Spatial control of m6A deposition on enhancer and promoter RNAs through co-acetylation of METTL3 and H3K27 on chromatin [RNA-seq]

Interaction between the m6A methyltransferase METTL3 and METTL14 is critical for METTL3 to deposit m6A on various types of RNAs. It is still intriguing whether there is spatial control of m6A deposition on different types of RNAs. Here, through genome-wide CRISPR/Cas9 screening, we find H3K27ac acetylase p300-mediated METTL3 acetylation suppresses the binding of METTL3 on H3K27ac-marked chromatin by inhibiting its interaction with METTL14. Consistently, p300 catalyzing the acetylation of METTL3 specifically occurs on H3K27ac marked chromatin. Disruptive mutations on METTL3 acetylation sites selectively promote the m6A of chromatin-associated RNAs from p300-bound enhancers and promoters marked by H3K27ac, resulting in transcription inhibition of ferroptosis-inhibition related genes. In addition, PAK2 promotes METTL3 acetylation by phosphorylating METTL3. Inhibition of PAK2 promotes the ferroptosis in a manner that depends on the acetylation of METTL3. Our study reveals a spatial-selective way to specifically regulate the deposition of m6A on enhancer and promoter RNAs. Overall design: The mRNA expression profile of METTL3-WT or METTL3-3KR A549 cells was generated by deep sequencing, in diplicate, using MGISEQ-2000RS platform on PE150 mode.

m6A甲基转移酶（m6A methyltransferase）METTL3与METTL14的相互作用，是METTL3在各类RNA上沉积m6A修饰的关键前提。目前学界仍有待阐明的科学问题是：不同类型RNA上的m6A沉积是否存在空间调控机制。本研究通过全基因组CRISPR/Cas9筛选发现，H3K27ac乙酰转移酶（H3K27ac acetylase）p300介导的METTL3乙酰化，可通过抑制METTL3与METTL14的相互作用，削弱METTL3结合H3K27ac标记染色质的能力。一致性实验结果显示，p300催化的METTL3乙酰化特异性发生在H3K27ac标记的染色质区域。METTL3乙酰化位点的破坏性突变，可选择性地促进p300结合的、带有H3K27ac标记的增强子与启动子区域的染色质相关RNA的m6A修饰，最终抑制铁死亡抑制相关基因的转录。此外，PAK2可通过磷酸化METTL3促进其乙酰化；抑制PAK2可通过依赖于METTL3乙酰化的途径促进铁死亡。本研究揭示了一种空间选择性调控m6A在增强子与启动子RNA上沉积的分子机制。总体实验设计：采用MGISEQ-2000RS平台，以PE150模式进行双重复深度测序，获取METTL3野生型（METTL3-WT）或METTL3-3KR突变型A549细胞的mRNA表达谱。