Disruption of cardiac Med1 inhibits RNA polymerase-II promoter occupancy and induces chromatin remodeling

The Mediator co-activator complex directs gene specific expression by binding distal enhancer-bound transcription factors through its Med1 subunit while bridging to RNA Polymerase-II (Pol-II) at gene promoters. In addition, Mediator scaffolds epigenetic modifying enzymes that determine local DNA accessibility. We previously found that deletion of Med1 in cardiomyocytes deregulates more than 5000 genes and promotes acute heart failure and hypothesize Med1 deficiency disrupts enhancer-promoter coupling. Using ChIP-seq, we find Pol-II pausing index is increased in Med1 knockout versus floxed control hearts primarily from decreased Pol-II occupancy at the majority of transcriptional start sites. Med1-dependent gene expression correlates strongly with histone H3 K27 acetylation while H3 K27 tri-methylated levels are increased and inversely correlate with absolute expression levels. Furthermore, Med1 deletion leads to dynamic changes in acetyl-K27 associated super-enhancer regions and their enriched transcription factor binding motifs that are consistent with altered gene expression. Our findings suggest that Med1 is important in establishing enhancer-promoter coupling in the heart by facilitating the recruitment of Pol-II to gene promoters, determining chromatin accessibility within genes and enhancer regions and altering transcription factor binding motifs that are likely important in directing gene-specific expression. Overall design: ChIP-sequencing data of RNA Pol-II, acetylated H3K27, and tri-metylated H3K27 from hearts of Med1 floxed and cardiac knockout mice.

Mediator共激活因子复合物通过其Med1亚基结合远端增强子结合的转录因子，同时与基因启动子处的RNA聚合酶II（RNA Polymerase-II，Pol-II）形成桥接，从而介导基因的特异性表达。此外，Mediator可作为表观遗传修饰酶的支架，调控局部DNA的可及性。本团队此前发现，心肌细胞中Med1的缺失会导致超过5000个基因的表达失调，并诱发急性心力衰竭，据此我们提出假说：Med1缺乏会破坏增强子-启动子耦合。通过染色质免疫沉淀测序（ChIP-seq）分析，我们发现与floxed对照小鼠心脏相比，Med1敲除小鼠心脏中的Pol-II暂停指数升高，这一现象主要源于绝大多数转录起始位点处的Pol-II占用率降低。Med1依赖性基因表达与组蛋白H3 K27乙酰化水平呈显著正相关；而H3 K27三甲基化水平升高，且与基因绝对表达水平呈负相关。此外，Med1缺失会导致乙酰化H3K27相关的超级增强子区域及其富集的转录因子结合基序发生动态变化，这与基因表达的改变相一致。我们的研究结果表明，Med1在心脏中增强子-启动子耦合的建立过程中发挥关键作用，具体机制包括促进Pol-II向基因启动子的招募、调控基因及增强子区域内的染色质可及性，以及改变可能对基因特异性表达至关重要的转录因子结合基序。整体实验设计：采集Med1 floxed对照与心肌特异性敲除小鼠心脏中RNA聚合酶II、乙酰化H3K27以及三甲基化H3K27的ChIP-seq数据。