The goal of these studies is to determine how the multilineage hematopoietic growth factor, granulocyte- macrophage colony-stimulating factor (GM-CSF), stimulates the proliferation and maturation of progenitor cells into specific myeloid elements. Our studies will focus on a class of genes known to play an important role in coordinating embryogenesis and differentiation in Drosophila and Xenopus, the homeobox genes. These genes encode transcription factors that contain a highly conserved 60-amino acid DNA- binding motif known as the homeodomain. Homeobox genes can both positively and negatively regulate the transcription of other genes, and consequently, they play critical roles in anteriorposterior axis formation, segmentation, and orchestrate the coordinated expression of genes required to generate complex structures. The focus of this grant application is to identify the homeobox genes that are involved in myeloid differentiation and to elucidate how interaction of GM-CSF with the myeloid progenitor cells affects the expression and action of these homeobox genes.
Specific Aim 1 - Identify and characterize homeobox genes involved in human myelopoiesis. Reverse transcription polymerase chain reaction (RT-PCR), using degenerate primers recognizing the homeodomain, is employed to identify the homeobox genes expressed in normal bone marrow progenitors stimulated by GM- CSF, in semi-solid media, to undergo two or three cell divisions. The expression of specific homeobox genes in myeloid progenitors will be confirmed using quantitative RT-PCR (and specific primers) of fluorescence-activated cell sorted (FACS) bone marrow subsets. Evidence supporting a functional role for these homeobox genes will be obtained using antisense oligonucleotides (ODN) to block their expression in in vitro assays. We have successfully employed these strategies to identify three HOX genes, and thus far, have obtained biological data supporting the role of two of them in myelopoiesis.
Specific Aim 2 - Employ biological assays to determine the functional role of specific homeobox genes in human myelopoiesis. Retroviral vectors will be used to enforce expression of specific homeobox genes in hematopoietic cell lines and primary hematopoietic progenitor cells. Cell lines will be assayed for their responses to multiple differentiation inducers. Transduced CD34+ progenitor cells will be assayed in long-term culture assays for their differentiation and proliferative potentials.
Specific Aim 3 - Identify target genes that are regulated by specific homeobox proteins during myelopoiesis. Homeobox genes identified in Aim 1 and shown to play a physiologic role in myelopoiesis in studies described in Aim 2 will be used to isolate target genes. Inducible plasmid vectors directing the expression of a specific HOX gene will be stably transfected into a model hematopoietic cell line; """"""""representational difference analysis"""""""" (RDA) will be used to identify target genes that are either activated or repressed by the expression of this homeobox gene.
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