The long term objective of this project is to determine mechanisms regulating expression of the histone genes during spermatogenesis. The immediate objective of this project is to determine the contributions made by transcriptional regulation and by posttranscriptional mechanisms such as mRNA stability in controlling the steady-state levels of histone H4t mRNA in various stages of germinal cell development. To meet these objectives, the following three specific aims are proposed. (1) The histone H4t promoter will be analyzed in order to establish the extent to which transcription regulates expression of the histone H4t gene during spermatogenesis. The degree to which transcription of the H4t gene is under stringent control and is downregulated in late pachytene spermatocytes and in early spermatids and the degree to which the gene is constitutively expressed during the transition from meiotic pachytene spermatocytes to postmeiotic early spermatids will be determined using in vitro transcription assays. (2) The stability of H4t mRNA will be measured in order to determine the degree to which mRNA stability regulates expression of the gene. H4t mRNA turnover rates will be measured by pulse chase experiments. The effects of 5'-leader region mutations and 3'-noncoding region mutations upon H4t mRNA stability will be examined. The H4t gene with a coding region mutation to be used as a marker will be placed under the control of an inducible promoter and transfected into specific cell types. The gene will be will be induced and the mutant mRNA levels and turnover rates will be followed. (3) Protein-DNA interactions will be examined in order to understand mechanisms by which the gene is transcribed also determine the tissue specificity of transcription and the level of transcription. DNA binding proteins will be identified, characterized, and purified and DNA-protein interactions within the promoter will be established. The proximal promoter and the distal upstream region of the promoter will be examined using in vitro and in vivo DNA footprinting. The proximal promoter region and upstream and downstream regions of the gene will be mapped for in vitro patterns of binding of nuclear proteins. Analysis of specific binding will be conducted by electrophoretic mobility shift assays and footprint analysis.
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