Regulation of proliferation and differentiation in the male germ line adult stem cell lineage

项目总结(上级项目)： 雄性生殖系成体干细胞系增殖分化的调控 从增殖到分化的转换是胚胎发育和细胞分化的关键调节点。 作为组织维持和修复的基础的成人干细胞谱系。此交换机的故障可能会导致 癌症的起源。我的实验室长期以来一直使用果蝇雄性生殖系作为模型来研究如何 自我更新、增殖和分化在成体干细胞谱系中受到调节。我们调查的几行字 最近开始集中在发育程序化背后的分子机制上 从有丝分裂增殖到减数分裂和分化的开始，涉及许多分子和 调节这一关键开关的细胞机制。我们发现RNA结合蛋白参与翻译 控制和选择性剪接自主地作用于细胞，以调节增殖的停止，并且 分化的进展需要相关的体细胞支持细胞进行沟通。我们发现 在特定的新生转录本被切割成3‘末端的位置上，有一个发育调节的开关， 导致产生具有缩短的3‘UTRs的新的mRNA异构体，控制着 在分化的精母细胞和增殖的精原细胞中表达的蛋白质。我们发现 染色质的戏剧性变化开启了2000多个新的启动子，这些启动子具有新颖的核心序列结构 当细胞启动精母细胞分化时，新的细胞类型特异性转录程序。其中一些最早的 在这种分化程序中打开的基因编码与染色质相关的蛋白，以防止虚假 打开通常隐蔽的启动子，从而防止与基因相关的大量错误表达 单元类型错误。其他随着分化开始上调的转录本编码特定细胞类型的翻译 推迟核心G2/M细胞周期机制的产生，以编程延长的G2减数分裂阶段的调节器 前期工作。在接下来的5年里，我们建议绘制出这些流程如何协作以形成法规 启动的电路随后执行从有丝分裂到减数分裂的切换。我们将研究RNA结合是如何 Bam和Bgcn蛋白通过抑制有丝分裂到分化的表达而触发从有丝分裂到分化的转换 选择性剪接因子How，通过免疫沉淀和RNA- SEQ，并评估它们在体内的功能，包括它们是否与邻近的躯体支持细胞通信。 我们将研究在新生阶段，由于备选3‘端切割位点的选择而导致的蛋白质表达的转换 转录产物是受调控的，并影响分化。我们将研究细胞类型的特定染色质是如何 将它们招募到特定基因座的调节器和蛋白质建立了新的转录程序来进行分化。 为了阐明发育程序如何重塑基本的细胞过程，如设置细胞周期 上调分化的特化细胞类型，我们将首先研究细胞类型特异的RNA结合蛋白 抑制、激活细胞周期蛋白B在减数分裂前期的翻译以及DAZ同源基因Boule如何调节 进入减数分裂。

PROJECT SUMMARY (of Parent Project): Regulation of proliferation and differentiation in the male germ line adult stem cell lineage The switch from proliferation to differentiation is a key regulatory point in both embryonic development and the adult stem cell lineages that underlie tissue maintenance and repair. Failure of this switch may contribute to genesis of cancer. My laboratory has long used the Drosophila male germ line as a model to investigate how self-renewal, proliferation and differentiation are regulated in adult stem cell lineages. Several lines of our inquiry have recently begun to converge on the molecular mechanisms underlying the developmentally programmed transition from mitotic proliferation to onset of meiosis and differentiation, implicating a number of molecular and cellular mechanisms in regulating this critical switch. We find that RNA binding proteins involved in translational control and alternative splicing act cell autonomously to regulate the cessation of proliferation and that progression of differentiation requires communication from associated somatic support cells. We discovered that a developmentally regulated switch in the site at which specific nascent transcripts are cut to form 3’ ends, leading to production of novel mRNA isoforms with shortened 3’UTRs, controls dramatic changes in the suite of proteins expressed in differentiating spermatocytes compared to proliferating spermatogonia. We found that dramatic changes in chromatin open over 2000 new promoters with novel core sequence structure to turn on the new cell type specific transcription program when cells initiate spermatocyte differentiation. Some of the earliest genes turned on in this differentiation program encode chromatin associated proteins that prevent spurious opening of normally cryptic promoters, thus preventing massive misexpression of genes associated with the wrong cell type. Other transcripts upregulated with differentiation onset encode cell type-specific translational regulators that delay production of core G2/M cell cycle machinery to program the extended G2 phase of meiotic prophase. Over the next 5 years, we propose to map how these processes collaborate to form the regulatory circuitry that initiates then executes the switch from mitosis to meiosis. We will investigate how the RNA binding proteins Bam and Bgcn trigger the switch from mitosis to differentiation by repressing expression of the alternative splice factor HOW, identify candidate substrates of HOW by immunoprecipitation followed by RNA- Seq, and assess their function in vivo, including whether they communicate with adjacent somatic support cells. We will investigate how the switch in proteins expressed due to alternative 3’ end cut site selection on nascent transcripts is regulated and influences differentiation. We will investigate how cell-type specific chromatin regulators and proteins that recruit them to specific loci set up the new transcription program for differentiation. To elucidate how the developmental program remodels fundamental cellular processes like the cell cycle to set up differentiation of specialized cell types, we will investigate how cell-type specific RNA binding proteins first repress, then activate translation of cyclin B during meiotic prophase and how the DAZ homolog Boule regulates progression into the meiotic divisions.