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、生物化学、遗传和显微镜分析产生了新颖和令人惊讶的结果。例如，我们发现了一个意想不到的发现，与普遍认为核外围是哺乳动物细胞中的抑制区室的观点相反，珠蛋白基因表达起始于核周边，然后基因重新定位于更中心的位置，在那里发生高水平表达。此外，我们还表明，珠蛋白基因座控制区（LCR）是这种重新定位所必需的。我们的工作还揭示了在红系或髓系中共调节的基因倾向于在基因组中聚集，并且在每个谱系中，不同的染色体倾向于基于共调节基因的染色体分布而关联。我们还发现MLL5是哺乳动物三胸组蛋白质的成员，在体外模型中对红细胞分化是必需的。我们现在提出的实验使用单基因座，基因组，蛋白质组学，遗传和高分辨率显微镜的方法来调查核定位，启动和维持转录状态之间的关系，MLL5调节红系分化的机制。具体而言，我们建议：1.确定分化过程中基因位点的外周定位与转录活性之间的关系。为了实现这一点，我们将使用高分辨率显微镜，诱变的天然？珠蛋白基因座，以及野生型（WT）和突变基因座在鼠红细胞生成过程中与核孔复合物（NPC）和核纤层的拴系。我们还将确定在红系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）的单等位基因表达模型，以确定顺式序列和反式因子，指定定位的非活性和活性等位基因在不同的核隔室。使用活细胞成像，我们将测试的假设，即在不同的隔室中的等位基因的维护涉及在转录状态的传播负责建立该状态的信号的情况下，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。为了检验MLL5功能是通过与不同蛋白质伴侣的相互作用介导的这一假设，我们将在体外和体内红系分化过程中鉴定MLL5相互作用蛋白，并使用基因组方法确定红细胞生成过程中MLL5复合物的功能。我们还将在果蝇中进行遗传和生物化学筛选，以确定进化上保守的dMLL5相互作用蛋白和调节途径，以补充并告知我们对小鼠MLL5功能的分析。结合起来，这些实验将导致更好地理解造血谱系中的基因激活和沉默。公共卫生相关性：基因在细胞核不同区室中的定位是基因被激活和沉默的一种机制，并调节各种细胞过程，包括分化。重要的是，一些恶性肿瘤，包括伯基特淋巴瘤，急性髓性白血病（AML）和乳腺癌被认为是由核组织的改变引起的。这些数据突出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.