Repression via Facultative Heterochomatin

描述(由申请人提供)：本提案研究表观遗传学的分子方面。表观遗传学包括在不改变细胞基因组DNA序列的情况下发生的基因表达谱的变化。这源于真核生物中通过一系列紧凑作用构建染色体DNA区域的动态过程。基因表达模式的改变是细胞分化和发育的关键，由子代细胞继承，从而维持特定细胞类型的完整性、规格和功能。这种表观形成过程中的无序性引起了扰动，这些扰动也是遗传的，并对正常的细胞特性造成破坏。将DNA包装成染色质的组蛋白受到翻译后修饰的影响，从而产生不同的染色质结构。虽然常染色质具有允许转录的松弛结构，但构成异染色质是密集堆积的，无法接触到。这项提议的重点是兼性染色质，它是抑制性的，但可以改变其性质并变得活跃。其目的是确定控制兼性异染色质形成的分子机制，以及确保其通过细胞分裂繁殖的表观遗传参数。我们关注影响兼性异染色质形成的三个主要活动：SIRT1，L3MBTL1(L1)和L3MBTL2(L2)，它们组成了定义的多蛋白复合体，通过它们的作用得以促进。SIRT1是一种NAD+组蛋白脱乙酰酶，在体内靶向组蛋白H4-赖氨酸16(H4K16)进行脱乙酰化，并与组蛋白H1B和组蛋白赖氨酸甲基转移酶SUV39H1相互作用。L1和L2都含有MBT结构域，与核小体结合，并与HP1伽马相关。L1还与组蛋白H1结合，并与两个抑制性组蛋白标记结合。L2与三个环指蛋白和E2F6结合，催化组蛋白H_2AK119的单核苷酸。因此，所有三个SirT1、L1和L2都显示了与兼性异染色质相关的组蛋白残基上的活性。我们将确定每个目标基因(全基因组芯片实验)，它们在目标基因上的定位是基因表达的函数，以及使用高度特异的抗体(免疫荧光和芯片分析)作为特定组蛋白修饰的函数，以及组蛋白H1和其他相互作用因素在染色质压缩分析中的作用(包括蔗糖梯度沉淀和电子显微镜)。

DESCRIPTION (provided by applicant): This proposal investigates molecular aspects of epigenetics. Epigenetics encompasses changes in gene expression profiles that occur without alterations in the genomic DNA sequence of a cell. This arises from the dynamic processes that structure regions of chromosomal DNA through a range of compaction in eukaryotes. The altered pattern of gene expression is pivotal to cellular differentiation and development and is inherited by daughter cells thereby maintaining the integrity, specifications, and functions for a given cell type. Aberrancies in this epigentic process gives rise to perturbations that are also inherited and disruptive to normal cellular properties. The histone proteins that package DNA into chromatin are subject to post- translational modifications that give rise to different chromatin structures. While euchromatin has a relaxed structure permissive to transcription, constitutive heterochromatin is densely packed and inaccessible. The focus of this proposal is facultative chromatin that is repressive, but can be altered in its properties and become active. The aims are to identify the molecular mechanisms controling the formation of facultative heterochromatin and the epigenetic parameters that ensure its propagation through cell divisions. We focus on three major activities that impact facultative heterochromatin formation: SirT1, L3MBTL1 (L1) and L3MBTL2 (L2) that comprise defined multiprotein complexes through which their role is facilitated. SirT1 is a NAD+ histone deacetylase that targets histone H4-lysine 16 (H4K16) for deacetylation in vivo, and interacts with histone H1b and the histone lysine methyltransferase Suv39h1. Both L1 and L2 contain MBT domains, bind to nucleosomes and associate with HP1 gamma. L1 also associates with histone H1 and binds to two repressive histone marks. L2 associates with three ring finger proteins and E2F6 and catalyzes monoubiquitination of histone H2AK119. Thus all three, SirT1, L1, and L2 exhibit activites on histone residues associated with facultative heterochromatin. We will identify the target genes for each (genome- wide ChIP on ChIP experiments), their localization on target genes as a function of gene expression and as a function of specific histone modifications using highly specific antibodies (immunofluoresecnce and ChIP analyses), and the role of histone H1 and other interacting factors in chromatin compaction analyses (including sucrose gradient sedimentation together with electron microscopy).