The dramatic cellular differentiation program of male gametogenesis depends on a robust, cell type specific transcription program initiated in meiotic prophase. We discovered that testis-specific homologs of general Po1II transcription machinery components regulate transcription of terminal differentiation genes in Drosophila spermatocytes. Tissue-specific forms of TAFs, TBP and subunits of TFIIA have also been implicated in spermatogenesis in mammals. We propose now to exploit the Drosophila system to investigate the mechanism by which testis TAFs selectively regulate transcription of spermatid differentiation genes. We found that the testis TAFs co-localize with and are required for localization of components of the Polycomb (Pc) transcriptional silencing machinery to the nucleolus in primary spermatocytes, suggesting that testis TAFs might allow expression of target genes by sequestering or antagonizing a negative regulator. We will determine if testis TAFs bind directly to Polycomb components or act in a TFIID-like or HAT-like complex and test genetically whether the Polycomb and trithorax regulatory complexes control expression of spermatid differentiation genes in primary spermatocytes. To investigate how the testis TAFs regulate gene expression, we will map cis-acting sequences that make target genes depend on the testis TAFs and determine whether these are likely to bind activators for expression in spermatocytes or repressors that must be overcome by the testis TAFs by mutation of key cis-acting motifs, testing occupancy of control regions by Polycomb subunits, testis TAFs, or other trans-acting regulators as appropriate by chromatin immune-precipitation (ChIP). To identify a possible partner or downstream factor that may act with the testis TAFs to regulate transcription of spermatid differentiation genes, we will investigate and clone mage, a gene with a similar mutant phenotype as the testis TAFs. Our proposed work will reveal molecular mechanisms that act at key points of the genetic regulatory network controlling terminal differentiation of male gametes and shed light possible roles for chromatin silencing and nuclear substructure in regulation of the primary spermatocyte transcription program.
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