Regulation of Spermatocyte Transcription by Testis TAFS

睾丸 TAFS 调节精母细胞转录

• 批准号: 7874882
• 负责人: MARGARET T FULLER
• 金额: $ 18.1万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7874882
• 关键词: Binding; Binding Sites; Biological Assay; Cell Nucleolus; Cell Nucleus; Chromatin; Chromatin Remodeling Factor; Cis-Acting Sequence; Complex; Dependence; Drosophila genus; Enhancers; Epitopes; Gametogenesis; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Germ Cells; Homologous Gene; Hybrids; Immunoprecipitation; Light; Mammals; Maps; Meiosis; Molecular; Mutation; Nature; Nuclear; PRC1 Protein; Phenotype; Polycomb; Prophase; Protein Binding; Regulation; Reporter; Role; Scanning; Spermatids; Spermatocytes; Spermatogenesis; Spermatogonia; Spermiogenesis; Staining method; Stains; System; Testing; Testis; Tissues; Trans-Activators; Transcription Coactivator; Transcription Factor TFIIA; Work; Yeasts; cell type; chromatin immunoprecipitation; fly; in vivo; male; mutant; programs; promoter; transcription factor

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.

描述（由申请人提供）：雄性配子发生的戏剧性细胞分化程序取决于在减数分裂前期启动的强大的、细胞类型特异性的转录程序。我们发现，一般 Po1II 转录机制组件的睾丸特异性同源物调节果蝇精母细胞中终末分化基因的转录。 TAF、TBP 和 TFIIA 亚基的组织特异性形式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 TAF 选择性调节精子细胞分化基因转录的机制。我们发现睾丸 TAF 与原代精母细胞中的 Polycomb (Pc) 转录沉默机制的组件共定位到核仁，并且是其定位所必需的，这表明睾丸 TAF 可能通过隔离或拮抗负调节因子来允许靶基因的表达。我们将确定睾丸 TAF 是否直接与 Polycomb 成分结合或在 TFIID 样或 HAT 样复合物中起作用，并从遗传学上测试 Polycomb 和 trithorax 调节复合物是否控制初级精母细胞中精细胞分化基因的表达。为了研究睾丸 TAF 如何调节基因表达，我们将绘制使靶基因依赖于睾丸 TAF 的顺式作用序列，并确定这些序列是否可能与精母细胞中表达的激活剂或抑制物结合，而睾丸 TAF 必须通过关键顺式作用基序的突变来克服这些抑制剂，测试 Polycomb 亚基、睾丸 TAF 或其他反式作用对控制区域的占用情况。 通过染色质免疫沉淀 (ChIP) 适当调节调节剂。为了确定可能与睾丸 TAF 一起作用来调节精子细胞分化基因转录的伙伴或下游因子，我们将研究并克隆 mage，这是一种与睾丸 TAF 具有相似突变表型的基因。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关键点的分子机制，并揭示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。