Chromatin Dynamics and Genome Regulation

Mammalian development results in the specification of over 200 different cell types from a single genome, with subsequent maintenance of cell identity in adult organisms. The genomes of eukaryotic cells are packaged into a dynamic chromatin structure, which allows cells to control the accessibility of all DNA encoded information. The selective incorporation of specialized histone proteins, or variants, into this dynamic genomic structure is an important feature of epigenetic regulation. A main focus of my lab is one such protein, the histone variant H3.3. The identification of mutations in H3.3 and associated proteins in human cancers and developmental disorders has heightened the pressing need to understand the role of this histone variant in normal development and adult homeostasis. Although H3.3 is critical to cellular function in multiple contexts, how H3.3 contributes uniquely to chromatin function is a long-standing, unanswered question in the field. While long associated with gene activation, recent studies establish that H3.3 also deposited at repetitive, heterochromatic regions of the genome, with deposition at each region facilitated by independent chaperone complexes. We still do not know how H3.3 is partitioned between its two chaperone complexes, or how this equilibrium influences cellular function. Once deposited, our studies and others have demonstrated that H3.3 influences the chromatin modification landscape at both euchromatin and heterochromatin. Our data suggest that H3.3 can perform this function directly at euchromatin via phosphorylation of a unique serine that influences the activity of a histone acetyltransferase or indirectly at heterochromatin through chaperone-mediated recruitment of a co-repressor complex. Despite these intriguing observations, we do not yet fully understand the detailed mechanisms by which H3.3 deposition influences chromatin states. Finally, we do not understand how H3.3 performs its myriad functions in the context of complex, multicellular organisms. The goals of this proposal are to: (1) understand how H3.3 chaperone complex equilibrium is established, and determine the effects of disequilibrium on cell function, (2) determine the molecular mechanisms by which H3.3 influences local chromatin landscapes, and how these events influence downstream genome usage, and (3) make use of our novel mouse models to understand the role of H3.3 in adult organisms, including adult stem cells. Our proposed research is significant because it will serve as a platform to understand epigenetic regulation of cell identity in both normal development and adult homeostasis, and by extension, developmental misregulation and disease states. Project Summary/Abstract