METTL14 is a chromatin regulator independent of RNA N6-methyladenosine (CUT & RUN)

METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC, also known as m6A writer) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on mouse embryonic stem cell (mESC) self-renewal. While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-indenepent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification and PRC2 complex. Mechanically, METTL14, but not METTL3, recognizes H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression . The regulation of H3K27me3 by METTL14 is essential to the transition of mESCs from self-renewal to differentiation. This work reveals a regulation mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance and differentiation of mESCs. Overall design: Examination of METTL3 and METTL14 genomic bindings in mESCs

METTL3与METTL14是构成核心异二聚体的两个组分，该异二聚体属于主要的RNA m⁶A甲基转移酶复合物（RNA m6A methyltransferase complex, MTC，亦称为m⁶A写入器），负责催化m⁶A修饰的安装。令人意外的是，METTL3或METTL14敲低对小鼠胚胎干细胞（mouse embryonic stem cell, mESC）的自我更新展现出截然不同的调控效应。尽管分别在Mettl3敲除与Mettl14敲除的mESC中均观察到程度相当的全局RNA m⁶A低甲基化水平，但Mettl14敲除会导致全局新生RNA合成量显著下降，而METTL3敲低则引发转录上调，这提示METTL14可能具备不依赖m⁶A的基因调控功能。
我们发现METTL14与抑制性组蛋白修饰——组蛋白H3第27位赖氨酸三甲基化（H3K27me3）及多梳抑制复合体2（PRC2）存在共定位现象。从分子机制层面来看，METTL14（而非METTL3）能够识别H3K27me3，并招募赖氨酸去甲基化酶6B（KDM6B）以诱导不依赖METTL3的H3K27me3去甲基化。因此，METTL14敲低会导致全局H3K27me3水平升高，同时伴随全局基因表达抑制。
METTL14对H3K27me3的调控对于小鼠胚胎干细胞从自我更新状态向分化状态的转变至关重要。本研究揭示了METTL14通过不同于METTL3且不依赖m⁶A的方式调控异染色质的机制，该机制对小鼠胚胎干细胞的转录调控、干性维持及分化过程均具有关键影响。
实验整体设计：在小鼠胚胎干细胞中检测METTL3与METTL14的基因组结合特征。