Regulation of Nucleosome Assembly

描述(由申请人提供)： 在真核细胞中，染色质调节基因表达并维持基因组的完整性。染色质的基本重复单位是核小体，由146个碱基对的DNA包裹在一个组蛋白八聚体周围，组蛋白八聚体包含一个(H3-H4)2四聚体和两个H_2A-H_2B二聚体。人们认为，组蛋白H3-H4组装成核小体是基因转录和DNA复制后核小体形成的限速步骤。核小体组装的错误调节导致基因组不稳定，并与衰老和癌症的发展有关。我们一直在研究H3-H4分子如何组装成核小体，这是一个仍然难以捉摸的研究领域。在哺乳动物细胞中，组蛋白H3有两种主要形式，典型的H3(也称为H3.1)和组蛋白H3变体H3.3。虽然H3.1和H3.3只有5个氨基酸的差异，但它们具有不同的功能，并通过不同的机制组装成核小体。H3.1-H4在复制偶联的核小体组装过程中被组蛋白伴侣CAF-1组装成核小体，H3.3-H4通过复制无关的核小体组装途径被HIRA组装成核小体。然而，目前尚不清楚H3.3和H3.1是如何通过不同的途径组装成核小体的，尽管它们的序列变异有限。我们已经有了一个显著的发现，H4丝氨酸47(H4S47ph)的磷酸化有助于H3.3-H4与Hira的联系。此外，该修饰还抑制了CAF-1与H3.1-H4的结合。基于这些结果，我们建议确定H4S47ph是如何受到H4S47激酶(Ak2)和磷酸酶的调节的，这两种酶参与细胞信号转导，以及这种修饰如何影响Hira在核小体组装和衰老相关异染色质焦点的形成中的作用。这些研究将提供一个将核小体组装与细胞信号联系起来的新范式，并揭示衰老的一种新的调节形式，衰老是一个对肿瘤抑制至关重要的过程。

DESCRIPTION (provided by applicant): In eukaryotic cells, chromatin regulates gene expression and maintains genome integrity. The basic repeating unit of chromatin is the nucleosome, consisting of 146 base pairs of DNA wrapped around a histone octamer containing one (H3-H4)2 tetramer and two H2A-H2B dimers. It is believed that the assembly of histones H3-H4 into nucleosomes is the rate-limiting step of nucleosome formation following gene transcription and DNA replication. Mis-regulation of nucleosome assembly results in genome instability and is associated with aging and the development of cancer. We have been addressing how H3-H4 molecules are assembled into nucleosomes, a research area that remains elusive. In mammalian cells, there are two major forms of histone H3, canonical H3 (also called H3.1) and the histone H3 variant H3.3. Though differing only by five amino acids, H3.1 and H3.3 have distinct functions and are assembled into nucleosomes via distinct mechanisms. H3.1-H4 is assembled into nucleosomes by the histone chaperone CAF-1 in a replication-coupled nucleosome assembly process, and H3.3-H4 is assembled into nucleosomes by HIRA through the replication-independent nucleosome assembly pathway. However, it remains unclear how H3.3 and H3.1 are assembled into nucleosomes via distinct pathways despite their limited sequence variation. We have made a remarkable discovery that phosphorylation of H4 serine 47 (H4S47ph), a modification whose function was not known previously, contributes to the association of H3.3-H4 with HIRA. In addition, this modification inhibits the binding of CAF-1 with H3.1-H4. Based on these results, we propose to determine how H4S47ph is regulated by the H4S47 kinase (Pak2) and phosphatases, enzymes involved in cell signaling and how this modification impacts HIRA's role in nucleosome assembly and the formation of senescence-associated heterochromatin foci. These studies will provide a new paradigm connecting nucleosome assembly with cell signaling and reveal a novel form of regulation of senescence, a process important for tumor suppression.