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的细胞多样性 动态平衡。ACD产生两个遗传上相同的子细胞，其细胞命运受信息影响 他们是继承人。如果没有适当地监管ACD，就会导致组织退化和癌症等疾病。一个老牌的 ACD模型为黑腹鼠雄性生殖系干细胞(GSCs)。我们实验室发现姐妹染色单体在 果蝇雄性GSCs根据它们的合成时间不同地合并组蛋白。然后这对姐妹 不对称遗传的。具有预先存在的组蛋白的姐妹分离到自我更新的GSC，而具有 新合成的组蛋白分离到正在分化的子细胞。这个不对称姐妹的极端例子 染色单体遗传使雄性GSCs成为研究不同的表观遗传信息如何建立的强大系统。 DNA复制可能导致姐妹染色单体上不对称的组蛋白沉积，因为这是当 大多数组蛋白被结合到染色质中。此外，DNA复制与DNA本身是不对称的 连续合成(领先链)或不连续合成(滞后链)。来自我们实验室的数据 证明了在GSCs中，旧的组蛋白循环到领先链，而落后的链结合了新的组蛋白。 类似地，在小鼠和酵母中观察到偏向的组蛋白掺入。有鉴于此，DNA复制可能具有 在建立姐妹染色单体不对称性中保守的，但被低估的作用。使用雄性果蝇的GSCs，这是 该提案提出，“DNA复制如何导致组蛋白不对称，从而影响细胞命运的决定？” 当领先和滞后的链在复制起始区和终止区切换时，还不清楚如何定位 复制分叉处的不对称性产生姐妹染色单体，大多数富含新的或旧的组蛋白。虽然这是 这项研究的长期目标是，有必要首先定义GSC中的表观遗传和DNA复制环境。 由于野生型组织中的GSC数量很少，基因组研究在历史上是具有挑战性的。然而，一个类似GSC的 肿瘤系统为使用这项技术提供了前所未有的机会。在K99期，GSC样瘤系统 将用于建立一种新的细胞特异性染色质免疫切割测序法来分析组蛋白 特别是在GSC中的修改。此外，将通过复制排序来定义复制时间。这个 这些实验的结果对于研究组蛋白群体在复制过程中的全基因组关联是不可或缺的 地区。此外，遗传学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中。这些研究将在R00阶段进行扩展，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 将进一步开发，以在体内创造一种新的可诱导生殖系分化的程序。这将推进我们的 能够研究细胞修饰时染色质模式、染色质可及性和基因表达的调节机制 他们的身份。总之，拟议的实验将为深入了解细胞的分子机制提供帮助。 复制偶联组蛋白掺入对命运决定的影响。

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.