK293T cells were harvested without cell sorting.
To detect methylation, 2 ug of gDNA from HCT-116 or HEK293T cells transfected with dCas9-DNMT3A-3L or dCas9-DNMT3A-3L plus guides were bisulfite converted using the EpiTect Bisulfite Kit (QIAGEN 59104). Converted gDNA was eluted in 20 uL H2O. Converted gDNA was PCR amplified with EpiMark® Hot Start Taq DNA Polymerase (NEB M0490) using 3 uL of converted gDNA as template and locus specific primers (Table S2). PCR was carried out as follows: 95° C. for 30 sec; 95° C. for 20 sec; 52° C. for 30 sec; 68° C. for 30 sec; repeat steps 2-4 45×; 68° C. for 5 min; Hold 4° C. Resultant amplicons were cleaned up using QIAquick PCR Purification Kit (QIAGEN 28106) and eluted in 20 ul H2O. Clean amplicons (3 uL) were subcloned using the pGEM-T Easy vector system (Promega A1360) and transformed into DH5α competent cells. Individual colonies were then picked, and colony PCR was carried out using GoTaq Green Master Mix (Promega M712) with the same primers originally used for the converted gDNA amplification. Colony PCR was carried out as follows: 95° C. for 2 min; 95° C. for 45 sec; 51° C. for 45 sec; 72° C. for 45 sec; repeat steps 2-4 29×; 72° C. 5 min; Hold 4° C. Resultant amplicons were then Sanger sequenced, and CpG methylation was detected as CpG sequences that were not converted to TpG. All converted DNA analyzed had >95% bisulfite conversion rate.
Hi-C Visualization
Hi-C datasets were visualized using the 3D Genome browser at http://www.3dgenome.org.
Visualization of ChIA-PET Interactions on the WashU Genome Browser
The output of origami was visualized in the WashU genome browser by converting the output of origami into a WashU compatible format using origami-conversion.
Topologically Associating Domain (TAD) Calls
TAD calls were taken from the TAD calls in (Dixon et al., 2012) from the H1 human embryonic cell line.
Quantification and Statistical Analysis
ChIP-Seq Data Analysis
ChIP-Seq datasets were generated for this study as well as collated from previous studies (Table S3), and were aligned using Bowtie (version 0.12.2) (44) to the human genome (build hg19, GRCh37) with parameter −k 1 −m 1 −n 2. We used the MACS version 1.4.2 (model-based analysis of ChIP-seq) (45) peak finding algorithm to identify regions of ChIP-seq enrichment over input DNA control with the parameter “--no-model--keep-dup=auto”. A p-value threshold of enrichment of 1e-09 was used. UCSC Genome Browser tracks were generated using MACS wiggle outputs with parameters “-w -S -space=50”. The browser snapshots of the ChIP-Seq binding profiles displayed throughout the study use read per kilobase per million mapped reads dimension (rpm/bp) on the y-axis.
Identification of Enhancers and Super-Enhancers
Enhancers and super-enhancers were identified using H3K27Ac ChIP-seq data as previously described (Hnisz et al., 2013). Briefly, enhancers were defined as H3K27Ac ChIP-Seq peaks identified using MACS. To identify super-enhancers, the H3K27Ac ChIP-Seq peaks (i.e. enhancers) were stitched together if they were within 12.5 kb, and the stitched enhancers were ranked by their ChIP-seq read signal of H3K27Ac, using the ROSE algorithm (https://bitbucket.org/young_computation/rose) (Lovén et al., 2013). ROSE separates super-enhancers from typical enhancers by identifying an inflection point of H3K27ac signal vs. enhancer rank (Hnisz et al., 2013; Lovén et al., 2013).
