Loss of MLL3/4 decouples enhancer H3K4 monomethylation, H3K27 acetylation, and gene activation [CUT&RUN]

Enhancers are essential in defining cell fates through the control of cell type specific gene expression. Enhancer activation is a multi-step process involving chromatin remodelers and histone modifiers. One of these steps is monomethylation of H3K4 (H3K4me1) by MLL3 (KMT2C) and MLL4 (KMT2D). MLL3/4 are thought to be critical for enhancer activation and cognate gene expression including through the recruitment of acetyltransferases for H3K27. Here we test this model by evaluating the impact of MLL3/4 loss on chromatin and transcription during early embryonic stem cell differentiation. We find that MLL3/4 activity is required at most if not all sites that gain or lose H3K4me1, but is largely dispensable at sites that remain stably methylated during this transition. This requirement extends to H3K27 acetylation (H3K27ac) at most transitional sites. Unexpectedly, many sites gain H3K27ac independent of MLL3/4 or H3K4me1 including enhancers regulating key factors in early differentiation. Furthermore, despite the failure to gain active histone marks at thousands of enhancers, transcriptional activation of nearby genes is largely unaffected, thus uncoupling the regulation of chromatin state from transcriptional changes during this transition. These data challenge current models of enhancer activation and imply distinct mechanisms between stable and dynamically changing enhancers. Moreover, the data suggest limited roles for many enhancers in transcriptional regulation in early ESC differentiation. Collectively, our study highlights gaps in knowledge about the steps and epistatic relationships of enzymes necessary for enhancer activation and cognate gene transcription. Overall design: CUT&RUN of four different cell lines in two different cuture conditions with two biological replicates cultured, collected, and prepared independently for H3K4m1 and a negative IgG control. Includes CUT&RUN for MLL4 for WT and DKO formative cells with two biological replicates cultured, collected, and prepared independently.

增强子（enhancer）通过调控细胞类型特异性基因表达，在细胞命运决定过程中发挥不可或缺的作用。增强子激活是一个多步骤过程，涉及染色质重塑因子与组蛋白修饰酶。该过程中的一个关键步骤是由MLL3（KMT2C）与MLL4（KMT2D）催化的H3K4单甲基化（H3K4me1）。既往研究认为，MLL3/4对于增强子激活及靶基因表达至关重要，其机制包括招募针对H3K27的乙酰转移酶。
本研究通过探究MLL3/4缺失在早期胚胎干细胞（embryonic stem cell, ESC）分化过程中对染色质与转录的影响，对该模型进行了验证。我们发现，绝大多数（乃至全部）发生H3K4me1水平改变的位点均依赖MLL3/4的活性，而在该分化过渡阶段保持稳定甲基化的位点则基本不需要MLL3/4的参与。这一依赖关系在绝大多数过渡位点的H3K27乙酰化（H3K27ac）中同样适用。
出乎意料的是，众多位点可独立于MLL3/4或H3K4me1获得H3K27ac修饰，其中包括调控早期分化关键因子的增强子。此外，尽管数千个增强子未能获得活性组蛋白修饰，其邻近基因的转录激活却基本不受影响，这打破了该过渡阶段中染色质状态调控与转录变化之间的固有关联。上述数据对当前的增强子激活模型提出了挑战，并提示稳定型与动态型增强子可能存在截然不同的调控机制。
此外，本研究数据表明，在早期ESC分化过程中，多数增强子在转录调控中仅发挥有限作用。综上，本研究揭示了当前学界在增强子激活及靶基因转录所需酶的调控步骤与上位性关系方面的认知空白。
实验设计概要：针对4种不同细胞系，设置2种不同培养条件，分别在每种条件下开展2次独立培养、收集与样本制备的生物学重复，采用CUT&RUN技术检测H3K4me1并设置IgG阴性对照。本研究同时针对野生型（wild type, WT）与双敲除（double knockout, DKO） formative细胞开展MLL4的CUT&RUN测序，同样设置2次独立培养、收集与样本制备的生物学重复。