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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

基本信息

批准号：
8665617
负责人：
Brian D Dynlacht
金额：
$ 9.37万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8665617
关键词：
Ablation Affect Binding Bypass Cell Cycle Chromatin Complex Coupled DNA DNA Methylation Data Data Set Deposition 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 Hurricane Immunoprecipitation Investigation Lead Link Lysine Maintenance Maps Mediating Methods Methylation Methyltransferase Molecular Profiling Mono-S Muscle Muscle Fibers Myoblasts PRC1 Protein Parents Phase Play Polycomb Process Proteins RBL2 gene Recruitment Activity Regulation Regulator Genes Retinoblastoma Retinoblastoma Protein Role Signal Transduction Staging Structure System Testing Time Tumor Suppressor Proteins Withdrawal Work base cdc Genes chromatin immunoprecipitation chromatin modification gene repression genome-wide histone methyltransferase histone modification mutant myogenesis novel research study

项目摘要

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 myotubespecific 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 (H3K27me3) 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 Ezh1, 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. Progress on both Aims was impacted by Hurricane Sandy. Funds are requested to continue the Aims listed above.

我们已经开始系统地研究与肌源性生长相关的所有染色质修饰，使用ChIP测序（ChIP-seq）和表达谱分析的组合来区分。在此，我们建议下面的目的是进一步剖析口袋蛋白参与细胞周期退出的机制。和差异化。在我们的第一个目标中，我们将通过以下方法研究肌原性分化过程中的基因调控：特别关注组蛋白H2 B泛素化（H2 BUb）在分化过程中的作用。我们将研究肌管中组蛋白串扰的程度，并研究消融RNF 20的影响，负责H2 B泛素化，分化。值得注意的是，我们发现H2 BUb基本上在分化过程中消失，因此我们将研究肌管特异性的潜在机制基础。失去H2 Bub。在我们的第二个目标中，我们将研究pRB和辅阻遏物在基因组范围内的作用，指导染色质修饰。我们将首先在分化的肌管中对pRB和p130进行ChIP-seq，使我们能够识别这些因素所限制的区域。我们还将在删除后执行表达式分析 pRB或p130的基因。通过合并这些数据，我们将确定染色质修饰和基因表达受到口袋蛋白的损失的影响。使用我们广泛的ChIPseq数据作为指导，我们将测试pRB指导H3 K27（H3 K27 me 3）三甲基化的假设，并询问Polycomb是否抑制复合物（PRC 2）参与，以及在多大程度上。我们将研究Ezh 2或替代品是否组蛋白甲基转移酶（HMT），如Ezh 1，可能参与H3 K27 me 3沉积。我们将确定 pRB是否影响任一或两种HMT的募集。收购我们广泛的ChIP-seq 数据集使我们能够在全基因组范围内研究pRB之间的关系，结合，染色质修饰和基因表达在发育相关的设置，从而提高我们对可逆和永久基因表达调控的理解，沉默、退出细胞周期和肌肉分化。这两个目标的进展受到以下因素的影响：飓风桑迪。需要资金来继续实现上述目标。