Contribution of DNA replication to epigenetic inheritance in a model multi-cellular organism

不对称细胞分裂（ACD）有助于发育和成体组织中的细胞多样性 体内平衡ACD产生两个遗传上相同的子细胞，它们具有受信息影响的不同细胞命运 他们继承。不能正确调节ACD会导致组织变性和癌症等疾病。一个既定 ACD模型为D.黑腹动物雄性生殖系干细胞（GSC）。我们的实验室发现， 果蝇雄性GSC差异纳入组蛋白的基础上，当他们被合成。姐妹篇俩当时 不对称遗传具有预先存在的组蛋白的姐妹分离为自我更新的GSC，而具有预先存在的组蛋白的姐妹分离为自我更新的GSC。 新合成的组蛋白分离到分化中的子细胞中。这个不对称姐妹的极端例子 染色单体遗传使得雄性GSC成为研究不同表观遗传信息如何建立的强大系统。 DNA复制可能有助于姐妹染色单体上的不对称组蛋白沉积，因为这是当 大多数组蛋白被掺入染色质中。此外，DNA复制与DNA本身是不对称的， 连续合成（前导链）或不连续合成（滞后链）。来自我们实验室的数据 证明了在GSC中，旧的组蛋白循环到前导链，而滞后链并入新的组蛋白。 类似地，在小鼠和酵母中观察到偏向性组蛋白掺入。鉴于此，DNA复制可能具有 在建立姐妹染色单体不对称性中保守但未被充分认识的作用。使用雄性果蝇GSC， 该提案提出：“DNA复制如何影响组蛋白不对称性，从而影响细胞命运决定？” 由于前导链和滞后链在复制起始区和终止区切换， 复制叉处的不对称性产生主要富集新或旧组蛋白的姐妹染色单体。虽然这是 为了实现这项研究的长期目标，有必要首先确定GSC中的表观遗传和DNA复制景观。 由于野生型组织中GSC的数量很少，基因组研究历来具有挑战性。然而，一个类似GSC的 肿瘤系统提供了前所未有的机会，使用这项技术。在K99期，GSC样肿瘤系统 将用于开发一种新的细胞特异性染色质免疫切割测序分析，以分析组蛋白 特别是在GSC中。此外，复制时间将通过复制测序来定义。的 这些实验的结果对于研究组蛋白群体在复制中的全基因组关联是不可或缺的。 地区此外，遗传实验将测试组蛋白伴侣是否有助于观察到的局部组蛋白不对称性 在GSC。这些研究将在R 00阶段扩大，以调查DNA复制的贡献- 在发育过程中，将组蛋白组装与体细胞表观遗传相结合。此外，GSC样肿瘤系统 将进一步发展，以创造一种新的诱导生殖细胞分化程序在体内。这将推动我们的 能够研究调节染色质模式，染色质可及性和基因表达的机制，作为细胞修饰 他们的身份总之，所提出的实验将提供深入了解细胞的分子机制， 受复制偶联组蛋白掺入影响的命运决定。

Asymmetric cell division (ACD) contributes to cellular diversity during development and adult tissue homeostasis. ACD generates two genetically identical daughter cells with distinct cell fates influenced by the information they inherit. Failure to properly regulate ACD leads to diseases such as tissue degeneration and cancer. An established model of ACD are D. melanogaster male germline stem cells (GSCs). Our laboratory discovered that sister chromatids in Drosophila male GSCs differentially incorporate histones based on when they were synthesized. The sisters are then asymmetrically inherited. The sister with preexisting histones segregates to the self-renewing GSC whereas the sister with newly synthesized histones segregates to the differentiating daughter cell. This extreme example of asymmetric sister chromatid inheritance makes male GSCs a powerful system to study how distinct epigenetic information is established. DNA replication likely contributes to asymmetric histone deposition on sister chromatids because this is when the majority of histones are incorporated into chromatin. Moreover, DNA replication is inherently asymmetric with DNA being synthesized either continuously (leading strand) or discontinuously (lagging strand). Data from our laboratory demonstrates that in GSCs, old histones recycle to the leading strand while the lagging strand incorporates new histones. Similarly, biased histone incorporation is observed in mouse and yeast. Given this, DNA replication may have a conserved, yet underappreciated role in establishing sister chromatid asymmetries. Using male Drosophila GSCs, this proposal asks, “How does DNA replication contribute to histone asymmetries that influence cell fate decisions?” As leading and lagging strands switch at replication initiation and termination zones, it is unclear how local asymmetries at the replication fork produce sister chromatids mostly enriched for either new or old histones. While this is the long-term goal of this research, it is necessary to first define the epigenetic and DNA replication landscapes in GSCs. Due to the small number of GSCs in wildtype tissue, genomic studies are historically challenging. However, a GSC-like tumor system provides the unprecedented opportunity to use this technology. In the K99 stage, the GSC-like tumor system will be used to develop a novel cell-specific Chromatin Immuno-Cleavage-sequencing assay to profile histone modifications specifically in GSCs. Further, replication timing will be defined through Replication-sequencing. The results of these experiments are integral to investigate genome-wide associations of histone populations in replicating regions. Further, genetic experiments will test if histone chaperones contribute to the local histone asymmetries observed in GSCs. These studies will be expanded upon during the R00 stage to investigate the contribution of DNA replication- coupled histone assembly to somatic epigenetic inheritance during development. Additionally, the GSC-like tumor system will be further developed to create a novel inducible germline differentiation procedure in vivo. This will advance our ability to study mechanisms that regulate chromatin patterns, chromatin accessibility, and gene expression as cells modify their identity. Altogether, the proposed experiments will provide insight into the molecular mechanisms underlying cell fate determination as influenced by replication-coupled histone incorporation.