Regulation of Spermatocyte Transcription by Testis TAFs

描述（由申请人提供）：编程细胞从增殖前体细胞分化的基因调控机制对于发育、组织稳态和癌症至关重要。使用戏剧性的细胞分化程序的精子发生作为模型干细胞谱系，我们试图了解独特的，细胞类型特异性转录程序在精母细胞阶段启动的程序细胞的终末分化。我们发现，睾丸特异性TAFs，同源的组件的一般PolII转录机器，打开精子细胞分化基因的表达，部分通过抵消沉默的Polycomb机器在前体细胞。紧接着的下一个挑战是发现睾丸TAF如何作用-直接在启动子处激活终末分化基因的稳健表达，以及通过隔离Polycomb转录沉默复合物在精母细胞核仁中。我们将测试tTAFs是否形成睾丸特异性SAGA样复合物，招募组蛋白去泛素化酶启动子近端停滞的聚合酶，允许延长靶转录物，或相反，在TFIID样复合物中与TBP起作用。我们将研究候选辅因子的作用，这些辅因子可能与我们在试验性RNAi筛选中确定的tTAF、介体亚基Med27和孤儿核激素受体Hr51一起起作用。由于tTAF发挥着如此关键的作用，因此对tTAF本身的细胞类型和阶段特异性表达进行精细编程的机制对于理解细胞分化的发育调控途径至关重要。我们将研究BTB-Zn指转录调节因子Lola的作用和作用方式，该因子是精母细胞中tTAFs表达所需的，其结合伴侣Jil 1 H3S10激酶是正确的减数分裂细胞周期进程和精子细胞分化所需的，如Lola。此外，在一个定向遗传筛选，我们将测试候选转录调节表达的开关从精原细胞到精母细胞，以确定额外的辅因子与tTAF的行为和阐明的监管机制，控制tTAF的表达。我们的工作在果蝇雄性生殖系中的tTAFs的作用机制和调节模式可能提供了一个范例，了解如何发展程序细胞类型的特定终末分化，通过抵消镇压的PcG在特定的靶基因。此外，了解果蝇减数分裂阻滞基因的作用方式可能会阐明人类减数分裂I成熟阻滞不育的机制。 公共卫生相关性：相关性的简短声明：这个项目的目标是了解基因调控机制，程序分化的雄性配子。在胚胎干细胞谱系和成体干细胞谱系中，在前体细胞中被Polycomb转录沉默机制抑制的终末分化基因必须在细胞分化时以细胞类型特异性和基因选择性的方式被稳健地激活。我们对果蝇睾丸特异性TAFs的作用和调节模式的分析结果将为核心基因调节复合物的细胞类型特异性替代形式如何控制细胞分化以及这些机制本身如何受生殖系发育程序控制建立范例。由于tTAF突变导致减数分裂I停滞和精子细胞分化失败，因此了解它们的调节和作用方式可能会阐明男性不育综合征减数分裂I成熟停滞的机制。

DESCRIPTION (provided by applicant): The gene regulatory mechanisms that program cell differentiation from proliferating precursor cells are fundamentally important for development, tissue homeostasis and cancer. Using the dramatic cellular differentiation program of spermatogenesis as a model stem cell lineage, we seek to understand the unique, cell type specific transcription program initiated in the spermatocyte stage that programs cells for terminal differentiation. We discovered that testis-specific TAFs, homologs of components of the general PolII transcription machinery, turn on expression of spermatid differentiation genes, in part by counteracting silencing by the Polycomb machinery in precursor cells. The immediate next challenge is to discover how the testis TAFs act - both directly at promoters to activate robust expression of terminal differentiation genes and in the spermatocyte nucleolus by sequestering the Polycomb transcriptional silencing complex. We will test whether the tTAFs form a testis-specific SAGA-like complex that recruits a histone deubiquitinating enzyme to promoter proximal stalled polymerases, allowing elongation of target transcripts, or instead act in a TFIID-like complex with TBP. We will investigate the role of candidate cofactors that may act with the tTAFs we have identified in a pilot RNAi screen, the mediator subunit Med27 and the orphan nuclear hormone receptor Hr51. Because the tTAFs play such a key role, the mechanisms that program the exquisitely cell type and stage specific expression of the tTAFs themselves are central for understanding the developmental regulatory pathway for cell differentiation. We will investigate the role and mode of action of the BTB-Zn finger transcriptional regulator lola, required for expression of tTAFs in spermatocytes, and its binding partner the Jil1 H3S10 kinase, which is required, like lola, for proper meiotic cell cycle progression and spermatid differentiation. In addition, in a directed genetic screen, we will test candidate transcription regulators expressed at the switch from spermatogonia to spermatocyte to identify additional cofactors that act with the tTAFs and elucidate the regulatory machinery that controls tTAF expression. Our work on the mechanism of action and mode of regulation of the tTAFs in the Drosophila male germ line may provide a paradigm for understanding how development programs cell-type specific terminal differentiation by counteracting repression by the PcG at specific target genes. In addition, understanding the mode of action of the meiotic arrest genes of Drosophila may illuminate mechanisms underlying meiosis I maturation arrest infertility in man. PUBLIC HEALTH RELEVANCE: Short statement of relevance: The goal of this project is to understand the gene regulatory mechanisms that program differentiation of male gametes. In both embryonic and adult stem cell lineages, terminal differentiation genes that are repressed by the Polycomb transcriptional silencing machinery in precursor cells must be robustly activated in a cell type specific and gene- selective manner as cell differentiate. Results of our analysis of the mode of action and regulation of the testis specific TAFs of Drosophila will establish paradigms for how cell type specific alternate forms of core gene regulatory complexes act to control cell differentiation, and how these mechanisms are themselves controlled by the germ line developmental program. Because mutations in the tTAFs cause meiosis I arrest and failure of spermatid differentiation, understanding their regulation and mode of action may illuminate mechanisms underlying the male infertility syndrome meiosis I maturation arrest.