Methods and compositions for altering function and structure of chromatin loops and/or domains

Abstract

Chromatin 3D structure modulating agents in the context of the present invention are intended to interfere or manipulate the function of loop anchor motifs, such as CTCF motifs. In certain example embodiments, the present invention may block formation of an loop anchor or chromatin domain or induce formation of a loop anchor or chromatin domain at a targeted genomic location. For instance, a loop anchor motif can be altered, such as by mutating (including inverting) a binding motif so as to remove such a motif, or by adding new binding motifs in new locations within a loop domain, so as to reduce the size of an existing loop, so as to modify the size of an existing loop, or combinations thereof. Alternatively, the chromatin 3D structure modulating agent may bind a target region and mask a loop anchor motif, thereby preventing a loop anchor or chromatin domain from forming. The chromatin 3D structure modulating agent may bind a target region and cause a loop anchor of chromatin domain to form.

What is claimed is: 
     
         1 . A method to modify one or more existing chromatin loops and contact domains in a target region of chromatin DNA inside the nucleus of a cell,
 wherein the target region of chromatin DNA is being extruded by each of the two subunit of a CCCTC-binding factor (CTCF) and/or cohesin-comprising extrusion complex in opposite direction with respect to the genome and wherein the target region of chromatin DNA comprises a forward and reverse CTCF or cohesin binding motif in convergent orientation on opposite strands of the extruded chromatin DNA,   said method comprising targeting one or more sequence-specific DNA targeting agents to a CTCF or cohesin binding motif or within 150,000 base pairs of a CTCF or cohesin binding motif in said target region in the nucleus of the cell, wherein the sequence-specific DNA targeting agents bind to a target site in the target region, to thereby prevent the extrusion of at least one chromatin strand through the extrusion complex.   
     
     
         2 . The method of  claim 1 , wherein said targeting comprises removal of one or more existing chromatin loops or contact domains, or the modification of one or more existing loops or contact domains. 
     
     
         3 . The method of  claim 1 , wherein the one or more sequence-specific DNA targeting agents are targeted to a location between the forward and reverse CTCF or cohesin binding motifs at an existing loop or contact domain boundary. 
     
     
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         6 . The method of  claim 1 , wherein said targeting being in a location within 1000 base pairs following an existing forward CTCF or cohesin binding motif. 
     
     
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         12 . The method of  claim 1 , wherein said one or more sequence-specific DNA targeting agents is selected from the group consisting of a catalytically inactive CRISPR/Cas system, a Cas protein, a zinc finger protein (ZFP), a zinc finger nuclease (ZFN), a transcription activator-like effector (TALE), a transcription activator-like effector nuclease (TALEN), and a meganuclease. 
     
     
         13 . The method of  claim 1 , wherein the CTCF or cohesin binding motif is the CTCF motif; and/or
 wherein the extrusion complex comprises one or more members selected from the group consisting of CTCF, SA1/2, Smc3, Smc1, cohesin and Rad21.   
     
     
         14 . (canceled) 
     
     
         15 . The method according to  claim 1 , wherein said method further comprises the step of performing in situ Hi-C on said cell prior to or following said targeting, optionally combined with HYbrid Capture on the, thereby generating an in situ Hi-C library,
 wherein in situ Hi-C comprises performing Hi-C in intact nuclei, and   wherein said in situ Hi-C method identifies target chromatin loop modification sites or monitors the result of chromatin loop or contact domain modification in the target region.   
     
     
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         18 . The method of  claim 1 , wherein said target region comprises genes the expression of which is to be modified, preferably wherein said proximity to the target region is less than 1000 base pairs. 
     
     
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         20 . The method of  claim 1 , wherein targeting the one or more sequence-specific DNA targeting agents to the nucleus of a cell comprises:
 delivering one or more vectors encoding the one or more sequence-specific DNA targeting agents; or   delivering a cell-permeable reagent comprising the one or more sequence-specific DNA targeting agents, preferably a pyrrole-imidazole polyamide.   
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the one or more sequence-specific DNA targeting agents bind to one or more existing CTCF or cohesin binding motifs. 
     
     
         23 . The method of  claim 1 , wherein the one or more sequence-specific DNA targeting agents comprise a DNA methyltransferase domain that methylates one or more existing CTCF or cohesin binding motifs in said target region. 
     
     
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         50 . The method of  claim 20 , wherein the one or more vectors are delivered in vivo. 
     
     
         51 . The method of  claim 20 , wherein the one or more sequence-specific DNA targeting agents are under the inducible control of a vector promoter, preferably, wherein the vector promoter is a tissue-specific promoter or a ubiquitous expression promoter. 
     
     
         52 . (canceled) 
     
     
         53 . The method of  claim 20 , wherein the vector is a viral vector, preferably, wherein the viral vector is selected from the group consisting of lentiviral, adenoviral, adeno-associated viral, and herpes simplex virus vectors. 
     
     
         54 . (canceled) 
     
     
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         56 . The method of  claim 1 , wherein the target region is associated with a disease, preferably,
 wherein the disease is associated with aberrant chromatin folding; or   wherein the disease is cancer, a genetic disease, or infectious disease, more preferably,   wherein the target region comprises an oncogene or tumor suppressor gene, more preferably, wherein a target region associated with aberrant expression of an oncogene is targeted, whereby expression of the oncogene is repressed; or wherein a target region associated with aberrant expression of a tumor suppressor is targeted, whereby expression of the tumor suppressor is activated; or   wherein the genetic disease selected from the disorders identified in Tables A B or C, preferably, wherein the genetic disease is a disorder associated with genomic imprinting, more preferably, wherein an imprinted gene is unsilenced or wherein a gene is silenced by establishing imprinting.   
     
     
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         66 . The method of  claim 1 , wherein the target region comprises a virus integration site of an infectious virus, preferably wherein the virus is a retrovirus, an adenovirus, an adeno-associated virus (AAV), a lentivirus or a herpesvirus. 
     
     
         67 . The method of  claim 1 , wherein the target region is associated with improved yields, disease resistance, drought resistance or salt tolerance in plant or animals. 
     
     
         68 . The method of  claim 1 , wherein the cells or population of cells are part of a mammal; or wherein the cells or population of cells are part of a plant. 
     
     
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         77 . The method of  claim 18 , further comprising detecting changes in gene expression and/or changes in cell phenotype after said targeting. 
     
     
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