Investigating the Regulatory Roles of Histone Chaperones in Cellular Plasticity

项目概要 组蛋白伴侣是功能和结构多样化的蛋白质。它们在染色质中发挥核心作用 DNA 过程中通过与组蛋白结合并促进核小体组装来组织和维持 复制、转录、重组和修复过程。此外，一些组蛋白伴侣进化了 独立于组蛋白结合的附加功能。因此，组蛋白伴侣对于细胞至关重要 增殖和有机体发育。有趣的是，在许多细胞命运中规避了这种致命性 变化范式揭示了组蛋白伴侣在细胞可塑性中的作用。例如，我们和其他人有 显示染色质组装因子 1 (CAF-1)，一种参与复制的组蛋白伴侣复合物 依赖的核小体组装和异染色质调节可防止细胞重编程。更多 最近，我们证明CAF-1通过抑制干细胞和祖细胞的谱系完整性 分化基因的转录。在这种情况下，CAF-1 控制染色质可及性 谱系特异性基因座的增强子/启动子元件并防止转录因子的异常结合。在 除了这些 CAF-1 敏感位点之外，我们还鉴定了可访问性受到干扰的异染色质位点 CAF-1 丢失后，尽管对细胞命运的影响未知。 CAF-1对局部常色和 考虑到 CAF-1 以序列独立的方式进行组装，异染色质位点很有趣 DNA复制过程中的核小体。 CAF-1 对细胞命运的如此深远的影响是否与其 核小体组装功能或其他非规范功能仍未被探索。此外，鉴于 随着组蛋白伴侣和相关组蛋白变体的不断增长，目前尚不清楚 CAF-1 是否 与其他组蛋白伴侣合作以维持谱系完整性。因此，功能和结构 组蛋白伴侣网络作为一个整体在细胞命运背景下的决定因素仍然很重要 问题。研究组蛋白谱系特定功能的分子机制 作为伴侣，我们将使用成熟的细胞命运改变范式结合基因编辑/RNAi， 多组学、生化和功能方法。具体来说，我们提出以下两项研究 方向：（1）研究组蛋白伴侣敏感染色质的调控机制和功能 位点，(2) 探究组蛋白伴侣的结构-功能关系以及它们的结构域如何 与控制细胞命运密切相关。短期内，我们计划剖析表观基因组和结构 CAF-1 的决定因素。从长远来看，我们计划将我们的分析扩展到其他组蛋白伴侣通路 以及它们如何与 CAF-1 合作来控制细胞命运。如果成功，我们的研究将为设计做出贡献 操纵组蛋白伴侣通路以控制健康和疾病中细胞命运的策略。

PROJECT SUMMARY Histone chaperones are functionally and structurally diverse proteins. They play a central role in chromatin organization and maintenance by binding to histones and facilitating nucleosome assembly during DNA replication, transcription, recombination and repair processes. Moreover, some histone chaperones evolved additional functions independent of histone binding. Histone chaperones are thus essential for cellular proliferation and organismal development. Intriguingly, circumventing this lethality in a number of cell fate change paradigms revealed roles of histone chaperones in cellular plasticity. For example, we and others have shown that the chromatin assembly factor 1 (CAF-1), a histone chaperone complex involved in replication dependent nucleosome assembly and heterochromatin regulation, prevents cellular reprogramming. More recently, we demonstrated that CAF-1 maintains lineage integrity of stem and progenitor cells by repressing the transcription of differentiation genes. In this context, CAF-1 controls chromatin accessibility at enhancer/promoter elements of lineage specific loci and prevents aberrant binding of transcription factors. In addition to these CAF-1 sensitive sites, we also identified heterochromatic loci whose accessibility is perturbed upon CAF-1 loss, albeit with unknown effects on cell fate. The influence of CAF-1 on local euchromatic and heterochromatic loci is intriguing given that CAF-1 acts in a sequence independent manner to assemble nucleosomes during DNA replication. Whether such profound effects of CAF-1 on cell fate are linked to its nucleosome assembly function or additional non-canonical functions remain unexplored. Moreover, given the growing repertoire of histone chaperones and associated histone variants, it remains unclear whether CAF-1 cooperates with other histone chaperones to maintain lineage integrity. Therefore, the functional and structural determinants of the histone chaperone network as a whole in the context of cell fate remain important open questions. To investigate the molecular mechanisms underlying the lineage specifying functions of histone chaperones, we will use well established cell fate change paradigms in combination with gene editing/RNAi, multi-omics, biochemical and functional approaches. Specifically, we propose the following two research directions: (1) Investigate the regulatory mechanisms and function of histone chaperone sensitive chromatin sites and, (2) interrogate the structure-function relationships of histone chaperones and how their domains are intimately linked to control cell fate. In the short-term, we plan to dissect the epigenome and structural determinants of CAF-1. In the long-term we plan to extend our analysis to other histone chaperones pathways and how they cooperate with CAF-1 to control cell fate. If successful, our studies will contribute to design strategies for manipulating histone chaperone pathways to control cell fate in health and disease.