Activation and Silencing of Gene Expression during Hematopoiesis--OLD

描述（由申请人提供）：我们对造血过程中差异基因调控的分子、生化、遗传学和显微分析取得了新颖而令人惊讶的结果。例如，我们做出了意想不到的发现，与流行的观念相反，核外围是哺乳动物细胞中的抑制室，？-珠蛋白基因的表达开始于核外周，在高水平表达发生的更集中的基因重新定位之前。此外，我们证明了？-珠蛋白基因座控制区（LCR）是这种迁移所必需的。我们的工作还揭示了在红系或髓系谱系中共同调控的基因倾向于聚集在基因组中，并且在每个谱系中，基于共同调控基因的染色体分布，不同的染色体倾向于关联。我们还发现，在体外模型中，哺乳动物三胸蛋白群的成员MLL5对红细胞分化至关重要。我们建议使用单位点、基因组学、蛋白质组学、遗传学和高分辨率显微镜等方法来研究MLL5在细胞核定位、转录状态启动和维持之间的关系，以及MLL5调控红细胞分化的机制。具体而言，我们建议：1。确定分化过程中外周定位与基因位点转录活性之间的关系。为了实现这一目标，我们将使用高分辨率显微镜，诱变原生？-珠蛋白位点，以及野生型（WT）和突变型位点在小鼠红细胞生成过程中对核孔复合物（NPC）和板的系缚。我们还将确定红细胞分化过程中外周定位和表达状态的全基因组改变。2. 确定细胞记忆的分子基础。我们将使用小鼠Toll样受体4 （Tlr4）单等位基因表达模型来鉴定顺式序列和反式因子，这些顺式序列和反式因子指定了不同核室中非活性和活性等位基因的定位。利用活细胞成像技术，我们将验证这样一种假设，即在没有负责建立这种状态的信号的情况下，不同区室中等位基因的维持参与了转录状态的传播，即所谓的“细胞记忆”。3. 确定分化过程中含mll5复合物的组成和功能。为了验证MLL5功能通过与不同蛋白伴侣的相互作用介导的假设，我们将在体外和体内鉴定红细胞分化过程中与MLL5相互作用的蛋白，并使用基因组学方法确定含有MLL5复合物在红细胞生成过程中的功能。我们还将在黑腹果蝇中进行遗传和生化筛选，以确定进化上保守的dMLL5相互作用蛋白和调节途径，以补充并为我们在小鼠中分析MLL5功能提供信息。结合起来，这些实验将导致对造血谱系中基因激活和沉默的更好理解。公共卫生相关性：基因在细胞核不同区室的定位是基因被激活和沉默的一种机制，包括分化在内的各种细胞过程受到调节。重要的是，包括伯基特淋巴瘤、急性髓性白血病（AML）和乳腺癌在内的几种恶性肿瘤被认为是由核组织的改变引起的。这些数据强调了理解基因调控和核组织之间关系的重要性。

DESCRIPTION (provided by applicant): Our molecular, biochemical, genetic and microscopic analyses of differential gene regulation during hematopoiesis have yielded novel and surprising results. For example, we made the unanticipated finding that, contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells, ?- globin gene expression initiates at the nuclear periphery prior to relocalization of the gene more centrally where high level expression occurs. Moreover, we showed that the ?-globin locus control region (LCR) is necessary for this relocation. Our work also revealed genes that are co-regulated in the erythroid or myeloid lineages tend to be clustered in the genome, and that in each lineage, distinct chromosomes tend to associate on the basis of the chromosomal distribution of co-regulated genes. We also discovered that MLL5, a member of the mammalian Trithorax group of proteins, is essential for erythroid differentiation in an in vitro model. We now propose experiments using single locus, genomic, proteomic, genetic and high-resolution microscopy approaches to investigate the relationships among nuclear localization, initiation and maintenance of transcription state, and the mechanism by which MLL5 regulates erythroid differentiation. Specifically, we propose to: 1. Determine the relationships between peripheral localization and transcriptional activity of gene loci during differentiation. To accomplish this, we will use a combination of high-resolution microscopy, mutagenesis of the native ?-globin locus, and tethering of wildtype (WT) and mutant loci to nuclear pore complexes (NPC) and lamina during murine erythropoiesis. We will also determine the genome-wide alterations in peripheral localization and expression state during erythroid differentiation. 2. Determine the molecular basis of cellular memory. We will use the mouse Toll like receptor 4 (Tlr4) model of monoallelic expression to identify cis-sequences and trans-factors that specify positioning of the inactive and active alleles in different nuclear compartments. Using live cell imaging, we will test the hypothesis that maintenance of the alleles in distinct compartments is involved in propagation of transcription state in the absence of the signals responsible for establishing that state, so called "cellular memory". 3. Determine the composition and function of MLL5-containing complexes during differentiation. To test the hypothesis that MLL5 functions are mediated via interactions with different protein partners, we will identify MLL5-interacting proteins during erythroid differentiation in vitro and in vivo, and, using genomic approaches, determine the function of MLL5 containing complexes during erythropoiesis. We also will perform genetic and biochemical screens in Drosophila melanogaster to identify evolutionarily conserved dMLL5 interacting proteins and regulated pathways to complement and inform our analysis of MLL5 functions in mice. In combination, these experiments will lead to a greater understanding of gene activation and silencing in the hematopoietic lineage. PUBLIC HEALTH RELEVANCE: Localization of genes in different compartments the nucleus is a mechanism by which genes are activated and silenced, and various cellular processes, including differentiation, are regulated. Importantly, several malignancies, including Burkitt lymphoma, acute myeloid leukemia (AML) and breast cancer are thought to arise from alterations in nuclear organization. These data highlight the importance of understanding the relationships between gene regulation and nuclear organization.