Histone chaperones and cell state regulation

修改后的项目摘要/摘要部分 动态平衡代表着适应不断变化的条件和保持总体稳定之间的基本平衡，而扰动会导致糖尿病、胰腺炎和癌症等疾病。表观遗传机制是动态平衡的核心，包括组蛋白变体和调节其沉积的伴侣复合体。组蛋白3.3(H3.3)是典型的组蛋白H3的替代变体，通过含有DAXX和ATRX的复合体沉积在异染色质中。当任何成分被删除时，小鼠早期胚胎死亡，以及人类癌症中反复出现的体细胞突变，强调了这种表观遗传调节轴的重要性。这包括43%的胰腺神经内分泌肿瘤中相互排斥的DAXX或ATRX功能丧失突变。对这种调节复合体及其组成部分的生理功能的了解仍处于初级阶段。新的证据表明，单个成分调节细胞分化状态，包括在体外建立和维持诱导的多能干细胞，以及在体内保护造血干细胞免受不适当的分化。PI最近的工作表明，Daxx限制了胰腺细胞的可塑性，并在体内维持内源性逆转录病毒(ERV)的沉默。这导致了中心假说：作为H3.3和异染色质的调节者，Daxx实施了一个强大的染色质环境，这对于维持转录状态和分化程序是重要的。该项目中提议的研究将结合全面的分子和细胞分析来剖析Daxx如何调节表观基因组，影响基因表达，并促进生理细胞状态。该项目将：确定体内表观基因组依赖于Daxx的调控(目标1)；并阐明体内胰腺损伤和恢复过程中依赖Daxx的细胞状态的变化(目标2)。PI最近建立了两个新的小鼠模型，分别取消了Daxx：ATRX和Daxx：H3.3的相互作用，后续研究将纳入这些创新的功能分离等位基因，以进一步剖析DAXX/ATRX/H3.3轴。此外，越来越多的数据表明，ERV抑制是Daxx的一项重要生理功能，并承认不同物种之间重复基因组的差异，拟议的工作将确定DAXX缺失如何影响人类基因组背景下的转录和细胞状态程序(目标3)。总而言之，该项目提出了一个创新的研究计划，将强大的遗传模型与全面的表观基因组和转录图谱相结合，以提供对Daxx/ATRX/H3.3复合体如何促进染色质维持和动力学以及扰动如何影响下游转录和表型状态的直接机械性见解。总而言之，这项工作有助于该项目的长期目标，即了解维持细胞特性和动态平衡的分子机制，以及当这些机制丢失时的下游病理后果。

Modified Project Summary/Abstract Section 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, and 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: 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 Daxx regulates the epigenome, impacts gene expression, and contributes to physiologic cell state. This project will: Define Daxx-dependent regulation of the epigenome in vivo (Aim 1); and Elucidate Daxx-dependent cell state changes in a time course of pancreatic injury and recovery in vivo (Aim 2). Two new mouse models have recently been generated by the PI that abrogate the Daxx:Atrx and Daxx:H3.3 interactions respectively and subsequent studies will incorporate these innovative separation-of-function alleles to further dissect the DAXX/ATRX/H3.3 axis. Additionally, 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 with 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 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.