The major hypothesis of this proposal is that certain HOX homeobox genes play a key role in regulating hematopoiesis. HOX genes are organized in four clusters (HOXA to HOXD) and encode transcription factors that play a critical role in determining body pattern and tissue fate during embryogenesis. Recent data suggest that a subset of these genes serves important regulatory functions in hematopoiesis. Several HOX A and B genes are expressed in primitive marrow cells. Certain HOX genes - HOXB3 and HOXB4 - are down-regulated within the CD34+ compartment, whereas other genes, HOXA9 and HOXA10, are down-regulated when cells leave the CD34+ compartment. When HOXB4 is over-expressed in mouse marrow, there is a marked expansion of stem cells, an observation with implications for marrow transplantation and gene therapy. Mice bearing homozygous deletions of HOXA9 show reductions in B cells and granulocytes. These data indicate that HOX genes influence early events in hematopoiesis. This collaborative effort, wedding two groups with complementary expertise, one in homeobox research and the other in hematopoietic stem cell studies, has three major goals: 1) To complete the stage- and lineage-specific patterns of expression in HOX genes in human and murine hematopoiesis using FACS fractionation and RT-PCR. Specific HOX gene transcripts expressed in marrow cells will be established by the characterization of cDNAs from the CD34+ marrow cDNA library. 2) To examine the biological effects of over-expression and loss of function of four specific HOX genes - HOXA9, HOXA10, HOXB3, and HOXB4 - on murine hematopoiesis. Mouse marrow transduced with retroviral vectors for each gene will be studied in clonogenic assays and transplantation experiments. Mice bearing homozygous deletions of each gene will be subjected to detailed hematologic evaluation, including histology, FACS analysis, and clonogenic assays.
This aim i ncludes studies of the effects of HOX gene expression on cellular physiology - proliferation, cell cycle distribution, requirements for growth factors, apoptosis, and adhesiveness - in cell lines and murine marrow cells transfected with various HOX constructs. Patterns of surface adhesion molecules in these transfected cells will be studied by FACS analysis. 3) To identify target genes of HOX proteins in blood cell using a) a focused approach of examining binding of HOX proteins to regulatory regions of adhesion molecule genes expressed in blood cells, and the effects of HOX gene expression on the transcription of a reporter gene containing CAM gene regulatory sequences, and b) a global approach using the differential display technique to identify early mRNA changes in cells transfected with HOX gene expression vectors.
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