The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.

对转录控制中新生 RNA 的机制了解仍然有限。在这里，通过高灵敏度甲基化刻录新生转录本测序 (MINT-seq) 方法，我们表征了新生 RNA 上 N6-甲基腺苷 (m6A) 的景观。我们发现转录调控元件（包括启动子上游反义RNA和增强子RNA（eRNA））产生的新生RNA上有大量但选择性的m6A沉积，这与它们的长度、m6A基序的包含和RNA丰度呈正相关。m6A-eRNA 标记高度活跃的增强子，它们招募核 m6A 读取器 YTHDC1 以依赖于其 C 末端固有无序区域和精氨酸残基的方式相分离成液体状冷凝物。m6A-eRNA/YTHDC1 缩合物与 BRD4 共激活剂缩合物共混合并促进其形成。因此，YTHDC1 的缺失减少了 BRD4 凝聚体及其对增强子的募集，导致增强子和基因激活受到抑制。我们认为 eRNA 的化学修饰与阅读器蛋白一起在增强子激活和基因转录控制中发挥广泛作用。