Transcriptional Control of Hemoglobin Synthesis

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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.