Investigating Rb/E2F control over gene expression during germline differentiation

研究种系分化过程中 Rb/E2F 对基因表达的控制

• 批准号: 8311943
• 负责人: Kathryn Elizabeth Gardner
• 金额: $ 4.92万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8311943
• 关键词: Adopted; Adult; Animal Model; Binding; Binding Sites; Biological; Biological Models; Caenorhabditis elegans; Cell Differentiation process; Cell Nucleus; Cells; Chromatin; Co-Immunoprecipitations; Complement; Complex; Coupled; Cues; Dependence; Development; Disease; Embryonic Development; Enzymes; Equilibrium; Excision; Exhibits; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Germ; Germ Cells; Germ Lines; Goals; Gonadal structure; Histocompatibility Testing; Histone H3; Histone H4; Histones; Homeostasis; Individual; Light; Link; Lysine; Maintenance; Malignant Neoplasms; Meiosis; Methodology; Methylation; Modeling; Modification; Molecular; Multienzyme Complexes; Nematoda; Oocytes; Organism; Pathway interactions; Pattern; Phenocopy; Population; RNA Interference; RNA Polymerase II; Recruitment Activity; Sequence Analysis; Site; Staging; Stem Cell Development; Stem cells; System; Tail; Techniques; Therapeutic; Time; Tissue-Specific Gene Expression; Tissues; Transcriptional Activation; Transcriptional Regulation; Transgenic Organisms; Tumor Suppressor Proteins; Type I Epithelial Receptor Cell; Undifferentiated; base; cell type; chromatin immunoprecipitation; design; gene repression; genome-wide; histone methyltransferase; human disease; human stem cells; in vivo; innovation; insight; loss of function; mutant; progenitor; programs; research study; response; spatiotemporal; stem; transcription factor; tumor

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. PUBLIC HEALTH RELEVANCE: The tumor suppressor Rb/E2F regulates gene expression to control differentiation of multiple cell types during development, and accordingly is frequently inactivated in tumors of diverse tissue origin. The proposed studies will delineate mechanisms by which the Rb/E2F pathway directs tissue-specific gene regulation during the progression from a progenitor cell type to a differentiated state using the model organism C. elegans. As specification and maintenance of cellular identity is critical for tissue homeostasis, elucidating mechanisms by which Rb/E2F functions during cell-type specification is highly significant and will provide a platform for developing more specific therapeutic approaches targeting components of the Rb pathway in the treatment of human diseases, such as cancer.

描述（由申请人提供）：个体细胞命运的建立和维持对于后生动物的正常发育至关重要。正常发育过程中细胞类型规范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，这是获得成体形式的多种组织类型所必需的。在发育过程中，Rb/E2F 调控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/E2F 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/E2F 通过利用其将各种染色质修饰酶招募到不同基因子集的能力来发挥作用，从而改变染色质景观，从而影响整个生物体成熟过程中的基因表达。该提案的目标是使用线虫秀丽隐杆线虫种系作为模型，剖析 Rb/E2F 在从干细胞到分化细胞的进展过程中定义基因表达程序的分子机制。线虫生殖系为此类分析提供了特别适合的体内系统，因为从干细胞到分化配子的生殖细胞发育的所有阶段都同时存在于成体性腺中。通过对当前染色质免疫沉淀结合深度测序 (ChIP-seq) 方法进行创新改造，将在种系的不同亚群中鉴定 Rb/E2F 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，Rb/E2F 靶基因的组蛋白甲基化谱也将被鉴定。此外，还将分析生殖细胞分化过程中组蛋白甲基转移酶 (HMT) 和去甲基化酶 (HDM) 向染色质的募集，以及这种募集对功能性 Rb 途径的依赖性。总之，这些分析将阐明 Rb/E2F 如何与组蛋白甲基化系统相互作用，从而在种系内的细胞分化过程中调节基因表达。总体而言，拟议的研究将为我们在确定的生物学背景下细胞类型规范过程中对 Rb 功能的理解提供重大进展。 公共健康相关性：肿瘤抑制因子 Rb/E2F 调节基因表达以控制发育过程中多种细胞类型的分化，因此在不同组织来源的肿瘤中经常失活。拟议的研究将利用模型生物线虫描述 Rb/E2F 通路在从祖细胞类型到分化状态的进展过程中指导组织特异性基因调控的机制。由于细胞身份的规范和维持对于组织稳态至关重要，因此阐明 Rb/E2F 在细胞类型规范过程中发挥作用的机制非常重要，并将为开发针对人类疾病（例如癌症）中 Rb 通路成分的更具体的治疗方法提供平台。