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

项目概要/摘要 从增殖到分化的转变是成体干细胞谱系中的关键调节点， 作为组织维持和修复的基础，不能干净地转换可能有助于癌症的发生。我 实验室长期以来一直使用果蝇雄性生殖系作为模型，研究自我更新， 在成体干细胞谱系中调节增殖和分化。我们的调查中有几条线索 最近集中在分子机制的发展编程过渡， 有丝分裂增殖到减数分裂和分化的开始，涉及许多分子和细胞 调节这一关键开关的机制。我们发现参与翻译控制的RNA结合蛋白 和选择性剪接自主地作用于细胞以调节增殖停止和 分化需要来自相关的体细胞支持细胞的通讯。我们发现， 某些新生转录物被切割形成3 '末端的发育调节的替代选择位点， 导致产生具有缩短的3'UTR的新mRNA同种型，控制了该套件中的显著变化。 与增殖的精原细胞相比，分化的精母细胞中表达的蛋白质。我们发现 染色质的巨大变化打开了2000多个具有新核心序列结构的新启动子 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。一些 在这个分化程序中最早启动的基因编码染色质相关蛋白， 通常隐藏的启动子的假开放，从而防止相关基因的大量错误表达， 错误的细胞类型。其他转录本在分化开始时上调，编码细胞类型特异性 延迟核心G2/M细胞周期机器产生以编程延长的G2的翻译调节因子 减数分裂前期。在接下来的5年里，我们计划绘制这些流程的协作方式， 形成调节电路，启动然后执行从增殖到分化的转换。我们将 研究RNA结合蛋白Bam和Bgcn如何触发从有丝分裂到减数分裂的转换， 抑制选择性剪接因子HOW的表达，鉴定HOW的候选底物， 免疫沉淀，然后RNA-Seq，并评估其在体内的功能，包括它们是否 与相邻的支持细胞沟通。我们将研究蛋白质表达中的开关如何 由于在新生转录物上的选择性3 '端切割位点受到调节并影响分化。 我们将研究分化程序是如何保持在前体细胞和细胞类型特异性 染色质调节因子和将它们募集到靶位点的蛋白质建立了新的转录程序， 分化为了阐明发育程序如何重塑细胞周期，我们将研究 细胞类型特异性RNA结合蛋白在减数分裂期间首先抑制，然后激活细胞周期蛋白B的翻译 以及DAZ同源物Boule如何调节减数分裂的进程。 .

Project Summary / Abstract The switch from proliferation to differentiation is a key regulatory point in the adult stem cell lineages that underlie tissue maintenance and repair, and failure to cleanly 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 converged 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 alternate choice of site at which certain 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 proliferation to differentiation. We will investigate how the RNA binding proteins Bam and Bgcn trigger the switch from mitosis to meiosis 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 the differentiation program is kept off in precursor cells and how cell-type specific chromatin regulators and proteins that recruit them to target loci set up the new transcription program for differentiation. To elucidate how the developmental program remodels the cell cycle, 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. .