The major focus of this proposal is to initiate a detailed investigation of Sp-mediated regulatory mechanisms tha are responsible for temporal gene activation and expression during germ cell differentiation. The CREB and the LDH genes constitute the major experimental paradigm for these studies on the regulation of temporal gen activation during the early stages of mouse spermatogenesis. The applicant?s hypothesis is that the temporal transactivation pattems observed for CREB and the respective functional LDH genes are determined by interactions of Sp transcription factors with GC-box cis-regulatory elements present in the individual promoters.
In Aim 1, cDNA corresponding to these Sp transcription factor variants identified in germ cells will be overexpressed in Sp-deficient Drosophila S2 cells, the Sp proteins purified and utilized to synthesize specific antibodies for immunohistochemical studies to determine their respective spatial and cell-type-specific expression pattems In Aim 2, transfection assays will be performed in the Drosophila Schneider S2 cells to demonstrate that these Sp factor variants are able to transactivate the CREB and LDH promoter/CAT reporter constructs. Although Sp transcription factors are necessary for transactivation of the CREB and LDH genes, optimal and sustained gene expression levels require other germ-cell-specific coactivators that interact with the Sp transcription factors.
In Aim 3, the stage and cell-type- specific coactivators interacting with the Sp transcription variants will be identified and isolated by utilizing yeast two-hybrid screening strategies. Antibodies will be produced and immunohistochemical studies performed to demonstrate that these coactivators are expressed in a stage and cell-type-specific manner during spermatogenesis. Further transfection assays will be performed to verify that these isolated cofactors are required to sustain optimal levels of CREB and LDH promoter directed CAT gene expression. These studies will provide important insights into the transcriptional basis for differential gene expression during the early stages of spermatogenesis. The knowledge gained from these studies will have applications in designing therapeutic interventions for the treatment of cellular pathologies that affect the male reproductive tract and for developing effective male contraceptives.
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