Mechanisms of chromatin regulation of transcription

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MECHANISMS OF CHROMATIN REGULATION OF TRANSCRIPTION PROJECT SUMMARY Over the past decade, the Poirier lab has worked to contribute to the fields of chromatin biology and transcription regulation by developing a research program that is focused on the mechanisms by which chromatin and nucleosome structural dynamics regulate genome accessiblity to transcription regulatory complexes. We have developed and applied on our own and through collaborations, a wide range of experimental tools that enable mechanistic studies including single molecule fluorscence, single molecule force spectroscopy, ensemble fluorescence, and histone chemical ligations. In combination, we have quantitatively investigated how chromatin regulators including histone post translational modifications (PTMs), PTM binding proteins (readers), chromatin remodelers, linker histones, and transcripton factors function to control genome accessiblity to chromatin regulatory complexes. Building off this work, we propose to investigate two central questions in the fields of chromatin biology and transcription in the coming 5 years: (i) How do pioneer transcription factors target their recognition sites within compact nucleosomes and chromatin, and then facilitate chromatin decompaction? (ii) How do epigenetic regulators function together to synergistically or antagonistically regulate genome accessibility to transcription regulatory complexes? Pioneer factors (PFs) are master regulators of cell differentiation, are correlated with nucleosome depletion, and somehow access their binding sites within compact chromatin that are inaccessible to canonical transcription factors (TFs). Furthermore, PF disfunction is strongly correlated with disease most notably cancer. By combining single molecule and ensemble studies we will directly test distinct mechanisms of PF function, and determine what differentiates PFs from canonical TFs. Furthermore, through collaborative work we will investigate the same PFs in vivo to determine how the PF mechanisms that emerge from our in vitro studies impact their functions in vivo. This first direction will provide key insights into how pioneer factors gain access to their sites within compact chromatin and what differentiates PFs from canonical TFs. Epigenetic regulators including histone PTMs, and their readers and writers are critical to organismal development, aging and numerous diseases including cancer. We and others have investigated these regulators individually, yet how they function in combination remains largely unknown. Leveraging our ability to prepare core histone and most recently linker histones with any combination of PTMs, histone H3 readers, and most recently biochemical quantities of endogenous human SAGA and ATAC complexes, we will use single molecule, ensemble and ES cell-based methods to determine how biologically relevant combinations of these regulators control accessibility to canonical TFs and influence PFs. This second direction will provide a mechanistic and functional foundation for understanding how key epigenetic transcriptional regulators differentially control chromatin dynamics, accessibility and transcription.