Transcriptional Control of Hemoglobin Synthesis

描述(由申请人提供)：本申请的目标是了解β-珠蛋白转录是如何在红细胞生成过程中受GATA-1和其他因素调节的。我们发现，GATA-1在内源性β-珠蛋白基因座上占据了GATA-1DNA结合基序(WGATAR)的一小部分，引发了多个分子事件，最终导致转录激活。动力学分析导致了一种新的多步活化模型的发展。提出严格检验该模型的目的如下：1.阐明β-珠蛋白基因座激活的多步骤途径。我们假设GATA-1介导的早期分子事件反映了GATA-1的直接作用，这是后续事件所必需的。我们将测试有关β-珠蛋白基因座调控复合体的组装/功能的机械性问题。这些研究将产生内源性基因座核蛋白结构的全面分子快照和基本的机制洞察。2.明确各个步骤在β-珠蛋白基因座激活中的重要性。在已经建立了GATA-1在激活过程中引发的某些事件的时间调节之后，有人建议进行研究，从分子上调节该机制的各个步骤。我们假设某些步骤是相互关联的，因此单个步骤的扰动将扰乱其余步骤的子集。这一假说将通过使用蛋白质突变体、RNAi和新型化学抑制剂来验证。3.分析GATA因子染色质占有率的分子决定因素。GATA-1占据了β-珠蛋白基因座和其他基因座的WGATAR基序的子集。我们假设存在一个GATA识别码(GRC)，其中包括蛋白质-蛋白质相互作用、邻近的顺式元件和染色质结构等参数决定占有率。我们建议进行定量染色质免疫沉淀(CHIP)分析，并将CHIP与微阵列芯片技术相结合，以全面确定内源性小鼠和人类基因座内和周围的占有率。通过汇编GRC参数数据库和计算/统计分析，将检验关于GATA因素占有率决定因素的假设。

DESCRIPTION (provided by applicant): The goal of this application is to understand how beta-globin transcription is regulated by GATA-1 and additional factors during erythropoiesis. We discovered that GATA-1 occupancy of a small subset of the GATA-1 DNA binding motifs (WGATAR) within the endogenous beta-globin locus instigates multiple molecular events, culminating in transcriptional activation. Kinetic analyses led to the development of a novel multi-step activation model. The following aims propose to rigorously test this model: 1. To elucidate a multi-step pathway of beta-globin locus activation. We hypothesize that GATA-1- mediated early molecular events reflect direct actions of GATA-1, which are required for subsequent events. We will test mechanistic issues regarding the assembly/function of regulatory complexes at the beta-globin locus. These studies will yield comprehensive molecular snapshots of the nucleoprotein structure of the endogenous locus and fundamental mechanistic insights. 2. To define the importance of individual steps in beta-globin locus activation. Having already established the temporal regulation of certain events instigated by GATA-1 during activation, studies are proposed to molecularly modulate individual steps of the mechanism. We hypothesize that certain steps are interlinked, and therefore perturbation of a single step will disrupt a subset of the remaining steps. This hypothesis will be tested through the use of protein mutants, RNAi, and novel chemical inhibitors. 3. To analyze the molecular determinants of GATA factor chromatin occupancy. GATA-1 occupies a subset of the WGATAR motifs of the beta-globin locus and additional loci. We hypothesize that a GATA Recognition Code (GRC) exists in which parameters, including protein-protein interactions, neighboring cis-elements, and chromatin structure, determine occupancy. We propose to conduct quantitative chromatin immunoprecipitation (ChIP) analysis and ChIP coupled to microarray chip technology to comprehensively determine occupancy within and surrounding the endogenous murine and human loci. Through the assembly of a database of GRC parameters and computational/statistical analysis, hypotheses regarding determinants of GATA factor occupancy will be tested.