In acute myelogenous leukemia (AML), undifferentiated blast cells are unable to mature normally into granulocytes and monocytes. This results in the failure to produce normal differentiated myeloid and non-myeloid hematopoietic cells, and eventual death to the patient. We hope that through the careful study of regulation of a myeloid specific gene we shall be able to understand normal myeloid differentiation and gain some insight into the block in differentiation which results in AML. Therefore, the specific aims of this grant proposal are to study the regulation of CDllb, the alpha chain of the Mol antigen. The expression of CDllb is specific for myeloid cells and is strongly upregulated during the course of myeloid differentiation. Using a cDNA clone that we have previously isolated, we shall first isolate and characterize genomic clones for CDllb. We shall then examine the mechanism of the up-regulation of expression, investigating the rate of transcription and stability of the messenger RNA. We shall then proceed with a set of experiments designed to analyze on a molecular level the controls which result in the up-regulation of CDllb during normal myeloid differentiation. We shall first look for changes in the chromatin structure surrounding the gene, which may affect the ability of transcriptional factors to interact with control elements, specifically looking for changes in DNAse I and methylation patterns during differentiation, which might affect the expression of CDllb. We shall then isolate and characterize the transcriptional control elements which flank the CDllb and are responsible for both the baseline level of transcription, and for the upregulation of this gene in myeloid cells. These studies shall initially be performed in myeloid cell lines which have been derived from patients with AML. In order to demonstrate that the control elements for CDllb defined in these cells are the same as those that exist in normal hematopoietic cells, we shall repeat these experiments using isolated myeloid precursors. Finally, once we have defined the DNA regulatory elements which control the expression of CDllb in myeloid cells, we shall characterize and purify transcriptional factors which may interact with these control elements to regulate the expression of these genes. These experiments shall provide_a fundamental ground work for understanding the expression and up-regulation of this myeloid specific gene and will, hopefully, serve as a model for both normal and abnormal gene regulation in myeloid cells.
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