Regulation of Spermatocyte Transcription by Testis TAFs

描述(由申请人提供)：从增殖的前体细胞编程细胞分化的基因调控机制对发育、组织动态平衡和癌症至关重要。利用精子发生的戏剧性细胞分化程序作为干细胞谱系的模型，我们试图了解在精母细胞阶段启动的独特的、细胞类型特定的转录程序，该程序为细胞的终末分化编程。我们发现，睾丸特异的TAFs，一般PolII转录机制组件的同源物，开启精子细胞分化基因的表达，部分是通过对抗前体细胞中Polycomb机制的沉默。迫在眉睫的下一个挑战是发现睾丸TAFs是如何作用的--既直接作用于启动子以激活末端分化基因的强劲表达，又通过隔离Polycomb转录沉默复合体在精母细胞核仁中发挥作用。我们将测试tTAFs是否形成睾丸特异的SAGA样复合体，招募组蛋白去泛素酶来启动近端停滞的聚合酶，从而延长目标转录，还是与TBP作用于TFIID样复合体。我们将研究可能与我们在试点RNAi筛查中确定的tTAFs、中介亚基Med27和孤儿核激素受体Hr51一起作用的候选辅助因子的作用。由于tTAFs发挥着如此关键的作用，因此对tTAFs本身的精细细胞类型和阶段特异性表达进行编程的机制对于理解细胞分化的发育调控途径是至关重要的。我们将研究在精母细胞中表达tTAFs所需的BTB-锌指转录调控因子LOLA及其结合伙伴JIL1 H3S10激酶的作用和作用方式，与LOLA一样，JIL1 H3S10激酶是正常的减数分裂细胞周期进展和精子细胞分化所必需的。此外，在定向遗传筛选中，我们将测试在从精原细胞到精母细胞的转换过程中表达的候选转录调控因子，以确定与tTAFs作用的其他辅助因子，并阐明控制tTAF表达的调控机制。我们在果蝇雄性生殖系中tTAFs的作用机制和调节方式的工作可能为理解发育如何通过对抗PcG对特定靶基因的抑制来编程细胞类型特定的末端分化提供了一个范例。此外，了解果蝇减数分裂停滞基因的作用方式可能有助于阐明人类减数分裂I成熟停滞不育的机制。 公共卫生相关性：简短的相关性声明：本项目的目标是了解控制雄配子分化的基因调控机制。在胚胎和成体干细胞谱系中，前体细胞中受Polycomb转录沉默机制抑制的末端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强有力地激活。我们对果蝇睾丸特异性TAFs作用模式和调控的分析结果将为细胞类型特定的核心基因调控复合体的替代形式如何作用于控制细胞分化，以及这些机制本身如何受到生殖系发育程序的控制建立范例。由于tTAFs的突变导致减数分裂I期停滞和精子细胞分化失败，了解tTAFs的调控和作用方式可能有助于阐明男性不育综合征减数分裂I期成熟停滞的机制。

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.