This application is to request a MERIT Award extension. Our progress validated our hypothesis that globin gene expression and heme biosynthesis are interlinked through GATA-1-dependent mechanisms.
Aim 1. To distinguish between models for how heme amplifies GATA-1 activity to coordinate hemoglobin biosynthesis and erythroid cell development/function. We will test whether heme permits or enhances the GATA-1-dependent subnuclear transition that expels target loci from the nuclear periphery (Model 1) or enhances GATA-1 activity subsequent to the transition (Model 2). If heme enhances the GATA-1-dependent subnuclear transition of Bach1-sensitive genes, we will analyze the relationship between Bach1 and other factors/coregulators that drive locus relocalization. As the relationship between subnuclear transitions and chromatin looping remains elusive, we will determine whether heme and Bach1 regulate GATA-1-mediated looping. If heme enhances activation subsequent to the transition, we will dissect late mechanistic steps.
Aim 2. To assemble activation and repression matrices and use these unique resources to elucidate how the GATA-1/heme circuit establishes a critical sector of the erythroid cell transcriptome. We hypothesize that GATA-1 target gene cohorts requiring unique ensembles of factors (including heme) and coregulators share common mechanisms/pathways. We assembled first-gen. matrices illustrating relationships between target gene expression and factor/coregulator requirements. Considerably expanded second-gen. matrices will be developed. Unraveling the mechanisms/pathways will yield vital insights into hemoglobin synthesis and erythroid cell development/function.
Aim 3. To use a synthetic biology approach involving cis-element engineering to ascertain how GATA-1-binding cis-elements control heme biosynthesis and erythroid biology. Using CRISPR/Cas9, we will rewire the cis-element circuitry controlling heme biosynthesis to determine why the Alas2 intron1 GATA-1-binding cis-element is much more important than the intron8 GATA-1-binding cis-element. We will test the hypothesis that the difference reflects intrinsic differences between the elements, or distinct flanking sequences render the elements differentially active at endogenous loci. We will generate G1E-ER-GATA-1 cells in which the cis-elements are swapped to determine if they retain or adopt new attributes at the ectopic chromosomal site. Concepts will be validated in primary erythroblasts and in vivo. These studies will establish rules governing cis-element function in erythroid cells, which will inform GATA factor-dependent mechanisms, biology, and pathologies.

Public Health Relevance

The proposed studies shall provide fundamental insights into mechanisms underlying disorders of hemoglobin synthesis, including thalassemias and anemias and diseases associated with aberrant heme biosynthesis, including porphyrias. Moreover, the work shall provide a conceptual framework for the design and implementation of translational and therapeutic strategies for these disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK050107-22
Application #
9540850
Study Section
Special Emphasis Panel (NSS)
Program Officer
Bishop, Terry Rogers
Project Start
2016-08-15
Project End
2021-07-31
Budget Start
2018-08-01
Budget End
2019-07-31
Support Year
22
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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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
Bresnick, Emery H; Hewitt, Kyle J; Mehta, Charu et al. (2018) Mechanisms of erythrocyte development and regeneration: implications for regenerative medicine and beyond. Development 145:
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
DeVilbiss, A W; Tanimura, N; McIver, S C et al. (2016) Navigating Transcriptional Coregulator Ensembles to Establish Genetic Networks: A GATA Factor Perspective. Curr Top Dev Biol 118:205-44

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