Regulation of Spermatocyte Transcription by Testis TAFS

描述(由申请人提供)：雄配子发生的戏剧性细胞分化程序依赖于减数分裂前期启动的强大的、细胞类型特定的转录程序。我们发现，在果蝇精母细胞中，睾丸特异的PO1II转录机制组分的同源物调控着末端分化基因的转录。组织特异性形式的TAFs、TBP和TFIIA亚单位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选择性调控精子细胞分化基因转录的机制。我们发现，睾丸TAFs与Polycomb(Pc)转录沉默机制的组成部分在初级精母细胞的核仁中共定位，并且是定位所必需的，这表明睾丸TAFs可能通过隔离或拮抗负调控因子来允许靶基因的表达。我们将确定睾丸TAFs是直接与Polycomb组分结合，还是以TFIID样或HAT样复合体的形式发挥作用，并从基因上测试Polycomb和三胸调节复合体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是如何调节基因表达的，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激活因子或抑制子在精母细胞或抑制子中表达，而睾丸TAFs必须通过关键的顺式作用基序的突变来克服这些顺式作用基序，通过染色质免疫沉淀(CHIP)测试多梳亚单位、睾丸TAFs或其他适当的反式调节因子对控制区的占据。为了确定一个可能与睾丸TAFs一起作用于精子细胞分化基因转录调控的伙伴或下游因子，我们将研究和克隆MAGE基因，该基因与睾丸TAFs具有相似的突变表型。我们的工作将揭示作用于控制雄配子末端分化的遗传调控网络关键点的分子机制，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.