Mechanisms for histone segregation at DNA replication forks and implications for epigenetic inheritance.

染色质中编码的表观遗传信息负责维持转录程序， 确定细胞特性并调节分化。在每一次有丝分裂细胞分裂中，这种表观遗传信息 必须忠实地传递到子代细胞，以保存特定的转录景观。在这方面的失败 这一过程损害了细胞身份的维持，并可能导致肿瘤的发生。组蛋白 翻译后修饰控制DNA与其他蛋白质之间的相互作用，如转录 因子，因此被认为是表观遗传标记。然而，这些组蛋白修饰是如何呈现的 在亲本核小体，在DNA复制剩余期间转移到领先和滞后链上 很大程度上是未知的。利用eSPAN新方法确定链特异性蛋白质在 复制分叉，我们最近展示了先导链DNA聚合酶的两个非必要亚基是如何 𝝴和McM_2-Ctf_4-Polα轴分别介导组蛋白向领先链和滞后链的转移。 这些重要的发现表明，女儿之间表观遗传信息的对称遗传 细胞需要几种不同因素的作用。然而，这些已知因素的突变会影响 亲代组蛋白转移对细胞存活率影响不大。因此，我假设一个复杂的网络 尚未被发现的蛋白质的数量控制着这一过程。该项目将重点放在识别和 亲代组蛋白向新生DNA链分离所涉及的所有因素的特征 使用遗传、生物化学和基因组方法的组合。我们的首要目标将是确定如何 保守的复制主调控因子增殖细胞核抗原在亲本核小体沉积中起作用 落后股(目标1)。我们的第二个目标是确定和描述在 使用候选和组合将表观遗传信息传递到子代DNA链 全基因组筛选方法(目标2)。此外，我们还将采用eSPAN方法来研究这一现象。 在新合成的DNA链上建立异染色质以进一步研究两者之间的关系 不对称双亲核小体分离和转录沉默缺失之间的关系。加在一起， 这个项目将揭示表观遗传的潜在机制，这是一个关键但很糟糕的问题 了解与癌症等几种疾病有关的过程。目标1将在年内启动和完成 在哥伦比亚大学组蛋白表观遗传学领域的领军人物张志国博士的指导下，K99阶段 大学，一所杰出的研究机构。目标2将在K99阶段结束时开始，但将在 R00独立阶段。来自这个奖项的资金和支持将使我能够专注于我的研究 同时让我有机会了解实验室管理、技术开发和数据 分析，成为一名成功的独立研究人员的关键技能。

Epigenetic information encoded in chromatin is responsible for sustaining transcriptional programs that determine cell identity and regulate differentiation. In every mitotic cell division, this epigenetic information must be faithfully transmitted to daughter cells to preserve specific transcriptional landscapes. Failure in this process compromises the maintenance of cell identity and can lead to tumorigenesis. Histone posttranslational modifications control the interactions between DNA and other proteins, like transcription factors, and are therefore considered epigenetic marks. However, how these histone modifications, present at parental nucleosomes, are transferred to both leading and lagging strands during DNA replication remains largely unknown. Using the novel method eSPAN to determine strand-specific protein enrichment at replication forks, we have recently shown how two non-essential subunits of leading strand DNA polymerase 𝝴 and the Mcm2-Ctf4-Pol α axis mediate parental histone transfer to leading and lagging strands, respectively. These important findings indicate that a symmetric inheritance of epigenetic information between daughter cells requires the action of several different factors. However, mutations at these known factors impacting parental histone transfer have a minor effect on cell viability. Therefore, I hypothesize that a complex network of proteins yet to be discovered regulates this process. This project will focus on the identification and characterization of all those factors involved in the segregation of parental histones to nascent DNA strands using a combination of genetic, biochemical and genome approaches. Our first aim will be to determine how the conserved master regulator of replication PCNA functions in deposition of parental nucleosomes to lagging strands (Aim 1). Our second aim is to identify and characterize additional factors playing a role in the transmission of epigenetic information to daughter DNA strands using a combination of candidate and genome wide screen approaches (Aim 2). Moreover, we will adapt the eSPAN method to investigate the re- establishment of heterochromatin on newly synthesized DNA strands to delve further into the relationship between asymmetric parental nucleosome segregation and loss of transcriptional silencing. Taken together, this project will shed light into the underlying mechanisms of epigenetic inheritance, a critical but poorly understood process involved in several diseases like cancer. Aim 1 will be started and completed during the K99 phase under the mentorship of Dr. Zhiguo Zhang, a leader in the field of histone epigenetics, at Columbia University, an outstanding research institution. Aim 2 will start at the end of K99 phase but will continue during the R00 independent phase. The funds and support from this award will allow me to focus on my research while giving me the opportunity to learn about laboratory management, technology development, and data analysis, critical skills to become a successful independent researcher.