Enhancer activation represents a critical step in eliciting the transcription of associated target genes. In addition to the key role of enhancers in normal development and cell-fate decisions, dysregulation of enhancers has been implicated in numerous diseases such as human malignancies and developmental defects. Since the identification of chromatin epigenetic mechanisms, the orchestration of histone modifications associated with active enhancers, in particular H3K27 acetylation (H3K27ac) and H3K4 monomethylation (H3K4me1), has been thought to govern enhancer activity in transcription. Despite proposals that active enhancers may generally arise from conversions of inactive enhancer states, such as primed (solely marked by H3K4me1) or poised (marked by H3K4me1 and H3K27me3) enhancers, emerging evidence indicates that a large number of active enhancers are established de novo during induction of gene activation. This strongly suggests that the deposition of H3K27ac and H3K4me1 at active enhancers could be coupled and involve the synergistic actions of multiple chromatin modifiers. Yet, the largely correlative studies to date have not revealed exactly how a naïve chromatin state on an enhancer is converted to an active state or the full repertoire of factors responsible for the establishment and maintenance of these unique epigenetic landscapes.

To address these fundamentally important questions, we conducted a combination of genetic models and genome-wide profiling (RNA-seq and ChIP-seq) in mouse ES cells (mESCs), cell-free (in vitro) chromatin-templated factor binding and transcription assays, and CRISPR-dCas9 mediated epigenetic editing in cells. Since MLL3 and MLL4 play redundant roles in the modulation of enhancer-associated H3K4me1 in mESCs, I focused primarily on MLL4. In this study, we uncovered a previously unrecognized reciprocal activation between MLL4 and p300 that drives a feed-forward regulatory loop to establish the “active enhancer landscape” in the mammalian epigenome. Notably, through a series of direct interactions with activators and cofactors, UTX was shown to play a key role in this process without using its demethylase activity. This work therefore defined a detailed mechanism for the role of UTX-MLL4-p300 regulatory network in the joint deposition of H3K4me1 and H3K27ac at active enhancers and provided mechanistic insights into the transition from naïve (unmarked) enhancers to an active state. Importantly, we demonstrated that the UTX H3K27 demethylase activity (previously thought to govern its function in transcriptional activation) appears to be dispensable for establishment/ maintenance of active enhancer function in ES cells. Instead, UTX appears to act primarily as a scaffold that by bridging MLL4 and p300 facilitates their recruitment and function during full activation of enhancer loci. We further showed that targeting of MLL4 (or UTX) with p300 to the endogenous enhancers (by CRISPR-dCas9-based approaches) induces long-range control of target gene transcription, raising the possibility that the interplay of MLL4/H3K4me1 and p300/H3K27ac could facilitate enhancer-promoter interaction. This study may open a new avenue for CRISPR-dCas9-mediated gene therapy through enhancer regulation.

⾃從染⾊質表觀遺傳機制之確⽴以來，與活性增強⼦ (active enhancer) 相關的組蛋⽩修飾-- 特別是H3K27⼄醯化（H3K27ac）和H3K4單甲基化（H3K4me1）--被認為與增強⼦活性以及基因轉錄激活有關; 然⽽對於細胞如何建⽴活性增強⼦景觀（即H3K27ac+ / H3K4me1+）的基本機制仍然不清楚。此外，儘 管MLL4和p300分別是哺乳動物增強⼦上H3K4me1和H3K27ac的關鍵組蛋⽩修飾酶，MLL4和p300之間是否具有協調增強⼦活性的潛在相互作⽤所知仍有限。為了解決這些表觀遺傳學的重要問題，本研究利⽤⼩⿏胚胎幹細胞（mESC）進⾏了遺傳模型和全基因組分析（RNA-seq 和 ChIP-seq)，並結合體外染⾊質模板基因轉錄 (in vitro chromatin-template transcription assay) 以及CRISPR-dCas9主導的細胞表觀遺傳編輯，確⽴了MLL4複合體和p300如何相互調節並建⽴活性增強⼦景觀的分⼦作⽤機制。我們發現MLL4單甲基轉移酶以及p300⼄醯轉移酶兩者的組蛋⽩修飾活性對於活性增強⼦景觀之建⽴都是不可或缺的要素，⽽且彼此之間存在⼀個正向循環調控機制。重要的是，MLL4複合體和p300被招募到增強⼦區域必須仰賴MLL4複合體內的UTX組成因⼦來完成; UTX可分別與轉錄激活因⼦、MLL4複合體、以及p300結合，並協助將MLL4以及p300帶往特定的增強⼦區域。有趣的是，以往UTX因具有H3K27去甲基酶活性，被認為可以拮抗Polycomb轉錄抑制複合體的功能⽽激活基因轉錄，但我們發現UTX在協同建⽴活性增強⼦景觀時，並不需要執⾏H3K27去甲基酶活性，⽽主要是充當⼀個⽀架，通過橋接MLL4和p300來促進它們在增強⼦上的募集與相關之組蛋⽩修飾。更重要的是，我們利⽤CRISPR-dCas9表觀遺傳編輯技術展⽰了將MLL4和p300誘導⾄細胞內⽣性增強⼦進⾏組蛋⽩修飾，可以激活遠端的標靶基因轉錄，以此奠定了CRISPR-dCas9表觀遺傳編輯技術可作為基因治療的理論基礎。