Regulation of Spermatocyte Transcription by Testis TAFS

描述（由申请人提供）：雄性配子发生的显著细胞分化程序依赖于减数分裂前期启动的稳健的细胞类型特异性转录程序。我们发现，睾丸特异性同源的一般Po1 II转录机制组件调节果蝇精母细胞终末分化基因的转录。组织特异性形式的TAF、TBP和TFIIA亚基也与哺乳动物的精子发生有关。我们现在建议利用果蝇系统来研究睾丸TAFs选择性调节精子细胞分化基因转录的机制。我们发现，睾丸TAFs的共定位和定位的Polycomb（Pc）转录沉默机制的组件在初级精母细胞的核仁所需的，这表明睾丸TAFs可能允许靶基因的表达，通过隔离或拮抗负调节。我们将确定睾丸TAFs是否直接结合到Polycomb组件或在TFIID样或HAT样复合物中起作用，并从遗传学上测试Polycomb和三胸调节复合物是否控制初级精母细胞中精子细胞分化基因的表达。为了研究睾丸TAFs如何调节基因表达，我们将绘制使靶基因依赖于睾丸TAFs的顺式作用序列，并确定这些顺式作用序列是否可能结合精母细胞中表达的激活因子或必须由睾丸TAFs通过关键顺式作用基序突变克服的阻遏因子，测试Polycomb亚基，睾丸TAFs，或其它反式作用调节剂（视情况而定）通过染色质免疫沉淀（ChIP）。为了确定一个可能的合作伙伴或下游因素，可能会与睾丸TAFs调节精子细胞分化基因的转录，我们将调查和克隆法师，一个基因与睾丸TAFs类似的突变表型。我们提出的工作将揭示作用于控制雄性配子终末分化的遗传调控网络的关键点的分子机制，并阐明染色质沉默和核亚结构在初级精母细胞转录程序调控中的可能作用。

DESCRIPTION (provided by applicant): 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.