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

雄性生殖系成体干细胞谱系增殖和分化的调节

• 批准号: 10160936
• 负责人: MARGARET T FULLER
• 金额: $ 85.15万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10160936
• 关键词: 3' Untranslated Regions; Alternative Splicing; Architecture; Biological; CSPG6 gene; Cell Communication; Cell Cycle; Cell Lineage; Cells; Chromatin; Communication; Cyclin B; Development; Drosophila genus; Ensure; Failure; G2 Phase; Gene Expression; Genes; Genetic Transcription; Germ Lines; Homologous Gene; Immunoprecipitation; Laboratories; M cell; Maintenance; Malignant Neoplasms; Maps; Meiosis; Messenger RNA; Mitosis; Mitotic; Modeling; Molecular; Process; Production; Proliferating; Prophase; Protein Isoforms; Proteins; RNA-Binding Proteins; Regulation; Site; Spermatocytes; Spermatogonia; Structure; Supporting Cell; System; Tissues; Transcript; Translations; adult stem cell; cell type; in vivo; male; novel; precursor cell; prevent; programs; promoter; recruit; repaired; self-renewal; transcriptome sequencing

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. .

项目概要/摘要 从增殖到分化的转变是成体干细胞谱系的关键调节点 是组织维护和修复的基础，如果不能干净地转换可能会导致癌症的发生。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自我更新、 成体干细胞谱系的增殖和分化受到调节。我们的几行询问有 最近集中于从发育程序转变背后的分子机制 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 调节这个关键开关的机制。我们发现RNA结合蛋白参与翻译控制 选择性剪接作用于细胞自主调节增殖的停止和增殖的进展 分化需要相关体细胞支持细胞的通信。我们发现一个 发育调控的位点替代选择，在该位点某些新生转录本被切割形成 3' 末端， 导致产生具有缩短的 3'UTR 的新型 mRNA 异构体，控制套件中的巨大变化 与增殖的精原细胞相比，分化的精母细胞中表达的蛋白质的数量。我们发现 染色质的巨大变化开启了 2000 多个具有新颖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 在此分化程序中最早开启的基因编码染色质相关蛋白，从而阻止 通常隐藏的启动子的虚假打开，从而防止相关基因的大量错误表达 细胞类型错误。随着分化开始而上调的其他转录物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扩展 G2 的翻译调节因子 减数分裂前期。在接下来的 5 年里，我们建议绘制这些流程如何协作的图 形成启动然后执行从增殖到分化的转换的调节电路。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变 抑制替代剪接因子 HOW 的表达，通过以下方式识别 HOW 的候选底物 免疫沉淀，然后进行 RNA-Seq，并评估它们的体内功能，包括它们是否 与邻近的体细胞支持细胞通讯。我们将研究蛋白质的转换如何表达 由于新生转录本上的选择性 3' 末端切割位点选择受到调节并影响分化。 我们将研究如何在前体细胞中阻止分化程序以及细胞类型特异性如何 染色质调节因子和将它们招募到目标位点的蛋白质建立了新的转录程序 差异化。为了阐明发育程序如何重塑细胞周期，我们将研究如何 细胞类型特异性 RNA 结合蛋白在减数分裂过程中首先抑制然后激活细胞周期蛋白 B 的翻译 前期以及 DAZ 同源物 Boule 如何调节减数分裂的进展。 。