The advent of genomic technologies has yielded abundant datasets documenting protein-chromatin interactions genome-wide. Translating genomic data into physiological mechanisms remains extremely challenging. The prior funding period built upon our foundation of a defined cohort of GATA-1/GATA-2 binding sites discovered via ChIP-seq and their functional attributes. Since GATA-1 replaces GATA-2 at these sites upon erythropoiesis, which often alters transcription, we designated these GATA switch sites. We developed evidence for an extremely innovative scenario in which two GATA switch sites at a locus (Gata2), both essential for embryogenesis, non-redundantly control distinct aspects of hematopoiesis - hematopoietic stem cell (HSC) generation from hemogenic endothelial cells (+9.5 site) or myeloid progenitor function (-77 site). This is the only example we are aware of in which two cis-elements at a locus exert essential, but distinct, functions in a multi-tiered developmental hierarchy and the only example of a cis-element required to generate a stem cell. As +9.5 and -77 alterations underlie non-malignant and malignant hematopoietic pathologies, the studies extend beyond understanding how master regulators establish/maintain genetic networks and how genetic/epigenetic mechanisms underlie stem/progenitor cell generation and function.
Aim 1 : To elucidate how a single cis-element controls HSC generation. We hypothesize that the multimeric complex on the +9.5 composite element in hemogenic endothelium ensures GATA-2 levels to establish/maintain a genetic network that promotes hematopoiesis. We will test models to explain how GATA-2 expression in +9.5-/- AGM stimulates HSC genesis. We will test whether signals targeting GATA-2 control HSC genesis in a +9.5- dependent manner and will use CRISPR/Cas9 to dissect how +9.5 regulates Gata2.
Aim 2 : To determine how two GATA switch sites at the same locus control distinct sectors in the hematopoietic hierarchy. +9.5 and -77 functions differ considerably. Hematopoietic clusters emerge from -77-/-, but not +9.5-/-, Aorta Gonad Mesonephros (AGM), consistent with a unique +9.5 role in HSC genesis. We will test the hypothesis that the two cis-elements use different mechanisms to control distinct components of hematopoiesis.
Aim 3 : To leverage rules governing +9.5 site function to discover novel regulators of hematopoiesis. Prioritization of 798 +9.5-like composite elements revealed a site that controls an unstudied gene encoding Sterile Alpha Motif protein 14. As Samd14 harbors a functional +9.5-like site, is expressed in hematopoietic stem/progenitor cells, has attributes distinct from known regulators of hematopoiesis, its downregulation reduces myeloerythroid progenitors, and we linked its function to that of Stem Cell Factor-c-Kit, we hypothesize that Samd14 is an important regulator of hematopoiesis. We will test models to explain how Samd14 regulates c-Kit signaling and whether it controls hematopoiesis via modulating this axis. In aggregate, the proposed studies will forge new paradigms of considerable fundamental and translational importance.
The proposal will lead to fundamental and applied knowledge, which can be used for innovate strategies to control blood cell development from stem and progenitor cells in normal and disease states. This knowledge will lead to the discovery of druggable targets and pathways (and the subsequent development of the respective drugs) that can be leveraged to develop efficacious treatments for nonmalignant and malignant disorders of the hematopoietic system. These disorders include ineffective erythropoiesis linked to anemia and myelodysplastic syndrome, primary immunodeficiency, and myeloid leukemia.
| 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 |
| Lu, Zhanping; Hong, Courtney C; Kong, Guangyao et al. (2018) Polycomb Group Protein YY1 Is an Essential Regulator of Hematopoietic Stem Cell Quiescence. Cell Rep 22:1545-1559 |
| 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 |
| 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 |
| Zhang, Jingfang; Kong, Guangyao; Rajagopalan, Adhithi et al. (2017) p53-/- synergizes with enhanced NrasG12D signaling to transform megakaryocyte-erythroid progenitors in acute myeloid leukemia. Blood 129:358-370 |
| Katsumura, Koichi R; Bresnick, Emery H; GATA Factor Mechanisms Group (2017) The GATA factor revolution in hematology. Blood 129:2092-2102 |
| Katsumura, Koichi R; Ong, Irene M; DeVilbiss, Andrew W et al. (2016) GATA Factor-Dependent Positive-Feedback Circuit in Acute Myeloid Leukemia Cells. Cell Rep 16:2428-41 |
| 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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