Structural and functional analysis of gene silencing

组蛋白将真核细胞核中的基因组DNA包装成染色质，染色质的结构控制着所有的DNA- 基础过程，包括转录，复制，重组和修复。一个主要的调节机制 染色质结构基于组蛋白的翻译后修饰，组蛋白通过酶沉积 或“写入器”，并被某些蛋白质的“读取器”结构域以高特异性和选择性识别，以介导 下游功能。作者和读者之间的相互作用是发展所必需的， 包括癌症在内的许多疾病。因此，染色质生物学的一个主要焦点是了解 控制染色质结构的机制，这是大大阻碍了相对较少的高分辨率 染色质结合蛋白质和蛋白质复合物的结构，其存款并识别组蛋白修饰。 真核生物基因组的大片段被包装成转录沉默的异染色质。 异染色质对于维持基因组完整性和防止染色体缺陷至关重要。有趣的是， 异染色质形成和DNA复制通常通过特定的染色质修饰来协调。 例如，组蛋白H4在赖氨酸20（H4 K20）处的不同甲基化状态调节不同的过程： H4 K20 me 3存在于异染色质中，而H4 K20 me 2存在于复制起点。甲基化 H4 K20由两种相关的组蛋白甲基转移酶SUV 4 - 20 H1和SUV 4 - 20 H2催化，但尚不清楚 这些酶是如何被募集到染色质中的，或者它们的催化活性是如何被调节的。此外，本发明还提供了一种方法， 异染色质的形成和复制可以通过阅读器来协调，例如Orc 1，它是异染色质的亚基。 起源识别复合物（origin recognition Complex，ORC）Orc 1利用其溴邻近同源（BAH）结构域与染色质结合， 其与组蛋白修饰和异染色质蛋白相互作用。所涉及的详细机制 这些方法在很大程度上是未知的。我们将使用结构和功能的方法来填补这一关键 知识差距，AIM 1侧重于阐明SUV 4 - 20 H酶的机制，AIM 2侧重于 了解Orc 1 BAH结构域的机制。我们将使用冷冻电镜来确定这些结构 作者和读者与核小体结合，并通过体外功能实验补充结构 和体内。复合体中SUV 4 - 20 H写入器和Orc 1 BAH结构域阅读器的结构和功能研究 将揭示组蛋白沉积和识别的一般原理 修改.我们提出的全面研究将为基础生物学提供重要的见解， 异染色质的形成和复制过程，并将提供宝贵的见解，如何放松管制 这些复合物的降解和由此导致的染色质结构畸变导致疾病。

Histones package genomic DNA in the eukaryotic nucleus into chromatin, whose structure controls all DNA- based processes, including transcription, replication, recombination, and repair. A major mechanism regulating chromatin structure is based on post-translational modifications of the histones, which are deposited by enzymes or “writers” and recognized with high specificity and selectivity by “reader” domains of certain proteins to mediate downstream functions. The interplay between writers and readers is essential for development and is perturbed in many diseases, including cancer. A major focus in chromatin biology is thus to understand the detailed mechanisms that control chromatin structure, which is greatly hampered by the relatively few high-resolution structures of chromatin-bound proteins and protein complexes that deposit and recognize histone modifications. Large segments of the eukaryotic genome are packaged into transcriptionally silent heterochromatin. Heterochromatin is critical to maintain genome integrity and to prevent chromosomal defects. Interestingly, heterochromatin formation and DNA replication are often coordinated through specific chromatin modifications. For example, different methylation states of histone H4 at lysine 20 (H4K20) regulate distinct processes: H4K20me3 is found in heterochromatin, whereas H4K20me2 is found at the origins of replication. Methylation of H4K20 is catalyzed by two related histone methyltransferases, SUV4-20H1 and SUV4-20H2, but it is not known how these enzymes are recruited to chromatin or how their catalytic activity is regulated. In addition, heterochromatin formation and replication can be coordinated through readers, such as Orc1, a subunit of the Origin Recognition Complex (ORC). Orc1 binds to chromatin using its bromo-adjacent homology (BAH) domain, which interacts with histone modifications and heterochromatin proteins. The detailed mechanisms involved in these processes are largely unknown. We will use structural and functional approaches to fill this critical knowledge gap, with AIM 1 focusing on elucidating the mechanisms of SUV4-20H enzymes and AIM 2 focusing on understanding the mechanisms of Orc1 BAH domains. We will use cryo-EM to determine structures of these writers and readers bound to nucleosomes and complement the structures with functional experiments in vitro and in vivo. The structural and functional studies of SUV4-20H writers and Orc1 BAH domain readers in complex with nucleosomes will uncover general principles that underlie the deposition and recognition of histone modifications. Our proposed comprehensive studies will provide crucial insights into the fundamental biological processes of heterochromatin formation and replication and will provide invaluable insights into how deregulation of these complexes and resulting aberrations in chromatin structure contribute to diseases.