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

描述（由申请人提供）：哺乳动物早期胚胎中的多能细胞经历异常快速的细胞增殖和生长。这种快速扩增对胚胎发育至关重要，并被捕获在培养的多能干细胞中，这些干细胞可以生长到非常高的数量，用于疾病建模方法。这种快速的细胞增殖产生了对细胞组分合成的高需求，这可能与多能细胞已经报道的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输出升高的分子机制，以及它们如何与发育相关，在很大程度上仍然是未知的。重要的是要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，因为转化细胞通常会吸收多能细胞染色质调节的方面。我们研究的长期目标是了解哺乳动物多能细胞的容许染色质状态的调节。本申请的具体目的是剖析染色质重塑Chd 1在转录输出和多能细胞发育中的作用。该提案将测试的假设，Chd 1保持一个允许的染色质状态所必需的高转录输出和哺乳动物多能细胞的快速增殖。具体目的是：1）探讨Chd 1在小鼠上胚层发育中的基本功能。初步的小鼠遗传数据揭示了Chd 1在植入后立即在多能外胚层中的重要作用，这是一个特别快速生长的阶段。突变的上胚层不生长，建立A/P模式或原肠胚，并表达较低水平的核糖体RNA（rRNA）。Chd 1在外胚层中的发育作用将被详细描述，包括体内转录输出的分析; 2）确定Chd 1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Chd 1靶基因处，Chd 1-/- ES细胞中的活性组蛋白标记和延长RNA聚合酶II减少。ChIP-Seq和ChIP-qPCR将用于获得Chd 1-/- 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以及RNA Pol I和II; 3）剖析ES细胞中转录输出的调控。初步分析显示，在Chd 1-/- ES细胞中，扩增速率较慢，mRNA和rRNA的表达水平较低。将量化Chd 1-/- ES细胞的每个细胞的转录输出，包括新生转录的分析，并将使用IP-MS和功能获得性分析对Chd 1的作用进行机械解剖。这项研究有望为快速增殖的哺乳动物多能细胞的转录输出调控提供新的见解，对发育和干细胞生物学，再生医学和癌症研究产生重大影响。

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.