Establishment of Heterochromatin

描述(申请人提供)：正常的细胞分化和发育广泛依赖于沉默机制。沉默的破坏可能导致遗传疾病、染色体丢失和肿瘤的发生。我们的长期目标是确定导致基因组沉默区域(异染色质)建立和维持的正常机制。这项提议的目标是定义着丝粒异染色质是如何建立的。异染色质建立研究的一个主要障碍是难以将建立和维持分开。为了避免这个问题，我们利用了分裂酵母的精致的遗传可塑性，其中沉默的染色质与哺乳动物有许多共同的特征，包括依赖类似的染色质标记系统，以及共同依赖非编码RNA和RNAi途径来进行沉默控制。通过对一种对异染色质组装至关重要的大分子复合体的鉴定，我们培育了一种新的酵母突变株，它可以保持预先组装的异染色质，但不能支持其从头建立。我们将使用这个突变体来筛选已知在异染色质稳定性中起作用的基因，以确定建立异染色质所需的基因。我们已经发现了一个编码组蛋白甲基转移酶CLR4(果蝇和哺乳动物中Suv39蛋白的同源物)的“建立”基因，它只在异染色质区域甲基化赖氨酸9(K9)上的组蛋白H3。我们预计，其他“建立”基因将调节CLR4的S向染色质的募集和/或其活性。其次，我们将确定建立异染色质所需的DNA序列。我们将使用高分辨率染色质免疫沉淀分析来确定CLR4活性的“热点”，并将评估这些序列是否足以建立从头开始的异染色质。我们将确定哪些建立基因通过哪些建立序列起作用。最后，我们将确定组蛋白H3的K9甲基化是否足以建立异染色质，以及结合这个标记的蛋白质在建立异染色质中所起的作用。这些研究将对决定异染色质建立的机制产生基本知识，并将为未来针对人类异常异染色质组装和疾病相关沉默缺陷的理解和开发治疗提供一个框架。与公共卫生相关：染色体特定区域的“关闭”或沉默对于有机体中细胞的正常生长与“启动”基因一样重要。沉默对于细胞类型的确定和所有细胞的正常分裂都很重要，因此不适当的沉默可能会导致遗传疾病和癌症。拟议的研究是直接相关的，因为它们将产生沉默的基本知识，并有助于指导未来的工作，以了解和开发治疗人类疾病相关沉默缺陷的方法。

DESCRIPTION (provided by applicant): Normal cellular differentiation and development rely extensively on silencing mechanisms. Disruption of silencing can lead to genetic disorders, chromosome loss and contribute to tumorigenesis. Our long term goal is to determine the normal mechanisms that lead to establishment and maintenance of silenced regions (heterochromatin) of the genome. The goal of this proposal is to define how heterochromatin at centromeres is established. A major impediment to research on the establishment of heterochromatin has been difficulty in separating establishment from maintenance. To circumvent this problem, we utilize the exquisite genetic tractability of the fission yeast, where silenced chromatin shares many features with mammals, including reliance on similar chromatin marking systems and a shared dependence on non-coding RNA and the RNAi pathway for silencing control. Through characterization of a macromolecular complex that is important for the assembly of heterochromatin, we generated a novel mutant strain of yeast that can maintain pre-assembled heterochromatin, but cannot support its de-novo establishment. We will use this mutant to screen genes that are known to play a role in heterochromatin stability to identify those that are required for heterochromatin establishment. We have already found that one "establishment" gene that encodes a histone methyltransferase, Clr4, (the homolog of Suv39 proteins in flies and mammals) which methylates histone H3 on lysine 9 (K9) only in regions of heterochromatin. We anticipate that other "establishment" genes will regulate Clr4's recruitment to chromatin and / or its activity. Second, we will determine the DNA sequences that are required for the establishment of heterochromatin. We will define "hot-spots" for Clr4 activity using high resolution chromatin immunoprecipitation analyses, and will assess whether these sequences suffice to establish de novo heterochromatin. We will determine which establishment genes work through which establishment sequences. Finally, we will determine if K9 methylation of histone H3 suffices to establish heterochromatin, and the role that the proteins that bind this mark play in the establishment of heterochromatin. These studies will yield a basic knowledge of the mechanisms that dictate heterochromatin establishment and will provide a framework for future work directed towards understanding and developing therapies for aberrant heterochromatin assembly and disease-related silencing defects in humans. PUBLIC HEALTH RELEVANCE: The "turning-off" or silencing of specific regions of chromosomes is as important for the normal growth of cells in an organism as is "turning-on" genes. Silencing is important for cell-type identity and for enabling all cells to divide normally, such that improper silencing can contribute to genetic disorders and cancer. The proposed studies are directly relevant as they will yield a basic knowledge of silencing and help direct future work towards understanding and developing therapies for disease- related silencing defects in humans.