Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (Kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops.
Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus-locus interaction stabilities. Compartmentalization is stable when fragments are over 10–25 kb. Fragmenting chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, while interactions for speckle and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics.
Chromosome conformation capture--based methods such as Hi--C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair--wise interactions). Here, we describe an updated protocol for Hi--C (Hi--C 2.0) that integrates recent improvements into a single protocol for efficient and high--resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (Kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra--chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and TADs, as well as high--resolution conformational features such as DNA loops.
SUMMARY
The fusion oncoprotein CBFβ-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here we demonstrate that CBFβ-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFβ-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers. There it represses MYC expression by mediating the replacement of SWI/SNF complex component BRG1 with polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFβ-SMMHC inhibitor with the BET- inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFβ-SMMHC drives leukemia maintenance, and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.
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