A systematic approach assigning new candidates to functional pathways in gene silencing and unraveling the role of replisome progression complex in heterochromatin inheritance

转录沉默的染色质是如何复制和表观遗传维持的，人们知之甚少。本研究旨在揭示在基因筛选中发现的复制体成分和其他候选物质对异染色质遗传的作用机制。多种因子和蛋白质复合物组装成在DNA合成和染色质复制过程中具有多种功能的复制体进展复合物。其中一些对异染色质维持也很关键。然而，尽管它们在酿酒酵母中复制过程中的功能已被广泛研究，但该模型系统缺乏规范的抑制性组蛋白修饰，并且对它们在异染色质方面的功能知之甚少。通过全基因组定量报告筛选，我的实验室在分裂酵母裂糖酵母（Schizosaccharomyces pombe）中发现了大量异染色质缺陷突变体。在本研究中，我们将重点研究一种功能未知的蛋白Dhm2和一些参与复制叉稳定性和组蛋白向子DNA链转移的复制体相关因子。我们未发表的研究结果表明，Dhm2在已知的异染色质通路中起冗余作用，而它的缺失会导致DNA损伤和与复制应激相关的表型。通过遗传学和蛋白质组学的结合，我们将验证Dhm2是一种新的复制因子的假设，并揭示其分子功能与异染色质沉默的联系机制。通过实现酿酒葡萄球菌特征的功能特异性突变，我们将进一步研究复制体相关因子如何促进酿酒葡萄球菌的异染色质维持。为此，我们将采用最先进的方法，使我们能够在复制过程中精确监测亲本核小体的遗传和组蛋白修饰。最后，通过功能基因组学和系统地评估成对遗传相互作用，我们寻求使用强大的E-MAP（上位迷你阵列分析）方法将其他候选者分配到异染色质沉默的分子途径和调控网络中。S. pombe是研究这一过程的理想模型系统，因为它在遗传上是可处理的，并且包含在S. cerevisiae中不存在的异染色质的保守标记。提出的工作将阐明异染色质的空间和时间调控，并为异染色质如何复制提供关键见解，这在过去几乎没有研究过。

How transcriptionally silent chromatin is replicated and epigenetically maintained is poorly understood. This proposal seeks to unravel the mechanisms by which replisome components and other candidates identified in genetics screens contribute to heterochromatin inheritance. A variety of factors and protein complexes assemble into the replisome progression complex that has multiple functions during DNA synthesis and chromatin replication. Several of them are also critical for heterochromatin maintenance. However, while their functions during replication have been extensively studied in Saccharomyces cerevisiae, this model system lacks canonical repressive histone modifications and little is known about their functions with respect to heterochromatin.Through genome-wide quantitative reporter screens, my lab has identified a large number of mutants with heterochromatin defects in the fission yeast Schizosaccharomyces pombe. In this proposal, we focus on a protein of unknown function, Dhm2, and several replisome-associated factors involved in replication fork stability and the transfer of histone proteins to the daughter DNA strands. Our unpublished results indicate that Dhm2 acts redundantly with known heterochromatin pathways, whereas its absence causes DNA damage and phenotypes associated with replication stress. Through a combination of genetics and proteomics, we will test the hypothesis that Dhm2 is a new replication factor and unveil the mechanism that links its molecular function to heterochromatin silencing. By implementing function-specific mutants characterized in S. cerevisiae, we will further investigate how replisome-associated factors contribute to heterochromatin maintenance in S. pombe. To this end, we will employ state-of-the-art approaches that allow us to monitor precisely the inheritance of parental nucleosomes and histone modifications during replication. Finally, through functional genomics and assessing systematically pairwise genetic interactions, we seek to assign other candidates to molecular pathways and regulatory networks in heterochromatin silencing using the powerful E-MAP (epistasis mini-array profiling) approach. S. pombe is an ideal model system to study this process, as it is genetically tractable and comprises conserved hallmarks of heterochromatin that are absent in S. cerevisiae. The proposed work will shed light on the spatial and temporal regulation of heterochromatin and provide critical insights into how heterochromatin is replicated, which has been scarcely studied in the past.