During the last year, we continued to define the molecular events that regulate hematopoietic stem and progenitor cell (HSPC) quiescence, survival, self-renewal and, myeloid cell lineage commitment and differentiation. We have focused our efforts on transcription factors since they are essential for stem cell growth and differentiation, and are frequently deregulated during the development of leukemias and lymphomas. We previously found that the helix loop helix (HLH) transcription factor, inhibitor of differentiation 1 (Id1), is induced during the early stages of myeloid development, and can instruct hematopoietic stem cells toward a myeloid versus erythroid and lymphoid cell fate suggesting that this gene and other family members Id2 and Id3 may regulate cell specification from HSPC. We have extended these studies to determine if Id1 is required for normal hematopoietic development using Id1-/- mice. Id-/- mice are viable and show no obvious defects. However, these mice have hematopoietic phenotypes including increased hematopoietic stem/progenitor cell cycling and defects in B cell and myeloid cell development. We determined that the hematopoietic phenotype observed in the Id1-/- mice was not intrinsic to the Id1-/- hematopoietic cells, but were due to defects in the microenvironment or niche. We have determined that mesenchymal stem and progenitor cell numbers are increased in bone marrow of Id1-/- mice, and that adipocyte development is enhanced in these cultures in vitro. Since these cells give rise to cells that contribute to the hematopoietic microenvironment or niche including adipocytes, osteoblasts, and chondrocytes, we have initiated studies to specifically delete Id1 in endothelial cells, osteoblasts and other stromal cell populations using a an Id1 conditional mouse model recently developed in our lab, and transgenic mouse strains that express cre recombinase in endothelial, adipocytes and osteoblasts cell lineages. Id3 protein is a negative regulator of T lymphoid development by antagonizing E protein function. However, it is not known if all Id proteins are physiologically required for T and B lymphocyte development. By analyzing Id2-/- mice we previously discovered that Id2 is a physiological regulator of B cell and erythroid cell development by negatively regulating functions of E2A and Pu.1 respectively. Gfi-1 is a transcription factor that is required for lymphoid, myeloid and stem cell development. However, the precise mechanism(s) by which Gfi-1 regulates hematopoiesis are currently not known. Over the last year, we discovered that Id2 is a direct transcriptional target of Gfi-1, and that Gfi-1 represses Id2 expression in B cell progenitors, which connects Gfi-1 to the B cell transcriptional network via Id genes. Future studies are planned to evaluate if the Id genes cooperate to regulate lymphoid cell development in primitive HSPC and their immediate progeny using Id2 and Id1 conditional mouse models generated in our lab. In other studies, we found that high levels of Id2 expression inhibit neutrophil differentiation and promote myeloid progenitor proliferation, suggesting that deregulation of Id2 might contribute to the myeloid defects observed in Gfi-1-/- mice. We confirmed this hypothesis by reducing the levels of Id2 in Gfi-1-/- mice, which rescued the myeloid hyperplasia observed in this mouse model, however, a complete rescue of differentiated myeloid cell development was not observed. It will be necessary to further reduce Id2 levels in these mice using the Id2 conditional mouse model. Thus, these studies link Gfi-1 and Id2 to normal myeloid development, and suggest a potential role for this gene in stem cells and disease. In this regard, Gfi-1-/- mice show a profound HSPC defect, therefore, studies are underway to evaluate if lowering Id2 levels in vivo could rescue this hematopoietic defect. We have found that over expression of Id1, Id2 and Id3 in HSPC results in a myeloproliferative disease (MPD) in mice transplanted with transduced HSPC. In addition, we determined that Id1 and Id2 are over expressed in many AML patient samples suggesting a role for these genes in hematopoietic malignancies. We have recently determined that over expression of Id1 promotes, while knock down of Id1 inhibits the growth of AML cell lines in vitro and in vivo suggesting that Id1 may represent a viable therapeutic target to treat hematopoietic malignancies. In an effort to identify novel transcriptional regulators of myeloid cell growth and differentiation, we have compared the global gene expression profile of undifferentiated and differentiating hematopoietic progenitor cells. We have identified a novel zinc finger transcription factor of unknown function, POGZ, which is down regulated during the early stages of myeloid development. We have generated a mouse strain with a targeted deletion of POGZ. POGZ-/- mice do not survive beyond the first few hours of life, and die at birth of unknown causes, suggesting that POGZ is essential for mouse survival. We have discovered that fetal liver hematopoietic cell development is impaired in POGZ-/- mice, which includes a dramatic decrease in cellularity. In addition, fetal thymic development is arrested at a very early stage of development suggesting that POGZ is required for thymic development. We have identified a putative consensus binding site for POGZ, and demonstrated that POGZ can enhance the reporter activity of one gene that contained this site in luciferase reporter assays. Future studies are planned to evaluate additional promoters and to mutate these sites. In addition, studies planned to compare gene expression by micro array analysis of POGZ-/- and POGZ+/+ tissue with genes that contain binding sites in their 3 3Kb promoters to identify potential direct targets of POGZ.
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