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Education Ph.D., National Institute of Immunology ResearchTranscriptional regulatory mechanisms determine cellular phenotypes by directing precise temporal patterns of gene expression. Mammalian spermatogenesis provides an excellent model system to study transcriptional control during differentiation of a defined cell lineage. Successful completion of spermatogenesis relies on sequential expression of characteristic groups of genes unique to each cell type; e.g., spermatogonium, spermatocyte or spermatid. This implies that distinct transcriptional control mechanisms may operate at different stages of the seminiferous epithelium cycle. We are interested in understanding the transcriptional control of spermatid gene expression. The haploid spermatids synthesize a number of unique proteins such as acrosomal and flagellar proteins, and enter a phase of biochemical and morphogenetic change, thus completing the differentiation into spermatozoa. We hypothesize that one or more key testis-specific transcription factors regulate the co-ordinate expression of a cohort of genes in the haploid spermatid to effect the terminal differentiation process. Currently, we are utilizing a gene encoding the acrosomal protein SP-10 as a model for studying regulation of post-meiotic, testis-specific gene expression. Messenger RNA for SP-10 is first detected in the round spermatids. Antibodies to SP-10 prevent fertilization in vitro, thus indicating a role for this protein in sperm-egg interaction. SP-10 is well characterized at the genomic level in both human and mouse. Promoter analysis of SP-10 gene is undertaken to identify the testicular transcription factors which govern post-meiotic gene expression. Transgenic mice have been generated using reporter gene constructs in which GFP (green fluorescent protein) cDNA is placed under the control of SP-10 promoter. Using this approach, the minimal SP-10 promoter sufficient to drive testis-specific gene expression has been identified. Gel-shift assays, DNAse Footprinting, in vitro Transcription, as well as Yeast one-hybrid system are being used to identify the cis-acting elements in SP-10 promoter and to isolate the cognate trans-acting factors. The long term goal of this study is to understand the extracellular signals and the transcriptional control mechanisms underlying the terminal differentiation process of male germ cells. Representative Publications 1. Reddi P.P., Marko Kallio, and John C. Herr.(1999) Green Fluorescent Protein as a Reporter for Promoter Analysis of Testis-Specific Genes in Transgenic Mice. Methods in Enzymology 302:272-284. |