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， 决定细胞身份并调节分化。在每一次有丝分裂中， 必须忠实地传递到子细胞，以保存特定的转录景观。失败在此 这一过程损害了细胞特性的维持，并可能导致肿瘤发生。组蛋白 翻译后修饰控制DNA和其他蛋白质之间的相互作用，如转录 这些因素，因此被认为是表观遗传标记。然而，这些组蛋白修饰如何呈现 在亲本核小体，在DNA复制过程中被转移到前导链和滞后链， 大部分未知。使用新方法eSPAN测定链特异性蛋白富集， 复制叉，我们最近已经表明，两个非必需亚基的前导链DNA聚合酶 α轴和Mcm 2-Ctf 4-Pol α轴分别介导亲本组蛋白转移到前导链和滞后链。 这些重要的发现表明，女儿之间的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影响 亲本组蛋白转移对细胞活力具有较小的影响。因此，我假设一个复杂的网络 尚未发现的蛋白质调控着这一过程。该项目将侧重于识别和 所有这些因素参与分离亲本组蛋白新生DNA链的表征 使用遗传学、生物化学和基因组方法的组合。我们的首要目标是确定 复制的保守主调节因子PCNA在亲本核小体沉积中起作用， 滞后股（目标1）。我们的第二个目标是确定和描述在这些因素中发挥作用的其他因素。 使用候选物和DNA的组合将表观遗传信息传递到子DNA链， 全基因组筛选方法（Aim 2）。此外，我们将采用eSPAN方法来调查重新- 在新合成的DNA链上建立异染色质，以进一步研究 不对称亲本核小体分离和转录沉默的丧失之间的关系。综合起来看， 这个项目将揭示表观遗传的潜在机制，一个关键的，但很差的 我们对癌症等多种疾病的发病过程有着深刻的理解。目标1将在 在哥伦比亚组蛋白表观遗传学领域的领导者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 R 00独立阶段。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，关键技能，成为一个成功的独立研究人员。

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.