pRB and changes in the chromatin landscape during myogenic differentiation

pRB 和生肌分化过程中染色质景观的变化

• 批准号: 8413005
• 负责人: Brian D Dynlacht
• 金额: $ 35.63万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413005
• 关键词: Ablation; Affect; Binding; Bypass; Cell Cycle; Cell Cycle Progression; Cells; Chromatin; Complex; Coupled; Cues; DNA; DNA Methylation; Data; Data Set; Deposition; E2F transcription factors; Enabling Factors; Enzymes; Epigenetic Process; Event; Excision; Funding; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Goals; Growth; Histone Deacetylase; Histone H2B; Histone H3; Histones; Immunoprecipitation; Investigation; Lead; Link; Lysine; Maintenance; Malignant Neoplasms; Maps; Mediating; Methods; Methylation; Methyltransferase; Modeling; Molecular Profiling; Mono-S; Muscle; Muscle Fibers; Myoblasts; PRC1 Protein; Phase; Play; Polycomb; Process; Proteins; RBL2 gene; Recruitment Activity; Regulation; Regulator Genes; Retinoblastoma; Retinoblastoma Protein; Role; Signal Transduction; Staging; Structure; System; Testing; Time; Tissues; Tumor Suppressor Proteins; Withdrawal; Work; base; cdc Genes; chromatin immunoprecipitation; chromatin modification; gene repression; genome-wide; histone methyltransferase; histone modification; mutant; myogenesis; novel; p107 protein; research study; retinoblastoma tumor suppressor

DESCRIPTION (provided by applicant): The retinoblastoma tumor suppressor (pRB) and two related proteins (P107 and P130; collectively referred to as pocket proteins) playa fundamental role in regulating proliferation, cell cycle progression, and cell cycle exit. pRB has also been shown to play an essential role in differentiation of several tissues, including muscle. Nevertheless, our understanding of the regulatory controls involving the transition from a proliferative to a quiescent and, finally, terminally differentiated state is incomplete. Our work during the previous funding period has focused on the unique roles for each of these pocket proteins in responding to growth arrest cues and in both promoting and maintaining differentiation of muscle. In addition, we have identified transcriptional and epigenetic regulatory mechanisms associated with cell cycle exit and terminal differentiation. In particular, we have begun a systematic investigation of all chromatin modifications associated with myogenic differentiation using a combination of ChIP-sequencing (ChIP-seq) and expression profiling. Here, we propose the following aims to further dissect the mechanisms underlying pocket protein involvement in cell cycle exit and differentiation. In our first Aim, we will examine gene regulation during myogenic differentiation by specifically focusing on the role of histone H2B ubiquitylation (H2BUb) during differentiation. We will examine the extent of histone cross-talk in myotubes and investigate the impact of ablating RNF20, the enzyme responsible for H2B ubiquitylation, on differentiation. Remarkably, we have found that H2BUb essentially disappears during differentiation, and therefore we will examine the underlying mechanistic basis for myotube-specific loss of H2Bub. In our second Aim, we will examine the genome-wide role of pRB and co-repressors in directing chromatin modifications. We will first perform ChIP-seq on pRB and P130 in differentiated myotubes, enabling us to identify regions bound by these factors. We will also perform expression profiling after removal of pRB or P130 from myotubes. By merging these data, we will determine how chromatin modifications and gene expression are affected by loss of pocket proteins. Using our extensive ChIPseq data as a guide, we will test the hypothesis that pRB directs tri-methylation of H3K27 (H3K27IDe3) and ask whether Polycomb repressive complex (PRC2) is involved and to what extent. We will examine whether Ezh2 or an alternative histone methyltransferase (HMT), such as Ezhl, may be involved in H3K27me3 deposition. We will determine whether pRB influences the recruitment of either or both HMTs. The acquisition of our extensive ChIP-seq dataset allows us an unprecedented ability to examine on a genome-wide scale the relationship between pRB binding, chromatin modifications, and gene expression in a developmentally relevant setting, thereby enhancing our understanding of regulatory controls that are essential for both reversible and permanent gene silencing, withdrawal from the cell cycle, and muscle differentiation.

描述（由申请人提供）： 视网膜母细胞瘤肿瘤抑制因子 (pRB) 和两种相关蛋白（P107 和 P130；统称为口袋蛋白）在调节增殖、细胞周期进展和细胞周期退出中发挥重要作用。 pRB 还被证明在包括肌肉在内的多种组织的分化中发挥重要作用。然而，我们对涉及从增殖状态转变为静止状态以及最终的终末分化状态的监管控制的理解是不完整的。我们在上一个资助期间的工作重点是这些口袋蛋白中的每一种在响应生长停滞信号以及促进和维持肌肉分化方面的独特作用。此外，我们还确定了与细胞周期退出和终末分化相关的转录和表观遗传调控机制。特别是，我们已经开始使用 ChIP 测序 (ChIP-seq) 和表达谱相结合，对与肌原性分化相关的所有染色质修饰进行系统研究。在这里，我们提出以下目标，以进一步剖析口袋蛋白参与细胞周期退出和分化的机制。在我们的第一个目标中，我们将通过特别关注组蛋白 H2B 泛素化 (H2BUb) 在分化过程中的作用来检查生肌分化过程中的基因调控。我们将检查肌管中组蛋白串扰的程度，并研究消除 RNF20（负责 H2B 泛素化的酶）对分化的影响。值得注意的是，我们发现 H2BUb 在分化过程中基本上消失，因此我们将检查肌管特异性 H2Bub 丢失的潜在机制基础。在我们的第二个目标中，我们将研究 pRB 和共阻遏物在指导染色质修饰中的全基因组作用。我们将首先对分化肌管中的 pRB 和 P130 进行 ChIP-seq，使我们能够识别受这些因素约束的区域。从肌管中去除 pRB 或 P130 后，我们还将进行表达谱分析。通过合并这些数据，我们将确定口袋蛋白丢失如何影响染色质修饰和基因表达。使用我们广泛的 ChIPseq 数据作为指导，我们将测试 pRB 指导 H3K27 (H3K27IDe3) 三甲基化的假设，并询问 Polycomb 抑制复合物 (PRC2) 是否参与以及参与程度如何。我们将检查 Ezh2 或替代组蛋白甲基转移酶 (HMT)（例如 Ezhl）是否可能参与 H3K27me3 沉积。我们将确定 pRB 是否影响其中一个或两个 HMT 的招募。获取我们广泛的 ChIP-seq 数据集使我们能够以前所未有的能力在全基因组范围内检查发育相关环境中 pRB 结合、染色质修饰和基因表达之间的关系，从而增强我们对调控控制的理解，这些调控对于可逆和永久性基因沉默、退出细胞周期和肌肉分化至关重要。