pRB and changes in the chromatin landscape during myogenic differentiation

我们已经开始对所有与肌源性有关的染色质修饰进行系统研究。 结合芯片测序(CHIP-SEQ)和表达谱分析进行差异分析。在这里，我们建议 以下内容旨在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，我们将通过以下方式来研究肌肉分化过程中的基因调控 特别是组蛋白H_2B泛素化(H_2BUb)在分化中的作用。我们将研究 组蛋白在肌管中的串扰程度以及消融RNF20酶的影响 负责H_2B泛素化，对分化负责。值得注意的是，我们发现H2BUb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 失去了H_2Bub。在我们的第二个目标中，我们将研究pRb和共抑制因子在全基因组中的作用 指导染色质修饰。我们将首先对pRb和在分化肌管中的p130执行芯片序列， 使我们能够确定受这些因素约束的地区。我们还将在删除后执行表情分析 PRb或p130的表达。通过合并这些数据，我们将确定染色质修饰和 基因表达受口袋蛋白丢失的影响。使用我们广泛的ChIPseq数据作为指导，我们将 检验pRb指导H3K27(H3K27me3)三甲基化的假设，并询问Polycomb 抑制性复合体(PRC2)被牵涉到什么程度。我们将研究Ezh2或替代方案 组蛋白甲基转移酶(HMT)，如Ezh1，可能参与H3K27me3沉积。我们将决定 PRB是否影响其中一名或两名HMT的招聘。收购我们广泛的芯片-SEQ 数据集使我们能够在全基因组范围内检查pRb和pRb之间的关系 在发育相关环境中的结合、染色质修饰和基因表达，从而 加强我们对可逆和永久基因所必需的调控的理解 沉默、退出细胞周期和肌肉分化。这两个目标的进展都受到以下因素的影响 飓风桑迪。请拨出资金以继续实现上述目标。

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.