Hematopoietic stem cell development and regeneration is controlled by a number of regulators including the WNT, BMP, and Notch pathways. In the previous grant, we found that the Wnt pathway was activated by PGE2, leading to enhanced homing and self-renewal of blood stem cells. We now wish to determine the mechanism by which signaling pathways such as the wnt-PGE2 pathway leads to these effects. Many signal transduction pathways terminate with specific signaling transcription factors, although the action of these DNA- binding proteins in stem and progenitor cells has not been defined. Our recent studies have demonstrated that signaling transcription factors bind adjacent to the cell-specific transcription regulators on lineage-specific genes in hematopoietic cells. We hypothesize that these signaling transcription factors reinforce the intrinsic hematopoietic program during regeneration, conferring an advantage to the recovering cells. We plan to use ChIPSeq technology to determine the state of chromatin and the characteristics of transcription factors binding on regions of lineage- and stage-specific genes. The recruitment and function of the signaling- and cell- specific transcription regulators will be evaluated during hematopoietic regeneration after injury from irradiation and during phenylhydrazine-induced anemia. Our work demonstrates that recruitment of signaling transcription factors occurs to blood-specific genes during differentiation of hematopoietic cells. Progenitor and precursor- expressed genes are co-bound by GATA2 and the BMP-stimulated transcriptional regulator SMAD1, but erythroid-specific genes in progenitors are not bound by SMAD1. As cells differentiate, there is a shift of SMAD1 from binding to progenitor genes to a position on erythroid genes adjacent to GATA1. The mechanism of the recruitment of SMAD1 from the progenitor to erythroid genes will be studied, identifying the factors that facilitate this process Transcription and chromatin factors required for stem and progenitor formation in zebrafish will be evaluated functionally for their ability to block or enhance SMAD1 binding to lineage-specific genes in murine and human hematopoietic cells. Chromatin factors associated with GATA1 and GATA2, or induced by GATA1 during differentiation will be tested for their ability to promote or prevent SMAD1 binding adjacent to the GATA-factors. Our studies will lead to a fundamental understanding of the biology of regeneration and may define methods that could be used in the clinic for the expansion of stem and progenitor cells for marrow transplantation or therapies for blood disorders such as thalassemia or sickle cell anemia.

Public Health Relevance

Blood stem cells have the ability to self-renew and differentiate, and have a remarkable capability to repopulate the immune system in a host that has been irradiated or treated with chemotherapy. We are examining the mechanism of the regeneration. We found that specific DNA-binding proteins are stimulated by developmental growth factors, and these signaling pathway transcription factors bind adjacent to the cell-specific factors on blood genes. By understanding the mechanism of this co-binding, we will be able to better manipulate the differentiation or regenerative process for therapies of sickle cell anemia and thalassemia, and other disorders that benefit from marrow or cord blood transplantation.

National Institute of Health (NIH)
Research Project (R01)
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Molecular and Cellular Hematology (MCH)
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Thomas, John
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Children's Hospital Boston
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