Activation and Silencing of Gene Expression during Hematopoiesis--OLD

描述(申请人提供)：我们对造血过程中差异基因调控的分子、生化、遗传和显微分析产生了新的和令人惊讶的结果。例如，我们做出了意想不到的发现，与哺乳动物细胞中核外周是一个抑制性隔间的流行观念相反，-珠蛋白基因的表达始于核外周，然后基因重新定位在发生高水平表达的更中心位置。此外，我们还证明了-珠蛋白基因座控制区(LCR)对于这种定位是必要的。我们的工作还揭示了在红系或髓系中共同调节的基因往往聚集在基因组中，并且在每个谱系中，不同的染色体往往根据共同调节基因的染色体分布而关联。我们还发现，MLL5是哺乳动物三胸蛋白家族中的一员，在体外模型中对红系分化是必不可少的。我们现在建议使用单基因座、基因组、蛋白质组、遗传学和高分辨率显微镜方法来研究核定位、转录状态的启动和维持之间的关系，以及MLL5调节红系分化的机制。具体地说，我们建议：1.确定分化过程中外周定位与基因座转录活性之间的关系。为了做到这一点，我们将结合使用高分辨率显微镜、天然珠蛋白基因座的突变以及野生型(WT)和突变基因座与小鼠红细胞生成过程中的核孔复合体(NPC)和板层的捆绑。我们还将确定红系分化过程中外周定位和表达状态的全基因组变化。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)模型的单等位基因表达来确定顺式序列和反式因子，这些顺式序列和反式因子指定不同核区中非活性和活性等位基因的定位。利用活细胞成像，我们将检验这样一种假设，即在没有负责建立转录状态的信号的情况下，不同区段中的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.确定含有MLL5的复合体在分化过程中的组成和功能。为了验证MLL5功能是通过与不同蛋白质伙伴相互作用而介导的假设，我们将在体外和体内鉴定与MLL5相互作用的蛋白质，并利用基因组方法确定含有MLL5的复合体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，以补充并为我们对小鼠MLL5功能的分析提供信息。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多了解。公共卫生相关性：基因在不同区域的定位核是一种机制，通过它基因被激活和沉默，包括分化在内的各种细胞过程受到调控。重要的是，包括Burkitt淋巴瘤、急性髓系白血病(AML)和乳腺癌在内的几种恶性肿瘤被认为是由于核组织的改变而引起的。这些数据突显了了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.