The research project described herein has been designed to provide a broad foundation in molecular biology for the principal investigator, Dr. Agura. It encompasses many current techniques including PCR, DNA sequencing, basic cloning, cell transfection, gel shift and DNAseI footprinting, DNAse hypersensitive site mapping and protein purification. The strategies proposed in this grant are current and have been used successfully in other laboratories around the world. Upon completion of this project, Dr. Agura will have sufficient expertise to compete successfully for other basic research grants. Scientific Goal: Identifying Factors Controlling Myeloid Gene Expression. The normal processes of myeloid differentiation are poorly understood especially in molecular genetic terms. This is paradoxical given the large number of cell lines produced, and cytogenetic abnormalities catalogued from, patients with acute myeloid leukemia. Some inroads have been made, however, by studying HL-60 cells. This promeylocytic line is a useful model system for studying the role of retinoids in normal myeloid maturation since it can be induced to differentiate with the Vitamin A derivative, retinoic acid (RA). The CD18 gene, which codes for leukocyte integrin beta chains, is a target for myeloid transcriptional regulators; it is strongly induced by RA in HL60 cells and is present in large quantities on mature neutrophils. Dr. Agura has analyzed the CD18 gene for the past 2 years and has recently identified its transcription start site and promoter. He has discovered that the CD18 promoter is unique: it completely lacks CAAT, TATA or other """"""""common"""""""" elements, yet drives tissue-specific and hormone (RA) inducible transcription. Dr. Agura is proposing to study the regulation of this unique gene to identify novel myeloid-specific and retinoid-inducible transcription factors. His approach will be to first, identify the genetic elements which participate in tissue-specific and RA-inducible transcriptional regulation. Then, he will identify myeloid proteins which bind those elements. He will specifically examine the role played by nuclear receptors for RA (RAR, RXR) but will also look for other factors which may confer myeloid-specific gene regulation or participate in the RA- inducible control of gene expression. These studies may contribute several important aspects to the understanding of normal and disordered eukaryotic gene regulation in myeloid lineages.
