靶向启动子DNA甲基化新方法的建立及其在研究DNA甲基化调控转录机制中的应用

DNA methylation is one of the best characterized epigenetic modifications that is generally associated with repression of gene transcription when occurred at CpG-island promoters. However the mechanisms by which promoter DNA methylation regulates gene transcription remain to be elucidated. One of the key issues is how to specifically target DNA methylation to gene promoter. We replaced the de novo methyltransferase DNMT3A’s ADD domain by the PHF2-PHD domain which specifically recognizes H3K4me3 modification which is enriched at promoter and referred this mutant as “DNMT3A-PHD”. Engineered DNMT3A acquires the binding ability towards H3K4me3. Furthermore DNMT3A-PHD can specifically bind to the H3K4me3 enriched promoters across the genome and establish DNA methylation at such regions. This tool helps us to study how DNA methylation regulates gene transcription and clarify the related molecular mechanisms. By induction of DNMT3A-PHD expression in HeLa cells, we find that extensive establishment of promoter DNA methylation inhibits global gene transcription, while a small subset of genes are activated. We have demonstrated that DNMT3A-PHD mediated global gene repression depends on its binding ability towards H3K4me3 and DNA methyltransferase activity. Previous studies indicate that DNA Methylation can directly recruit transcriptionally repressive methyl-CpG (mCpG)-binding proteins (MBPs), interact with histone modifications and interfere with the binding of transcriptional factors to DNA. In regard to these aspects, we will use this system to further study the molecular mechanism of promoter DNA methylation in gene transcriptional regulation.

DNA甲基化是一种重要的表观遗传修饰。研究表明基因启动子区域的DNA甲基化常与转录抑制相关，但启动子DNA甲基化调控基因转录的具体分子机制仍有待阐明，其中的一个关键问题是如何特异地将DNA甲基化靶向基因启动子区域。我们通过构建起始性DNA甲基转移酶DNMT3A与能特异性识别H3K4me3修饰的PHD结构域的融合蛋白，成功将DNMT3A特异性靶向具有H3K4me3修饰的基因启动子区域，并实现启动子区域的DNA甲基化。我们发现基因组启动子区域DNA甲基化的增加会导致大量基因转录水平下降，一小部分基因的表达升高。本项目将利用该体系，围绕启动子DNA甲基化招募甲基化CpG结合蛋白（MBPs）、影响组蛋白修饰（乙酰化、甲基化）、改变转录因子结合DNA等方面，深入研究启动子DNA甲基化调控基因转录的分子机制。

DNA甲基化是一种重要的表观遗传修饰，研究表明基因启动子区域的DNA甲基化常与转录抑制相关，但启动子DNA甲基化调控基因转录的具体分子机制仍有待阐明。其中的一个关键问题是如何特异性将DNA甲基化靶向基因启动子区域。我们通过构建起始性DNA甲基转移酶DNMT3A与能特异性识别H3K4me3修饰的PHD结构域的融合蛋白，成功将DNMT3A特异性靶向具有H3K4me3修饰的基因启动子区域，并实现启动子区域的DNA甲基化。我们发现基因组启动子区域DNA甲基化的增加导致大量基因转录水平下降，一小部分基因的表达升高。本项目利用该体系，围绕启动子DNA甲基化招募甲基化CpG结合蛋白（MBPs）、影响组蛋白修饰（乙酰化、甲基化）、改变转录因子结合DNA等方面，深入研究启动子DNA甲基化调控基因转录的分子机制。研究结果表明：1.组蛋白去乙酰化、H3K4me3水平下调及p53激活不是DNMT3A-PHD介导基因转录抑制的充分条件；2.MBPs家族蛋白不是启动子DNA甲基化抑制基因转录的必要条件；3.DNA甲基化可能直接抑制部分转录因子结合启动子DNA；4.DNA甲基化可能间接影响TFIID、PolII复合体结合启动子DNA；5. DNA甲基化促进启动子区域核小体占位是启动子DNA甲基化抑制基因转录的主要原因。.综上，在本项目的支持下，我们建立了DNMT3A-PHD靶向启动子DNA甲基化这一实验体系，该系统对于深入研究如何特异性甲基化基因组启动子区域的DNA、启动子区域DNA甲基化如何调控基因转录以及相关分子机制具有重要的学术价值。此外，通过分子机制的探究，我们发现启动子DNA甲基化通过影响核小体占位调控基因转录，这对于理解表观遗传调控与基因转录之间的关系十分重要。