4C Analysis
The 4C-seq samples were first processed by removing their associated read primer sequences from the 5′ end of each FASTQ read. To improve mapping efficiency of the trimmed reads by making the read longer, the restriction enzyme digest site was kept on the trimmed read. After trimming the reads, the reads were mapped using bowtie with options -k 1-m 1 against the hg19 genome assembly. All unmapped or repetitively mapping reads were discarded from further analysis. The hg19 genome was then “digested” in silico according to the restriction enzyme pair used for that sample to identify all the fragments that could be generated by a 4C experiment given a restriction enzyme pair. All mapped reads were assigned to their corresponding fragment based on where they mapped to the genome. The digestion of a sample in a 4C experiment creates a series of “blind” and “non-blind” fragments as described (van de Werken et al., 2012b). In a perfect experiment, we should have only observed reads at non-blind fragments, and reads at blind fragments exhibit a much higher experimental variability than non-blind fragments, so we only used the reads from non-blind fragments for further analysis. To normalize the distribution of different samples, we quantile normalized all non-blind fragments in each sample together. If no reads were detected at a non-blind fragment for a given sample when reads were detected in at least one other sample, we assigned a “0” to that non-blind fragment for the sample(s) missing reads. After normalization, we then smoothed the normalized profile of each sample using a 10 kb running mean at 1000 bp steps across the genome. After smoothing, for each condition we combined the replicates of a condition by taking the mean signal of each bin across all replicates of the condition.
HiChIP Data Analysis
The HiChIP samples were processed by removing their associated read primer sequences from the 5′ end of each FASTQ read. Read pairs were separated and separate reads were mapped using bowtie with options -k 1-m 1 against the hg19 genome assembly. Al unmapped or repetitively mapping read were discarded from further analysis. The hg19 genome was then divided in 50 kb bins and reads were joined back together in pairs (Paired End Tag PET). For every pair of bins the number of PETS joining them was then calculated. These data were then further analyzed by the ORIGAMI pipeline to identify significant bin to bin interaction pairs.
ChIA-PET Data Analysis and ORIGAMI Description
We development a new software pipeline and analytical method called origami to process ChIA-PET. The software and releases can be found at https://github.com/younglab/origami using version alpha20160828. Each ChIA-PET data sets was processed as follows: the reads were first trimmed and aligned using origami-alignment, which trims the ChIA-PET linker if present and aligns trimmed PETs. PETs not having a linker were discarded from further analysis. Each end of a PET with a linker sequence were separately mapped to the hg19 genome assembly using bowtie with the following options: -v 1-k 1-m 1. After alignment, the separated PETs were re-paired in the final BAM output. After repairing, all duplicated PETs within the data were removed, since these were believed to be PCR duplicates. Peaks were called on the re-paired ChIA-PET reads using MACS1 v1.4.2 with the following parameters: --nolambda -nomodel -p 1e-9. The ChIA-PET data analyzed with their corresponding linker sequence can be found in Table S4.
We developed a novel analytical method to analyze ChIA-PET data that used a two-component Bayesian mixture model to accurately identify in vivo interactions from the ChIA-PET data by accurately estimating the difference between the biological signal and technical and biological noise by controlling for error within the ChIA-PET protocol and linear genomic distance. We defined an in vivo interaction as two regions of the genome brought together in the nucleus longer than expected at random given the linear genomic distance between those two region. Our intuition was that true in vivo interactions would follow one distribution where experimental noise would arise from a separate distribution, and these two groups could be learned from the data using a mixture model. After alignment, we defined a set of putative contacts where a putative contact was any two MACS1 peaks linked together by at least one mapped PET. The PET count for a putative interaction was the total number of unique PETs mapped at both ends of the putative contact. All putative contacts and their PET count were used in the estimation. We estimated the distributions two-component mixture model (described more below) from these putative contacts using origami-analysis. We specifically designed the model to have the second mixture component represent the distribution of the in vivo interactions, and we tested whether the estimated group means (described below) were significantly different to validate that model found at least two different groups. After the estimation of each component, for each putative contact we estimate the posterior probability of whether the putative contact was within the distribution of the second component. If this posterior probability was greater than 0.9, we called this putative contact an in vivo interaction. We used this threshold because it was a good balance between what be believed to be a high true positive rate while minimizing the false positive rate in each sample analyzed (although we believe that are a few in vivo interactions below this threshold as well). In general, we often displayed all putative contacts within the MYC TAD by this posterior probability, eliminating the need for a specific cutoff threshold.
