Stability of the folded genome

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PROJECT SUMMARY Perturbations in normal gene expression arising from defects in genome organization can lead to cellular dysfunctions linked to aging and various disease states. The mammalian genome is generally organized into chromosomes, compartments, topological associating domains (TADs) and loops. Although TAD and loop formation have been extensively studied, little is known about the processes that drive nuclear compartment formation. It has been proposed that microphase phase separation drives the association of genomic domains of similar chromatin state, resulting in the formation of either type A (active chromatin) or B (inactive chromatin) compartments. However, identifying factors involved has been limited by a lack of tools capable of quantifying the biophysical properties driving this phenomenon. Mammalian heterochromatin protein 1 (HP1) α and HP1β bind constitutive heterochromatin and are known to facilitate the bridging of nucleosomes, suggesting that these proteins play a key role in heterochromatin compartmentalization. Although a recent study has demonstrated that heterochromatin compaction is independent of HP1α, work from our collaborators suggest that this protein is required to stabilize interactions between heterochromatic loci. Interestingly, HP1 proteins and several of their interacting partners can bind RNAs. Independent of HP1 function, specific RNA transcripts are known to play important roles in the formation and maintenance of spatial genome organization and perhaps microphase separation, notably at nucleoli, speckles, and the inactive X chromosome of female cells. We recently developed liquid chromatin Hi-C (LC-Hi-C), which allows quantification of chromatin interaction stability measurements genome-wide. Briefly, isolated nuclei are subject to in situ restriction digestion. Digestion of the genome into a specific fragment size distribution results in the loss of low density/unstable interactions whereas higher density/stable interactions are maintained, which is quantifiable by genome-wide chromosome conformation capture (Hi-C). This technique reveals that the dissolution kinetics of chromatin interactions vary widely between A and B compartments as well as compartmental substructures. The development of “in situ LC-HiC” in Aim 1 will allow stability measurement on mitotic chromosomes, streamline the existing protocol and allow the study of smaller cell populations. Aim 2 will assess contributions of (HP1) α and HP1β to stability of heterochromatic interactions. In Aim 3, LC-Hi-C will allow identification of genomic regions destabilized by RNA depletion. Candidate factors contributing to stability will then be identified using in situ chromatin-associated RNA sequencing (iMARGI) and validated by perturbation followed by LC-Hi- C. Taken together, this study aims to measure the dynamics of chromatin interactions and to provide new mechanistic insight as to how the genome is organized throughout the cell cycle.