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.
| Peng, Yajing; Shapiro, Samantha L; Banduseela, Varuna C et al. (2018) Increased transport of acetyl-CoA into the endoplasmic reticulum causes a progeria-like phenotype. Aging Cell 17:e12820 |
| Liu, Jinhua; Li, Yapu; Tong, Jingyuan et al. (2018) Long non-coding RNA-dependent mechanism to regulate heme biosynthesis and erythrocyte development. Nat Commun 9:4386 |
| Katsumura, Koichi R; Mehta, Charu; Hewitt, Kyle J et al. (2018) Human leukemia mutations corrupt but do not abrogate GATA-2 function. Proc Natl Acad Sci U S A 115:E10109-E10118 |
| McIver, Skye C; Hewitt, Kyle J; Gao, Xin et al. (2018) Dissecting Regulatory Mechanisms Using Mouse Fetal Liver-Derived Erythroid Cells. Methods Mol Biol 1698:67-89 |
| Tanimura, Nobuyuki; Liao, Ruiqi; Wilson, Gary M et al. (2018) GATA/Heme Multi-omics Reveals a Trace Metal-Dependent Cellular Differentiation Mechanism. Dev Cell 46:581-594.e4 |
| Mehta, Charu; Johnson, Kirby D; Gao, Xin et al. (2017) Integrating Enhancer Mechanisms to Establish a Hierarchical Blood Development Program. Cell Rep 20:2966-2979 |
| Hewitt, Kyle J; Katsumura, Koichi R; Matson, Daniel R et al. (2017) GATA Factor-Regulated Samd14 Enhancer Confers Red Blood Cell Regeneration and Survival in Severe Anemia. Dev Cell 42:213-225.e4 |
| Katsumura, Koichi R; Bresnick, Emery H; GATA Factor Mechanisms Group (2017) The GATA factor revolution in hematology. Blood 129:2092-2102 |
| Liu, Peng; Sanalkumar, Rajendran; Bresnick, Emery H et al. (2016) Integrative analysis with ChIP-seq advances the limits of transcript quantification from RNA-seq. Genome Res 26:1124-33 |
| Gao, Xin; Wu, Tongyu; Johnson, Kirby D et al. (2016) GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis. Stem Cell Reports 6:368-82 |
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