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 also found that 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. The hematopoietic microenvironment is a complex mixture of cells that includes endothelial cells, mesenchymal stem and progenitor cells and their differentiated progeny, adipocytes, osteoblasts, smooth muscle, and chondrocytes. Therefore, we developed and tested a conditional mouse model of Id1, and 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. Since Id genes are know to compensate for each other, we are currently evaluating if Id3 compensates for loss of Id1 by deleting both genes intrinsically and extrinsically using conditional mouse models and transgenic mice that express cre in specific cell lineages. 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. We have also 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, and have completed studies demonstrating that Id2 connects Gfi-1 to the erythroid transcription factor network. 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. Current efforts are focused on determining if knock down of Id1 in AML patient samples can inhibit leukemic cell growth in vitro and in vivo when transplanted into SCID mouse recipients. 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. We have found that loss of Pogz expression leads to a decrease in Bcl11a and KLF2 expression, and that embryonic globin genes are not silenced. We found that Bcl11a is directly regulated by Pogz in promoter reporter assays, and that POGZ is bound to the Bcl11a promoter using ChIP assays. Gain and loss of POGZ expression studies have confirmed that POGZ is a pathway that regulates Bcl11a gene expression. Future studies are aimed at confirming that this novel pathway repress embryonic globin gene expression.
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