During the last year, we continued to define the molecular events that regulate hematopoietic stem cell (HSC) 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. 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. To determine which stromal cell lineages are affected by the loss of Id1, we developed an Id1 conditional knock out mouse model to specifically delete Id1 in endothelial cells, osteoblasts and other stromal cell populations. We have established mouse strains with germ line transmission of the Id1 conditional allele. Future experiments are planned to cross the Id1 conditional knock out mice to transgenic mice that specifically express cre recombinase in endothelial, adipocytes and osteoblasts cell lineages.<BR><BR><BR><BR><BR> Id proteins (Id1, Id2 and Id3) are negative regulators of lymphoid development by antagonizing E protein function. However, it is not known if all Id proteins are physiologically required for 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. Furthermore, we found that the B cell defects in Gfi-1 mice can be rescued by reducing the levels of Id2 in Gfi-1-/- mice. Collectively, these studies provide a mechanistic insight into how Gfi-1 regulates cell fate and the development of B cells. In addition, these results importantly bridge Gfi-1 to the B cell transcriptional network via Id genes. We also found that Id2 expression was up-regulated in purified myeloid progenitors from Gfi-1-/- mice indicating that repression of Id2 by Gfi-1 is required for normal myeloid development. In other studies, we found that high levels of Id2 expression inhibit neutrophil differentiation and promote myeloid progenitor proliferation, suggesting that deregulation of Id2 contributes 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. 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. <BR><BR><BR><BR><BR> 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 discovered that POGZ binds DNA through a consensus sequence found in numerous promoters, including those promoters that regulate genes that are required for T cell development. We have developed a mouse strain to conditionally remove POGZ in specific tissues and hematopoietic lineages. POGZ will be deleted in adult hematopoietic cells using the mx1-cre and vav-cre transgenic mice, in T cells using Lck-cre and B cells using CD-19-cre. <BR><BR><BR><BR><BR>
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