For the analytical model, we wanted to build a model that was able to estimate and control for noise arising from the ChIA-PET protocol and linear genomic distance. The linear genomic distance is a potential source of noise in the data because regions of the genome closer together in linear genomic distance are on average more likely to have more frequent interactions by chance than regions of the genome farther away from each other, as observed in Hi-C data. We assume that these two sources of noise are independent Poisson processes from each other (since we are measuring the interaction frequency through PET counts), which appears to be a valid assumption in practice (Phanstiel et al., 2015). With this in mind, the parameters within the model were as follows:
Pi—the count of DNA mapped sequences/reads measured at position i, where i ∈{1 . . . N} and N is the total number of positions measured
Zi—a latent variable having a value of either 0 or 1 measuring whether the measurement in Pi came from a technical artifact (0) or in vivo biology (1)
Gij—a latent variable, where j ∈{0, 1}, measuring the number of counts observed for sample i if were part of component j
Bij—a latent variable measuring the number of counts observed for sample i if were part of component j as a function of the bias due to the genomic distance that sample i spans in the genome (where the distance is assumed to be a constant di for that sample i)
Rij—a latent variable measuring sum of the two independent processes Gij and Bij for sample i if were part of component j
λj—a parameter describing the mean of latent variable G.j for all samples, and we guarantee that λ1>λ0 to maintain identifiability of each component
vi (d)—a parameter describing the mean of the latent variable B.j at distance d
wij—a parameter describing the binomial probability that sample I is part of component j
ai, bi—a set of constants on the prior distribution of wi1 to adjust our prior belief in sample i based on our understanding of the biology we have already validated in lab
More specifically, the model is parametrized as follows:
The distribution of the parameters and hyperparameters were simulated by Markov Chain Monte Carlo (MCMC) using either Gibbs sampling or the Metropolis-Hastings algorithm as appropriate. To speed up the simulation between G.j and Bij, the G.j parameter is updated first. Then the Bij parameters is updated using the difference between the PETs for G.j and the number of PETs observed for each contact according to the component they are assigned to in that iteration. Additionally, the mean of G0 is enforced to be less than the mean of G1, although in practice the mean of G1 was always strictly greater than the mean of G0 during the MCMC run so this was never a problem.
The parameter vj (d) is the mean of the Poisson process estimating the biological bias from the linear genomic distance between the two ends of the putative contact as a function of this distance. To simplify processing, we estimated this function at each iterative using a smoothed cubic spline regression for putative contacts within group j. This approximation worked well by generating trends consistent with the power-law decay observed in Hi-C data sets.
The priors a.i and b.i are set to be minimally informative as possible. The a.i hyperparameter is the frequency of the number of contacts sharing one of the same anchors that have a strictly lower measured PET count than the putative contact i. The b.i hyperparamer is set to be the frequency of putative contacts sharing the same anchor that have strictly higher number of observed PETs linking the anchors plus the ratio of the multiplication of the depth of reads at both anchors of the putative contact divided by the median depth across all putative contacts floored at 0. We found setting the priors with a non-informative Beta distribution (i.e., Beta(1,1)) would also generally call the same in vivo interactions but call many more interactions from the putative contacts, where we believed many more of these were artifacts. Hence, we found this minimally informative prior to be more useful for us biologically.
Each run of origami-analysis was for 1,000 iterations with a 100 step burn-in period. We chose this number of iterations because the model tended to converge fairly quickly given the complexity of these ChIA-PET data sets. The output of origami is the estimated posterior probability that the putative contact arose from the distribution estimated for the second mixture component, which is assumed to model in vivo interactions within the ChIA-PET experiment. Accordingly, putative contact with a posterior probability closer to 1 are believed to be more likely to be in vivo interactions.
CTCF Motif Analysis
CTCF motifs were called in the human and mouse genomes (using hg19 and mm9 assemblies, respectively) using fimo (Grant et al., 2011). The CTCF motif from the JASPAR CORE 2014 database was used. The fimo p-value threshold was set to 1e-2 and the max-stored-scores parameter set to 100000000. To rank the importance of individual CTCF motifs, the motifs within the targeted CTCF peak upstream of MYC were ranked by their score within the score column in the GFF output of fimo.
