Structural and functional analysis of gene silencing

组蛋白将真核细胞中的基因组DNA包装成染色质，染色质的结构控制着所有的DNA- 基于过程，包括转录、复制、重组和修复。一个主要的调控机制 染色质的结构是基于组蛋白的翻译后修饰，组蛋白是由酶沉积的 或“编写者”，并被某些蛋白质的“阅读器”结构域高度特异性和选择性地识别以介导 下游功能。作家和读者之间的相互作用对发展是必不可少的，并受到干扰 在许多疾病中，包括癌症。因此，染色质生物学的一个主要焦点是理解详细的 控制染色质结构的机制，而染色质结构受到相对较少的高分辨率 染色质结合的蛋白质和蛋白质复合体的结构，可沉积和识别组蛋白修饰。 真核基因组的大片段被包装成转录沉默的异染色质。 异染色质是维持基因组完整性和防止染色体缺陷的关键。有趣的是， 异染色质的形成和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(H4K20)的不同甲基化状态调节不同的过程： H4K20me3存在于异染色质中，而H4K20me2存在于复制的起始处。甲基化 H4K20由两个相关的组蛋白甲基转移酶SUV4-20H1和SUV4-20H2催化，但尚不清楚 这些酶是如何被招募到染色质中的，或者它们的催化活性是如何被调节的。此外, 异染色质的形成和复制可以通过读取器来协调，例如ORC1，它是 原产地识别复合体(ORC)。ORC1通过其溴相邻同源(BAH)结构域与染色质结合， 它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 这些过程在很大程度上是未知的。我们将使用结构和功能方法来填补这一关键 知识鸿沟，目标1侧重于阐明SUV4-20H酶的机制和目标2聚焦 关于理解ORC1BAH结构域的机制。我们将使用冷冻-EM来确定这些分子的结构 作者和读者与核小体结合，并通过体外功能实验补充结构 在活体内。复合体中SUV4-20H编写器和ORC1BAH域读取器的结构和功能研究 核小体将揭示组蛋白沉积和识别的一般原理 修改。我们提议的全面研究将提供对基础生物学的重要见解 异染色质形成和复制的过程，将为如何放松管制提供宝贵的见解 这些复合体和由此导致的染色质结构的异常会导致疾病。

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.