Transcriptional networks orchestrate stem cell differentiation into blood cells. Master regulators of hematopoiesis, including GATA-2, establish these networks, which are disrupted in leukemias. GATA-2 is required for the genesis and/or survival of multipotent hematopoietic precursors, while erythropoiesis is associated with reduced GATA-2 and increased GATA-1. GATA-1 directly represses Gata2 transcription through GATA switches in which GATA-1 replaces GATA-2 from chromatin sites. We will test hypotheses regarding mechanisms and consequences of GATA switches.
Specific Aim 1 - To test models for how physiological levels of GATA-2 that control hematopoiesis are established and regulated in vivo. We deleted a GATA switch site from the Gata2 locus (-1.8 kb), and analysis of the mutant mice revealed normal Gata2 activation early in embryogenesis, normal Gata2 repression as development proceeds, but reactivation thereafter. The -1.8 kb site requirement for maintaining repression represents the first example of a cis-element that maintains versus initiates repression in vivo. Gata2 reactivation in -1.8 kb mice is associated with impaired erythropoiesis. We will dissect how the -1.8 kb site maintains repression and test whether a distinct Gata2 enhancer (+9.5 kb) is required for induction of Gata2 transcription.
Specific Aim 2 - To determine how GATA factors select chromatin target sites. We hypothesize that GATA-2 levels decline during erythropoiesis to permit unopposed GATA-1 occupancy of GATA switch sites. Computational approaches will be used to mine our genome-wide GATA factor ChIP-seq datasets to define molecular determinants of GATA factor chromatin occupancy. We shall test whether the -1.8 kb site deletion, which reactivates Gata2, creates a reverse GATA switch in vivo.
Specific Aim 3 - To establish the molecular basis for defective Gata2 repression by a GATA-1 mutant associated with the development of human megakaryoblastic leukemia. We found that the leukemogenic mutant of GATA-1 ( 1-83) is a hyperactive activator, but severely impaired in its capacity to repress Gata2. We hypothesize that 1-83 is defective in recruiting critical co-repressors, and residues 81-85 constitute a Retinoblastoma Protein-binding motif. We shall test whether 1-83 is compromised in specific steps leading to Gata2 repression. These studies will elucidate mechanisms that control normal levels of a master regulator of hematopoiesis, how GATA factors select sites in a complex genome, and how a leukemogenic GATA-1 mutation dysregulates Gata2 expression and function. As GATA switches involving other GATA factors are likely to occur in a wide spectrum of cells, the results are expected to have broad biological and pathophysiological importance.

Public Health Relevance

This competitive renewal application seeks to renew our grant Hematopoietic Regulation via GATA Switches. Members of the GATA factor family of transcription factors (GATA-1-6) regulate critical aspects of mammalian development and are dysregulated in human diseases including leukemias. These factors bind to a simple DNA motif distributed abundantly within genomes, but our studies revealed that GATA-1 and GATA-2 occupy only a small fraction of the total GATA motifs in cells. GATA-1 and GATA-2 can occupy the same sites of target genes at distinct stages of development, inducing distinct functional outputs. Mechanisms controlling chromatin site selection and differential activities of GATA factors are poorly understood. We aim to elucidate these mechanisms, which will yield fundamental insights into the control of hematopoiesis, hematologic malignancies involving GATA factor dysregulation, and diverse developmental processes. In the long-term, we aim to develop creative strategies to translate knowledge on GATA factor mechanisms into novel therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK068634-09
Application #
8610293
Study Section
Hematopoiesis Study Section (HP)
Program Officer
Bishop, Terry Rogers
Project Start
2004-07-01
Project End
2016-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
9
Fiscal Year
2014
Total Cost
$290,689
Indirect Cost
$94,939
Name
University of Wisconsin Madison
Department
Pharmacology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Sanalkumar, Rajendran; Johnson, Kirby D; Gao, Xin et al. (2014) Mechanism governing a stem cell-generating cis-regulatory element. Proc Natl Acad Sci U S A 111:E1091-100
Yamazaki, Hiromi; Suzuki, Mikiko; Otsuki, Akihito et al. (2014) A remote GATA2 hematopoietic enhancer drives leukemogenesis in inv(3)(q21;q26) by activating EVI1 expression. Cancer Cell 25:415-27
DeVilbiss, Andrew W; Sanalkumar, Rajendran; Johnson, Kirby D et al. (2014) Hematopoietic transcriptional mechanisms: from locus-specific to genome-wide vantage points. Exp Hematol 42:618-29
Gao, Xin; Johnson, Kirby D; Chang, Yuan-I et al. (2013) Gata2 cis-element is required for hematopoietic stem cell generation in the mammalian embryo. J Exp Med 210:2833-42
Linnemann, Amelia K; O'Geen, Henriette; Keles, Sunduz et al. (2011) Genetic framework for GATA factor function in vascular biology. Proc Natl Acad Sci U S A 108:13641-6
Lee, Hsiang-Ying; Johnson, Kirby D; Fujiwara, Tohru et al. (2009) Controlling hematopoiesis through sumoylation-dependent regulation of a GATA factor. Mol Cell 36:984-95
Scherzer, Clemens R; Grass, Jeffrey A; Liao, Zhixiang et al. (2008) GATA transcription factors directly regulate the Parkinson's disease-linked gene alpha-synuclein. Proc Natl Acad Sci U S A 105:10907-12