Histone chaperones and cell state regulation - PROJECT SUMMARY Homeostasis represents an essential balance between adjusting to changing conditions and maintaining overall stability, with perturbations contributing to diseases including diabetes, pancreatitis and cancers. Epigenetic mechanisms are central to homeostasis, including histone variants and the chaperone complexes that mediate their deposition. Histone 3.3 (H3.3) is a replacement variant for canonical histone H3 and is deposited in heterochromatin by a complex containing DAXX and ATRX. The importance of this epigenetic regulatory axis is emphasized by the early embryonic lethality of mice when any component is deleted, along with recurrent somatic mutations in human cancers. This includes mutually exclusive loss-of-function mutations in DAXX or ATRX in 43% of pancreatic neuroendocrine tumors. The understanding of the physiologic functions of this regulatory complex and its component parts remains in its infancy. Emerging evidence indicates individual components regulate cellular differentiation states, including contributing to the establishment and maintenance of induced pluripotent stem cells in vitro and safeguarding hematopoietic stem cells against inappropriate differentiation in vivo. Recent work by the PI demonstrates that Daxx restricts cellular plasticity in the pancreas and maintains endogenous retroviral (ERV) silencing in vivo. This leads to the central hypothesis of the proposed research: As a regulator of H3.3 and heterochromatin, Daxx enforces a robust chromatin landscape that is important for the maintenance of transcriptional states and differentiation programs. The proposed studies in this project will combine comprehensive molecular and cellular analysis to dissect how this complex regulates the epigenome, impacts gene expression, and contributes to physiologic cell state. To interrogate the requirements for specific protein:protein interactions, two new mouse models have been generated that abrogate the Daxx:Atrx and Daxx:H3.3 interactions respectively. These models will be used to: Define the specific contributions of Atrx and H3.3 to Daxx-dependent regulation of the epigenome in vivo (Aim 1); and determine the requirements for Atrx and H3.3 in Daxx-dependent regulation of pancreatic cell state in vivo (Aim 2). As mounting data suggests ERV repression is an important physiological function of Daxx and acknowledging the differences in repeat genomes between species, the proposed work will determine how DAXX loss affects transcriptional and cell state programs in the context of a human genome (Aim 3). Collectively, this project proposes an innovative research program that integrates powerful genetic models, in vivo structure-function analysis and comprehensive epigenomic and transcriptomic profiling to provide direct mechanistic insight into how the Daxx/Atrx/H3.3 complex contributes to chromatin maintenance and dynamics, and how specific perturbations impact downstream transcriptional and phenotypic states. Collectively, this work contributes to the project’s long-term goal of understanding the molecular mechanisms that maintain cellular identity and homeostasis, and the downstream pathological consequences when these mechanisms are lost.