Identification of Genes with a Putative Enhancer Docking Site
Genes with a putative enhancer docking site were identified by filtering the list of all 26,801 annotated genes down to those which occurred within a constitutive insulated neighborhood, had a constitutive CTCF site within 2.5 kb of the TSS, and exhibited differential enhancer usage across the cell lines HCT-116, Jurkat, and K562. Constitutive insulated neighborhoods were identified as follows. First, CTCF binding sites and cohesin binding sites were identified in HCT-116, Jurkat, and K562 cells. Cohesin ChIA-PET in the three cell types were processed with the Oragami pipeline as described below, and two CTCF bound sites that are connected by a cohesin ChIA-PET interaction were annotated as CTCF-CTCF/cohesin interactions in each cell type (i.e. insulated neighborhoods). Insulated neighborhoods were scored as constitutive across two cell types if they had a reciprocal overlap of at least 80% of the length of the interaction. The ChIA-PET datasets are likely not saturated, suggesting that not every interaction found within a cell will be potentially represented in the dataset. Therefore, we defined constitutive insulated neighborhoods as the set of insulated neighborhoods from all three cell types that were found overlapping in at least two of the three cell types. Conserved CTCF sites at gene's TSSs were defined as those sites that occur within 2.5 kb of the TSS and overlap by at least lbp across all 3 cell types. A gene was considered to use differential enhancers if in one of the cell types there was an enhancer present within the confines of the gene's constitutive insulated neighborhood that was not present in at least one of the other two cell types. Applying these three filters results in 1,725 genes that may utilize an enhancer docking site.
Data and Software Availability
ORIGAMI: https://github.com/younglab/origami using version alpha20160828.
The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) under ID code GSE92879
The HiChIP data have been deposited in the Gene Expression Omnibus (GEO) under ID codes GSE92881
The 4C-seq data have been deposited in the the Gene Expression Omnibus (GEO) under ID code GSE92880 Software and Algorithms
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Claims
1. A composition comprising
- a. a catalytically inactive site specific nuclease fused to an effector domain having methylation activity or one or more nucleic acids encoding the same; and
- b. one or more guide sequences homologous to at least a portion of promoter region CTCF binding site of a gene or one or more nucleic acids encoding one or more guide sequences homologous to at least a portion of the promoter region CTCF binding site.
2. The composition of claim 1, wherein the gene is selected from an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S 1.
3.-7. (canceled)
8. The composition of claim 1, wherein the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS.
9.-11. (canceled)
12. The composition of claim 1, wherein the one or more guide sequences comprise a sequence homologous to a sequence selected from the group consisting of SEQ ID NOS. 1-8.
13. The composition of any one of claim 1, wherein the composition is for treating cancer.
14.-26. (canceled)
27. A method for methylating a promoter region CTCF binding site of a gene in a cell comprising introducing into the cell:
- a. a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and
- b. one or more guide sequences homologous to at least a portion of the promoter region CTCF binding site,
- thereby methylating the promoter region CTCF binding site.
28. The method of claim 27, wherein the gene is selected from an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1.
29.-32. (canceled)
33. The method of claim 27, wherein the effector domain having methylation activity is DMNT3A-3L.
34. The method of claim 27, wherein the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS.
35.-37. (canceled)
38. The method of claim 27, wherein the one or more guide sequences comprise a sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS. 1-8.
39. (canceled)
40. The method of claim 27, wherein the cell is a human cell.
41. The method of claim 27, wherein the cell is contacted in vivo in a subject a mouse cell.
42. The method of claim 27, wherein the cell is a cancer cell or a pre-cancerous cell.
43. The method of claim 42, wherein the cancer cell is a colorectal cancer cell, a leukemia cell, or a breast cancer cell.
44. The method of claim 27, wherein the expression of the gene in the cell is reduced.
45. The method of claim 27, wherein the expression of mRNA of the gene is reduced by about 50% or more.
46.-63. (canceled)
64. The method of claim 41, wherein the subject has cancer and reducing the expression of the gene in the subject treats the cancer.
65. The method of claim 64, wherein the gene is MYC.
66. The method of claim 64, wherein the cancer is colorectal cancer, leukemia, or breast cancer.
67.-88. (canceled)
89. A method of screening for an anti-cancer compound comprising,
- a. contacting a cell with a test agent; and
- b. measuring methylation in a promoter region CTCF binding site of a gene,
- wherein the test agent is identified as a candidate anti-cancer compound if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent is modulated as compared to the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent.
90.-95. (canceled)
Type: Application
Filed: Dec 12, 2017
Publication Date: May 14, 2020
Inventors: Jurian Schuijers (Somerville, MA), Abraham S. Weintraub (Somerville, MA), John C. Manteiga (Boston, MA), Richard A. Young (Boston, MA)
Application Number: 16/469,131
