Mechanisms of Epigenetic inheritance

 描述(申请人提供)：在细胞周期的S阶段如何遗传表观遗传状态是染色质和表观遗传学领域最具挑战性的问题之一。DNA复制偶联的核小体组装在DNA复制和DNA修复之后的表观遗传中起着重要作用。参与复制偶联核小体组装的大多数基因的突变会导致酵母和哺乳动物细胞中异染色质转录沉默的缺陷和基因组的不稳定。我们一直在研究在酵母和人类细胞中复制DNA后核小体是如何形成的，并对这一过程做出了多项重大贡献。然而，亲代组蛋白(H3-H4)2四聚体如何被转移到复制的DNA仍然知之甚少，这阻碍了我们对表观遗传信息传递到子代细胞的理解。理解亲代组蛋白(H3-H4)2组装的主要挑战是缺乏跟踪这一过程的方法。尽管存在这一挑战，我们还是开发了eSPAN(蛋白质相关新生DNA浓缩和测序)方法，该方法可以识别蛋白质是与DNA复制叉的领先链还是滞后链结合。这种方法使我们第一次能够监测新合成的和亲本的组蛋白(H3-H4)2在DNA复制叉的领先和滞后链上的核小体组装。在这项建议中，我们将阐明亲本(H3-H4)2在DNA复制后组装成核小体的分子机制，以及如何结合遗传、生化和基因组方法在酵母和人类细胞的有丝分裂细胞分裂过程中遗传表观遗传标记。总之，我们的研究应该会对理解核小体组装和表观遗传产生深远的影响。

 DESCRIPTION (provided by applicant): How epigenetic states are inherited during S phase of the cell cycle is one of the most challenging questions in the chromatin and epigenetic fields. DNA replication-coupled nucleosome assembly plays an important role in epigenetic inheritance following DNA replication and DNA repair. Mutations of most, if not all, genes involved in replication-coupled nucleosome assembly result in defects in transcriptional silencing at heterochromatin and genome instability in both yeast and mammalian cells. We have been studying how nucleosomes are formed following DNA replication in yeast and human cells and have made multiple significant contributions to this process. However, how parental histone (H3-H4)2 tetramers are transferred to replicating DNA is still poorly understood, which hinders our understanding of transmission of epigenetic information into daughter cells. The major challenge to understanding parental histone (H3-H4)2 assembly is a lack of methods to track this process. Despite this challenge, we have developed the eSPAN (enrichment and Sequencing Protein- Associated Nascent DNA) method that can discern whether a protein binds to leading or lagging strands of DNA replication forks. This method enables us for the first time to monitor nucleosome assembly of both newly synthesized and parental histone (H3-H4)2 onto leading and lagging strands of DNA replication forks. In this proposal, we will elucidate molecular mechanisms whereby parental (H3-H4)2 are assembled into nucleosomes following DNA replication and how epigenetic marks are inherited during mitotic cell division in both yeast and human cells using a combination of genetic, biochemical and genomic approaches. Together, our studies should have a profound impact on the understanding of nucleosome assembly and epigenetic inheritance.