KLF4与组蛋白修饰酶协同调控血管平滑肌细胞特异基因表达的分子机制

Cell-type-specific gene expression in eukaryotes depends on the interaction of transcription factors with enhancers/promoters, and methylation and acetylation of histones are also required for cell-type-specific gene expression. We have demonstrated that transcription factor KLF4 can bind to TCE element on the enhancer/promoter of VSMC-specific genes, simultaneously, KLF4 recruits histone acetyltransferase p300 to these enhancers/promoters to acetylate histone H3. However, the following issues remain unknown: (i) When KLF4 recognizes the enhancer/promoter of VSMC-specific genes, whether it can also recruit histone methyltransferases; (ii) What are the histone modification patterns that underlie VSMC-specific gene activation; (iii) Whether there is a co-dependency between the methylation and acetylation of histones. In order to explore these problems, in present study, we shall investigate whether the methylation of histones on active enhancers/promoters of VSMC-specific genes depends on KLF4, determine subunits of MLL/Set1 complexes that interact with KLF4 and methylate histone H3, define the histone modification patterns that underlie VSMC-specific gene activation, as well as explore the relationship between the methylation and acetylation of histones, elucidate the alteration in the interaction of KLF4 with subunits of MLL/Set1 complexes in vascular remodeling. Our study will uncover a novel mechanism whereby KLF4 modulates vascular homeostasis and remodeling.

细胞类型特异基因的表达依赖于转录因子与增强子/启动子的相互作用以及增强子/启动子区的组蛋白甲基化和乙酰化。我们证实，KLF4在与血管平滑肌细胞（VSMC）特异基因增强子/启动子结合的同时，将组蛋白乙酰转移酶p300募集到该类基因的增强子/启动子上对组蛋白H3进行乙酰化。但目前尚不清楚①KLF4在识别VSMC特异基因增强子/启动子的同时，是否也能募集组蛋白甲基转移酶；②VSMC特异基因激活所依赖的组蛋白修饰谱型；③组蛋白甲基化与乙酰化之间是否存在依存关系。为弄清这些问题，本项目研究VSMC特异基因增强子/启动子区的组蛋白甲基化是否依赖于KLF4，确定KLF4通过与甲基转移酶MLL/Set1中的哪个亚基结合而对其发挥募集作用；明确VSMC特异基因激活所依赖的组蛋白修饰谱型及两种修饰的依存关系；阐明KLF4与MLL/Set1相互作用的改变与血管重构的关系，提出血管稳态与重构的新机制。

血管平滑肌细胞（VSMC）通过表达SM α-actin和SM22α等VSMC特异基因使细胞处于分化表型；当血管受损时，VSMC通过下调其特异基因表达而发生表型转化，VSMC表型转化是血管重构性疾病的先决条件。研究表明，VSMC特异基因表达除取决于KLF4和myocardin等转录因子对VSMC特异基因的激活外，组蛋白修饰也起着必不可少的作用。然而，目前尚不清楚①KLF4 如何协同调节VSMC特异基因表达和组蛋白修饰；②伴随α-actin表达所产生的circACTA2在VSMC表型转化中的作用；以及③受KLF4调控的其他蛋白质是否参与VSMC表型转化过程。本项目对这些尚待解决的科学问题进行研究。我们发现，KLF4分子中的C末端DNA结合结构域通过与p300或 MLL/Set1复合物中的催化亚基结合，介导MLL/Set1和p300对α-actin启动子区的H3K4和H3K27进行甲基化和乙酰化修饰，H3K4甲基化和H3K27乙酰化修饰是TGF-β诱导VSMC分化所必需的。TGF-β介导的 NRG-1-ICD 在促进α-actin基因转录过程中可形成circACTA2。circACTA2通过吸附miR-548f-5p，解除其对α-actin基因表达的阻抑，促进α-actin表达和VSMC分化。发表该论文的同期杂志刊登了题为《平滑肌分化调控形成完整的环路》的述评文章，指出该研究在平滑肌分化调控机制方面所做出的原创性发现，对该成果的临床意义进行了评论。我们还发现，除VSMC特异基因外，NMHC IIA表达也受KLF4的调节。KLF4负调控NMHC IIA表达，后者通过调节细胞骨架组构参与VSMC分化表型的维持。此外，我们的研究还发现，circACTA2 通过与 CDK4 mRNA 竞争性结合 ILF3使CDK4 mRNA与 ILF3 结合减少，CDK4 mRNA因稳定性降低而使其蛋白水平下调，进而导致细胞周期阻滞和细胞衰老。本项目还对在心血管功能调节中发挥重要作用的KLF5和KLF3进行了研究，部分揭示了KLF5和KLF3在VSMC自噬和炎症反应中的作用及其机制。本研究为防治血管重构性疾病提供了新靶点，对研发抗血管重构新型药物具有重要指导意义。