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 larger than 10-25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas 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.

Development and application of genomic approaches based on 3C methods combined with increasingly powerful imaging approaches have enabled high-resolution genome-wide analysis of the spatial organization of chromosomes in genome function. In this thesis, I first describe an updated protocol for Hi-C (Hi-C 2.0), integrating recent improvements that significantly contribute to the efficient and high-resolution capture of chromatin interactions. Secondly, I present an assessment of the epigenetic landscape and chromosome conformation around the MYC gene in acute myeloid leukemia (AML) cells before and after small molecule, AI-10-49, treatment. MYC is up-regulated upon inhibition of the RUNX1 repressor by the fusion oncoprotein CBFβ-SMMHC. Treatment of AML cells with AI-10-49 blocks the RUNX1-CBFβ-SMMHC interaction, restoring RUNX1 at MYC regulatory elements. We demonstrate that the established loop is maintained and exchange between activating and repressive chromatin complexes at the regulatory elements, rather than altered chromatin topology, mediates disruption of target gene expression. Finally, Hi-C interaction maps represent the population-averaged steady-states. To understand the forces that promote and maintain the association of loci with specific sub-nuclear structures genome-wide, we developed liquid chromatin Hi-C. Detection of intrinsic locus-locus interaction stabilities and chromatin mobility are enabled by fragmenting chromosomes prior to fixation and Hi-C, thus removing strong polymeric constraints. Nuclear compartmentalization was found to be stable for average fragment lengths are 10-25 kb while fragmentation below 6kb led to a gradual loss of spatial genome organization. Dissolution kinetics of chromatin interactions vary widely for different domains and are analyzed in detail in the final chapter of this thesis., with lamin-associated domains being most stable, and speckle-associated loci most dynamic.

The fusion oncoprotein CBFbeta-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 CBFbeta-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFbeta-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFbeta-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 CBFbeta-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

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.

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.

Jumonji domain-containing protein 6 (JMJD6) is a nuclear protein involved in histone modification, transcription and RNA processing. Although JMJD6 is crucial for tissue development, the link between its molecular functions and its roles in any given differentiation process is unknown. We report that JMJD6 is required for adipogenic gene expression and differentiation in a manner independent of Jumonji C domain catalytic activity. JMJD6 knockdown led to a reduction of C/EBPbeta and C/EBPdelta protein expression without affecting mRNA levels in the early phase of differentiation. However, ectopic expression of C/EBPbeta and C/EBPdelta did not rescue differentiation. Further analysis demonstrated that JMJD6 was associated with the Ppargamma2 and Cebpalpha loci and putative enhancers. JMJD6 was previously found associated with bromodomain and extra-terminal domain (BET) proteins, which can be targeted by the bromodomain inhibitor JQ1. JQ1 treatment prevented chromatin binding of JMJD6, Ppargamma2 and Cebpalpha expression, and adipogenic differentiation, yet had no effect on C/EBPbeta and C/EBPdelta expression or chromatin binding. These results indicate dual roles for JMJD6 in promoting adipogenic gene expression program by post-transcriptional regulation of C/EBPbeta and C/EBPdelta and direct transcriptional activation of Ppargamma2 and Cebpalpha during adipocyte differentiation.