pRB and changes in the chromatin landscape during myogenic differentiation

描述(由申请人提供)： 视网膜母细胞瘤肿瘤抑制因子(PRB)和两个相关蛋白(P107和P130，统称为Pocket蛋白)在调节细胞增殖、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几种组织的分化中起着至关重要的作用。然而，我们对监管控制的理解是不完整的，这些监管控制涉及从增殖状态向静止状态过渡，最后是终极分化状态。我们在前一个资助时期的工作集中在这些口袋蛋白在响应生长停滞信号以及在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末端分化相关的转录和表观遗传调控机制。特别是，我们已经开始使用芯片测序(CHIP-SEQ)和表达谱相结合的方法对与肌源性分化相关的所有染色质修饰进行系统研究。在这里，我们提出以下目的，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H_2B泛素化(H_2BUb)在分化过程中的作用来研究成肌细胞分化过程中的基因调控。我们将检查肌管中组蛋白的串扰程度，并研究去除RNF20对分化的影响。RNF20是负责H 2B泛素化的酶。值得注意的是，我们发现H_2BUb在分化过程中基本上消失了，因此我们将研究肌管特异性丢失H_2Bub的潜在机制。在我们的第二个目标中，我们将研究全基因组中pRb和共抑制物在指导染色质修饰中的作用。我们将首先在分化的肌管中对pRb和P130进行芯片序列分析，使我们能够确定这些因素结合的区域。我们还将在从肌管中移除pRb或P130后进行表达谱分析。通过合并这些数据，我们将确定口袋蛋白的丢失如何影响染色质修饰和基因表达。以我们广泛的ChIPseq数据为指导，我们将检验PRB指导H3K27(H3K27IDe3)三甲基化的假设，并询问是否涉及多梳抑制复合体(PRC2)以及在多大程度上参与。我们将研究Ezh2或另一种组蛋白甲基转移酶(HMT)，如Ezh1，是否可能参与H3K27me3的沉积。我们将确定PRB是否会影响其中一名或两名HMT的招聘。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与发育相关的环境中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的基因沉默、退出细胞周期和肌肉分化都是必不可少的。

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.