Role of Chd1 in the transcriptional output and development of pluripotent cells

描述(申请人提供)：早期哺乳动物胚胎中的多能细胞经历了异常快速的细胞增殖和生长。这种快速扩张对胚胎发育至关重要，并被培养的多能干细胞捕获，这些干细胞可以培养到非常高的数量，用于疾病建模方法。如此快速的细胞增殖产生了对细胞成分合成的高需求，这可能与已报道的多能细胞所允许的染色质景观和高转录输出相结合。然而，这种允许的染色质状态和转录产量增加背后的分子机制，以及它们如何与发育相关，在很大程度上仍不清楚。重要的是填补这一基本的生物学空白，以确保基于多能干细胞的治疗的有效性和安全性。填补这一空白也可能揭示癌症治疗的新方法，因为转化的细胞经常参与多能细胞染色质调节的各个方面。我们研究的长期目标是了解哺乳动物多能细胞染色质状态的调节。本应用的具体目的是分析染色质重构体Chd1在多能细胞转录输出和发育中的作用。这一提议将检验Chd1保持一种允许的染色质状态的假设，这对哺乳动物多能细胞的高转录输出和快速增殖是必不可少的。其具体目的是：1)剖析Chd1在小鼠上皮细胞发育过程中的重要作用。初步的小鼠遗传学数据显示，Chd1在着床后立即的多能性上皮细胞中具有重要的作用，这是一个特殊的快速生长阶段。突变的上胚层不生长，不形成A/P模式或原肠，并且表达较低水平的核糖体RNA(RRNA)。Chd1在上胚层中的发育作用将被详细描述，包括对体内转录输出的分析；2)确定Chd1在ES细胞染色质景观中的作用。初步的ChIP-Seq和ChIP-qPCR数据显示，在与细胞生长相关的高表达Chd1靶基因处，Chd1-/-ES细胞中活性的组蛋白标记和延长的RNA聚合酶II减少。ChIP-Seq和ChIP-qPCR将用于获得Chd1-/-ES细胞染色质状态的全面图像，包括核小体定位、与转录相关的组蛋白标记和RNA POL I和RNA POL II；3)剖析ES细胞转录输出的调节。初步分析显示，在Chd1-/-ES细胞中，扩增速度较慢，mRNAs和rRNA的表达水平较低。Chd1-/-ES细胞的每个细胞的转录输出将被量化，包括对新生转录的分析，并将使用IP-MS和功能增益分析对Chd1的作用进行机制剖析。这项研究有望为快速增殖的哺乳动物多能细胞转录输出的调控提供新的见解，对发育和干细胞生物学、再生医学和癌症研究具有重大影响。

DESCRIPTION (provided by applicant): Pluripotent cells in the early mammalian embryo undergo unusually rapid cell proliferation and growth. This rapid expansion is essential for embryonic development and is captured in cultured pluripotent stem cells, which can be grown to very high numbers for disease modeling approaches. Such rapid cell proliferation generates a high demand for synthesis of cellular components, which may be coupled with the permissive chromatin landscape and high transcriptional output that has been reported for pluripotent cells. However, the molecular mechanisms that underlie this permissive chromatin state and elevated transcriptional output, and how they relate to development, remain largely unknown. It is important to fill this basic biology gap in order to ensure the efficacy and safety of pluripotent stem cell-based therapies. Filling this gap may also reveal novel approaches to cancer therapies, because transformed cells often co-opt aspects of chromatin regulation of pluripotent cells. The long-term goal of our research is to understand the regulation of the permissive chromatin state of mammalian pluripotent cells. The specific objective of this application is to dissect the role of the chromatin remodeler Chd1 in the transcriptional output and development of pluripotent cells. This proposal will test the hypothesis that Chd1 maintains a permissive chromatin state essential for the high transcriptional output and rapid proliferation of mammalian pluripotent cells. The specific aims are: 1) To dissect the essential function of Chd1 in development of the mouse epiblast. Preliminary mouse genetic data reveal an essential role for Chd1 specifically in the pluripotent epiblast immediately after implantation, a stage of particulary rapid growth. The mutant epiblast does not grow, establish A/P patterning or gastrulate, and expresses lower levels of ribosomal RNA (rRNA). The developmental role of Chd1 in the epiblast will be characterized in detail, including analyses of transcriptional output in vivo; 2) o determine the role of Chd1 in the chromatin landscape of ES cells. Preliminary ChIP-Seq and ChIP-qPCR data reveal reductions in an active histone mark and elongating RNA Polymerase II in Chd1-/- ES cells at highly expressed Chd1 target genes associated with cellular growth. ChIP-Seq and ChIP-qPCR will be used to obtain a comprehensive picture of the chromatin state of Chd1-/- ES cells, including nucleosome positioning, histone marks associated with transcription and RNA Pol I and II; 3) To dissect the regulation of transcriptional output in ES cells. Preliminary analyses reveal a slower expansion rate and lower levels of expression of mRNAs and rRNA in Chd1-/- ES cells. The transcriptional output per cell of Chd1-/- ES cells will be quantified, including analyses of nascent transcription, and a mechanistic dissection of the role of Chd1 will be carried out using IP-MS and gain-of-function analyses. This research is expected to provide novel insights into the regulation of the transcriptional output of rapidly proliferating mammalian pluripotent cells, with significant impact in Developmental and Stem Cell Biology, Regenerative Medicine and Cancer Research.