Nuclear organization and its role in gene regulation

SUMMARY: My lab’s work has been at the forefront of studies showing that nuclear organization and long-range chromatin interactions play an essential role in recombination and gene regulation. We combine molecular and imaging (DNA/RNA FISH) approaches with in house generated computational pipelines, and are thus one of a handful of labs that has expertise in both the experimental and analytical aspects of chromosome folding. In this application we will extend our work to focus on two main interlinked problems of significant biological importance: Project 1: Understanding bookmarking in the context of mitotic chromatin folding. In mitosis (M), the promoters of M-phase active genes are “bookmarked” maintaining the accessibility of some regulatory elements. This provides a mechanism for the rapid activation of a subset of genes, allowing cells exiting from mitosis to preserve a memory of the epigenetic program of the previous cell cycle. Since regulatory factors associate with chromatin with distinct affinities, some factors will be retained on M-phase chromatin better than others, and furthermore, because binding occurs in a dynamic manner, sites at which factors remain bound will not be uniform across a population of cells. The inefficiency of bookmarking, combined with cell-to-cell variability, imparts daughter cells with a degree of epigenetic plasticity, enabling them to alter their phenotype in response to environmental signals, which can have a significant impact on developmental and biological processes. However, little is known about the mechanisms underlying this process, and in particular how condensin II-mediated chromatin folding during mitosis impacts accessibility. Project 2: Mechanisms underlying the chromatin organization and gene regulation of quiescent cells. Cells can adapt and survive under conditions of stress, toxicity, nutrient or growth factor depletion/and chemical insult, by exiting the cell cycle and entering a reversible dormant state known as quiescence (G0). Previous studies showed that CTCF/cohesin-mediated TAD structure is restored after entry into G1, but chromosome structure in mammalian G0 cells has not been studied carefully using modern molecular methodologies. Gene expression is globally repressed in G0 cells, but we now know that quiescent cells actively transcribe specific genes. Notably, chromatin in quiescent cells is predominantly compact, as in M phase. Accordingly, we hypothesize that cells exiting M into G0 partially preserve the compact organization of mitotic cells by retaining condensin II-mediated loops. The goal of our future studies is to determine whether (i) condensin II binding at the start of M-phase functions to bookmark a subset of active promoters, (ii) whether there is variability in the sites that are bookmarked between individual cells, and (iii) whether condensin II- mediated chromatin looping and bookmarking are mechanistically linked to the unique genome organization and transcriptional programs of G0 cells. Given our combined experimental and computational expertise in the field of nuclear organization, we are uniquely positioned to address these timely, relevant questions.