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.
The proposed research aims to provide a mechanistic understanding of how the genome regulates its RNA output to support the very rapid proliferation of pluripotent cells in the mammalian embryo. Such fundamental knowledge is relevant to the mission of the NIH because it may contribute to harnessing the potential of pluripotent stem cells in Regenerative Medicine and may reveal potential weak points of rapidly proliferating cancer cells.
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