Investigating Rb/E2F control over gene expression during germline differentiation

描述（由申请方提供）：个体细胞命运的建立和维持是后生动物正常发育的基础。在正常发育过程中，细胞类型特化的关键是转录因子和染色质修饰酶的协调努力，以通过空间和时间建立精确的转录谱，这是实现成体形式的许多组织类型所必需的。在发育过程中，Rb/E2 F调节复合物调节基因表达，以在多种细胞类型中建立增殖和分化之间的平衡。然而，Rb/E2 F控制组织特异性基因表达的确切机制仍然知之甚少。越来越多的证据表明，Rb/E2 F的功能是通过使用其招募不同的染色质修饰酶的能力，以不同的基因子集，改变染色质景观，从而影响基因表达在整个成熟的生物体。本提案的目标是剖析Rb/E2 F定义的基因表达程序的分子机制，在从干细胞分化的细胞使用线虫线虫的生殖系作为模型。梭由于从干细胞到分化的配子的生殖细胞发育的所有阶段同时存在于成体性腺中，因此线虫生殖系为这种分析提供了特别适合的体内系统。通过对目前的染色质免疫沉淀结合深度测序（ChIP-seq）方法进行创新性调整，将在生殖系的不同亚群中鉴定Rb/E2 F靶基因及其转录状态。鉴于组蛋白尾部甲基化与转录激活和抑制之间的密切联系，还将鉴定Rb/E2 F靶基因的组蛋白甲基化谱。此外，组蛋白甲基转移酶（HMTs）和脱甲基酶（HDMs）的招聘染色质，以及这种招聘功能Rb途径的依赖性，在生殖细胞分化将进行分析。总而言之，这些分析将阐明Rb/E2 F如何与组蛋白甲基化系统相互作用，以调节种系内细胞分化期间的基因表达。总体而言，拟议的研究将提供显着的进步，我们的理解Rb功能在细胞类型的规范在一个定义的生物背景。

DESCRIPTION (provided by applicant): Establishment and maintenance of individual cell fates are fundamental for proper metazoan development. Essential to cell type specification during normal development is the coordinated effort of transcription factors and chromatin modifying enzymes to establish the precise transcriptional profiles through space and time that are necessary to achieve the numerous tissue types of the adult form. During development, the Rb/E2F regulatory complex modulates gene expression to establish the balance between proliferation and differentiation in multiple cell types. However, the exact mechanism(s) by which Rb/E2F controls tissue-specific gene expression remain poorly understood. An expanding body of evidence indicates that Rb/E2F functions by using its ability to recruit various chromatin modifying enzymes to distinct subsets of genes to alter the chromatin landscape, thus impacting gene expression throughout maturation of the organism. The goal of this proposal is to dissect the molecular mechanisms by which Rb/E2F defines gene expression programs during the progression from stem to differentiated cell using the germ line of the nematode Caenorhabditis elegans as a model. The C. elegans germ line offers a particularly well-suited in vivo system for such analyses, as all stages of germ cell development from stem cell to differentiated gamete are simultaneously present within the adult gonad. By applying innovative adaptations to current chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq) methodologies, Rb/E2F target genes and their transcriptional state will be identified in distinct subpopulations of the germ line. Given the intimate link between methylation of histone tails with transcriptional activation and repression, histone methylation profiles at Rb/E2F target genes will also be identified. Additionally, the recruitment of histone methyltransferases (HMTs) and demethylases (HDMs) to chromatin, as well as the dependence of such recruitment upon a functional Rb pathway, during germ cel differentiation will be analyzed. Altogether, these analyses will elucidate how Rb/E2F interacts with histone methylation systems to modulate gene expression during cellular differentiation within the germ line. Overall, the proposed studies will provide significant advancements in our understanding of Rb function during cell type specification in a defined biological context.