ChrRNA-seq, ATAC-seq and Chrm6A-seq of ESCs and differentiated cardiomyocytes with or without METTL3. ChrRNA-seq, ATAC-seq and Chrm6A-seq of ESCs and differentiated cardiomyocytes with or without METTL3

Transcriptional programming plays a key role in determining the cell state. Timely reconfiguration of chromatin structure and attenuation of pluripotent genes are required for efficient embryonic stem cell (ESC) differentiation. Here, we identify METTL3, a core N6-methyladenosine (m6A) catalyzing enzyme, as a crucial modulator of dynamic transcription and chromatin accessibility upon ESC-derived cardiac differentiation. Genome-wide analysis of chromatin-associated RNAs revealed that depletion of METTL3 failed to dramatically attenuate the transcription of pluripotent genes, as well as activate nascent cardiomyocyte-specific transcripts upon differentiation. Consistently, ATAC-seq analysis showed that loss of METTL3 markedly attenuated the dynamic alteration of chromatin accessibility at both promoters and gene bodies, resulting in reduced sensitivity of ESC chromatin structure to cardiac differentiation signal. Furthermore, using chromatin-associated m6A sequencing, we found that nuclear m6A underwent a dramatic increase upon differentiation, which correlates with the decrease of chromatin accessibility. Collectively, our findings reveal that METTL3 and nuclear m6A epitranscriptome couples with chromatin state to ensure transcriptional regulation of cell fate transition. Overall design: ChrRNA-seq, ATAC-seq and Chrm6A-seq of ESCs and differentiated cardiomyocytes with or without METTL3

转录编程在细胞状态决定过程中发挥关键作用。高效完成胚胎干细胞（ESC）分化，需要及时重编程染色质结构并下调多能性基因的表达。本研究鉴定出METTL3——一种核心的N6-甲基腺嘌呤（m6A）催化酶——是胚胎干细胞向心肌细胞分化过程中动态转录与染色质可及性的关键调控因子。全基因组水平的染色质相关RNA分析显示，敲除METTL3后，不仅无法显著下调多能性基因的转录，同时也未能激活分化过程中心肌细胞特异性的新生转录本。与之一致的是，ATAC-seq分析表明，METTL3缺失会显著减弱启动子区域与基因体区域染色质可及性的动态变化，最终导致胚胎干细胞染色质结构对心肌分化信号的响应敏感性降低。进一步通过染色质相关m6A测序，我们发现细胞核内的m6A在分化过程中显著升高，该变化与染色质可及性的降低显著相关。综上，本研究揭示METTL3与细胞核m6A表观转录组通过耦联染色质状态，保障细胞命运转变过程中的转录调控。实验整体设计：对存在或缺失METTL3的胚胎干细胞及其分化获得的心肌细胞，分别开展ChrRNA-seq、ATAC-seq与Chrm6A-seq测序。