Transcriptional landscape of the human cell cycle

Steady-state gene expression across the cell cycle has been studied extensively. However, 2 transcriptional gene regulation and histone modification dynamics at different cell cycle stages is3 largely unknown. By applying a combination of GRO-seq, RNA-seq and histone modification 4 ChIP-seq, we depicted a comprehensive transcriptional landscape at G0/G1, G1/S and M phases 5 of breast cancer MCF-7 cells. Importantly, GRO-seq and RNA-seq analysis identified different 6 cell cycle regulated genes, suggesting a lag between transcription and steady-state expression 7 during the cell cycle. Interestingly, we identified genes actively transcribed at early M phase that 8 are longer in length and have low expression, which is accompanied by global increase of active 9 histone modifications of H3K4me2 and H3K27ac. In addition, we identified 2,440 cell cycle 10regulated enhancer RNAs (eRNAs) that are strongly associated with differential active 11transcription but not stable expression levels across the cell cycle. Motif analysis on dynamic 12eRNAs predicted KLF4 as a key regulator of G1/S transition, which was experimentally 13validated. Altogether, our combined analysis characterized the transcriptional and histone 14modification profile of the human cell cycle and identified novel dynamic transcriptional 15signatures across the cell cycle. MCF7 cells are synchronized to three different cell cycle stages: G0/G1, S and M phase. Global nuclear run-on followed by RNA sequencing (GRO-seq), ChIP-seq for H3K27ac and H3K4me2 modification were carried out in the synchronized cells. Dnase-seq were performed in asynchronized MCF7 cells.

细胞周期中的稳态基因表达已得到广泛研究。然而，不同细胞周期阶段的转录调控与组蛋白修饰动态变化仍未得到充分阐明。本研究联合应用全局核run-on测序（Global Nuclear Run-On Sequencing, GRO-seq）、RNA测序（RNA-seq）以及组蛋白修饰染色质免疫共沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq），全面刻画了乳腺癌MCF-7细胞在G0/G1、G1/S及M期的转录调控图谱。值得注意的是，GRO-seq与RNA-seq分析鉴定出了不同的细胞周期调控基因，这提示细胞周期中转录与稳态表达之间存在滞后效应。有趣的是，我们鉴定出在早M期活跃转录的基因，这类基因长度较长且表达量较低，同时伴随H3K4me2与H3K27ac这两种活性组蛋白修饰的全局水平升高。此外，我们鉴定出2440个细胞周期调控的增强子RNA（enhancer RNAs, eRNAs），它们与细胞周期中活跃转录的差异变化显著相关，但与稳态表达水平无关。对动态eRNAs进行基序分析后，预测KLF4是G1/S期转换的关键调控因子，这一结果已通过实验得到验证。综上，本研究的联合分析阐明了人类细胞周期的转录与组蛋白修饰图谱，并鉴定出细胞周期中全新的动态转录特征。本研究将乳腺癌MCF-7细胞同步化至三种不同的细胞周期阶段：G0/G1、S及M期。对同步化后的细胞开展了GRO-seq以及针对H3K27ac、H3K4me2修饰的ChIP-seq实验。此外，在未同步化的MCF-7细胞中开展了DNase I超敏感位点测序（DNase-seq）。