Investigating the Regulatory Roles of Histone Chaperones in Cellular Plasticity

项目摘要 组蛋白伴侣是功能和结构多样的蛋白质。它们在染色质中起着核心作用 通过结合组蛋白和促进核小体组装来组织和维持DNA 复制、转录、重组和修复过程。此外，一些组蛋白伴侣进化 独立于组蛋白结合的额外功能。因此，组蛋白分子伴侣对于细胞增殖和分化至关重要。 增殖和生物体发育。有趣的是，在许多细胞命运中， 改变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 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， 依赖于核小体组装和异染色质调节，阻止细胞重编程。更 最近，我们证明CAF-1通过抑制干细胞和祖细胞的分化来维持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， 在一些实施方案中，所述方法包括在谱系特异性基因座的增强子/启动子元件上进行转录，并防止转录因子的异常结合。在 除了这些CAF-1敏感位点外，我们还鉴定了可及性受到干扰的异染色质基因座 在CAF-1损失后，尽管对细胞命运的影响未知。CAF-1对局部常染色质和 异染色质基因座是有趣的，因为CAF-1以不依赖于序列的方式进行组装， 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 核小体组装功能或另外的非典型功能仍然未被探索。此外，鉴于 随着组蛋白伴侣蛋白和相关组蛋白变体的不断增加，目前还不清楚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 组蛋白伴侣网络的决定因素作为一个整体在细胞命运的背景下仍然是重要的开放 问题.探讨组蛋白谱系特异性功能的分子机制 伴侣，我们将使用完善的细胞命运改变范例与基因编辑/RNAi相结合， 多组学、生物化学和功能方法。具体而言，我们提出以下两项研究 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）询问组蛋白伴侣的结构-功能关系以及它们的结构域是如何 与控制细胞命运密切相关。在短期内，我们计划解剖表观基因组和结构 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途径 以及它们如何与CAF-1合作来控制细胞命运。如果成功，我们的研究将有助于设计 操纵组蛋白伴侣途径以控制健康和疾病中的细胞命运的策略。

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.