Repression via Facultative Heterochomatin.

描述(申请人提供)：多细胞生物体的复杂性以细胞特化和复杂的组织和器官结构为特征，这是因为有一个有效的索引系统，根据细胞类型和发育阶段跟踪基因组的哪些部分是活跃的或被抑制的。这种“表观遗传”信息(独立于DNA序列)不仅需要被准确地建立，而且还必须被传递给细胞的后代。这必须通过许多分子机制来实现，其中许多机制影响染色质的结构，特别是组蛋白。细胞识别的关键是“兼性异染色质”的建立和维持，即通过形成抑制转录的染色质结构(S)，使特定细胞系中的特定基因沉默。这项建议的目的是扩大我们之前对兼性异染色质潜在的分子机制的研究。如何实现选择性？什么染色质结构包含表观遗传信息？这种信息是如何通过细胞分裂传递的？我们将通过传统和创新的生物化学方法回答这些和其他问题。在目标1中，我们将扩大我们对PrC2的研究，PrC2是一种中央染色质调节因子，在过去的几年中，我们积累了大量的分子信息。我们将进一步研究：EZH1，一个甲基转移酶活性较低的EZH2同源物的功能；可选的PRC2复合体亚基在细胞分化和癌症中的作用；以及两个新的EZH2磷酸化位点对PRC2活性、染色质定位以及与非编码RNA(NcRNAs)相互作用的作用。在目标2中，我们将使用TIRF显微镜和最先进的质谱仪来确定同一核小体中的姐妹组蛋白是否带有任何对称的翻译后修饰，这将是“真正的”表观遗传标记的有力候选者，因为它们可能在DNA复制时被同等分离。在目标3中，我们将扩大我们对MBT蛋白的研究，这是一个未被研究的染色质结合蛋白家族；特别是我们将研究SFMBTS和SCMH1/L2的生化和细胞功能，它们包含MBT重复序列，在遗传学上被归类为多梳组基因。Pr-Set7在保持染色质结构、保护DNA损伤或发出DNA损伤信号中的作用，它与增殖细胞核抗原的相互作用，以及它与同源组蛋白阅读器L3MBTL1的相互作用，将在Aim 4中进行研究，以及这种相互作用传递表观遗传信息的可能性。在目标5中，我们将继续和扩大我们对SirT3的研究，Sirt3是一种迷人的Sirtuin，在线粒体功能和核转录调控中具有双重作用。我们将探索这种联系，以及SirT3的翻译后修饰和相互作用。 与公共健康相关：发展多细胞生物体的一个关键是维持不同基因表达模式所产生的不同细胞特性；细胞通过将基因组中未使用的部分组装成一种称为“异染色质”的抑制性结构来跟踪这些特性。当这一过程受到损害时，细胞可以获得新的身份，通常会转化为癌细胞。这项建议的目的是更好地了解这一过程中涉及的分子机制，以及它们在癌症等疾病中的作用。

DESCRIPTION (provided by applicant): The complexity of multi-cellular organisms characterized by cell specialization and sophisticated tissue and organ architecture is possible because of an efficient indexing system that keeps track of which sections of the genome are active or repressed, according to cell type and developmental phase. This "epigenetic" information (independent of DNA sequence) not only needs to be accurately established, but must also be transmitted to the cell's progeny. This must be achieved through a number of molecular mechanisms, many of which impact the structure of chromatin, particularly histones. Key to cell identity is the establishment and maintenance of "facultative heterochromatin", i.e. the silencing of specific genes in certain cell lineages through the formation of chromatin structure(s) repressive to transcription. The goal of this proposal is to expand our previous studies on the molecular mechanisms underlying facultative heterochromatin. How is selectivity achieved? What chromatin structures contain epigenetic information? How is this information transmitted through cell division? We will answer these and other questions through traditional and innovative biochemical approaches. In aim 1, we will expand our studies on PRC2, a central chromatin regulator on which we have accumulated much molecular information in the last years. We will study further: the function of EZH1, an EZH2 homolog with less pronounced methyltransferase activity; the effects of optional PRC2 complex subunits in cell differentiation and cancer; and the role of two novel EZH2 phosphorylation sites on PRC2 activity, chromatin localization, and interaction with non-coding RNAs (ncRNAs). In aim 2, we will employ TIRF microscopy and state-of-the-art mass spectrometry to determine whether sister histones within the same nucleosome carry any symmetrical post-translational modification, which would be strong candidates for "true" epigenetic marks as they may be equally segregated upon DNA replication. In aim 3, we will expand our studies on MBT proteins, an understudied family of chromatin binders; in particular we will investigate the biochemical and cellular function of SFMBTs and SCMH1/L2, which contain MBT repeats and were genetically classified as Polycomb group genes. The role of Pr-SET7 in preserving chromatin structure, protecting from or signaling DNA damage, its interaction with PCNA and its interaction with the cognate histone reader, L3MBTL1, will be investigated in aim 4, as well as the potential for this interaction to transmit epigenetic information. In aim 5, we will continue and expand our studies on SirT3, a fascinating Sirtuin with a dual role in mitochondrial function and regulation of nuclear transcription. We will explore this connection, as well as post-translational modifications and interactors of SirT3. PUBLIC HEALTH RELEVANCE: A key to development of multi-cellular organisms is the maintenance of distinct cell identities resulting from different patterns of gene expression; cells keep track of these by assembling unused parts of the genome in a repressive structure called "heterochromatin". When this process is compromised, cells can acquire a new identity, often transforming into cancer cells. The aim of this proposal is to better understand the molecular mechanisms involved in this process, and their role in disease such as cancer.