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.
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)