CpG islands and GC content dictate nucleosome depletion in a transcription independent manner at mammalian promoters (RNA-seq)

One clear hallmark of mammalian promoters is the presence of CpG islands (CGIs) at more than two thirds of genes whereas TATA boxes are only present at a minority of promoters. Using genome-wide approaches, we show that GC content and CGIs are major promoter elements in mammalian cells, able to govern open chromatin conformation and support paused transcription. First, we define three classes of promoters with distinct transcriptional directionality and pausing properties which correlate with their GC content. We further analyze the direct influence of GC content on nucleosome positioning and depletion, and show that CGIs correlate with nucleosome depletion both in vivo and in vitro. We also show that transcription is not essential for nucleosome exclusion but influences both a weak +1 and a well-positioned nucleosome at CGI borders. Altogether our data support the idea that CGIs have become an essential feature of promoter structure defining novel regulatory properties in mammals. Nucleosome density and positioning were studied by high-throughput sequencing of DNA previously treated with Mnase. In parallel, chIPseq for PolII and H3K27ac were performed in mouse and human with different conditions to assess a potential effect of transcription on nucleosomes properties. To investigate transcription at promoters, we analyzed together with genome-wide Pol II accumulation by ChIP-Seq, paused bidirectional transcripts associated with transcription start sites (TSS RNAs).哺乳动物启动子的一个显著特征是，超过三分之二的基因的启动子区域存在CpG岛（CpG Islands，CGIs），而TATA框（TATA box）仅在少数启动子中出现。本研究通过全基因组层面的实验手段证实，GC含量与CpG岛是哺乳动物细胞中的核心启动子元件，能够调控开放染色质构象并维持暂停转录状态。首先，我们根据转录方向性与暂停特性的差异，将启动子划分为三类，且这些特性与其GC含量紧密相关。随后，我们进一步分析了GC含量对核小体定位与缺失的直接影响，证实CpG岛无论在体内还是体外实验中，均与核小体缺失存在显著关联。此外，我们还发现转录并非核小体排除的必要条件，但会影响CpG岛边界处的弱结合+1核小体与定位清晰的核小体。综上，本研究数据支持以下观点：CpG岛已成为哺乳动物启动子结构的核心特征，赋予其全新的调控特性。本研究通过对经Mnase处理后的DNA进行高通量测序，分析了核小体密度与定位特征；与此同时，我们在小鼠与人类细胞中针对不同实验条件开展了Pol II与H3K27ac的ChIP-seq实验，以评估转录对核小体特性的潜在影响。为探究启动子区域的转录情况，我们结合全基因组范围的ChIP-seq检测到的Pol II富集情况，分析了与转录起始位点（Transcription Start Site，TSS）相关的暂停双向转录本（TSS RNAs）。