Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability (RNA-Seq). Mus musculus

Histone modifications and DNA methylation represent two distinct modes of varying epigenetic landscapes, but whose exact interrelationship remains unclear. Previous studies have shown that histone H3 lysine 4 trimethylation (H3K4me3) inhibits the binding of de novo DNA methyltransferases (Dnmt) through the ATRX-DNMT3-DNMTL (ADD) domain, thus protecting H3K4me3 marked CpG islands (CGI) from DNA methylation. In addition to H3K4me3, we identified antagonistic relationship between H3T3 phosphorylation and the binding of the ADD domain to the unmodified H3 N-terminus. To assess the physiological relevance of these restrictions, we engineered the wild-type ADD domain of Dnmt3a (WT) to permit additional binding to either H3K4me3 (WWD) or H3T3ph (R) and stably introduced FLAG-tagged, full-length normal or mutant Dnmt3a2 into ESCs lacking all Dnmts (TKO; triple knock-out of Dnmt1, Dnmt3a, and Dnmt3b) using the PiggyBac transposon system. For each WT-, WWD-, and R-Dnmt3a2, we generated bulk and clonally-derived ESC lines. We then employed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) to identify the genomic distribution of full-length WT-, WWD-, R-Dnmt3a2, and the H3K4me3 distribution. In parallel, we quantitatively measured genome-wide CpG (cytosine) methylation at base-pair resolution using an enhanced form of reduced representation bisulfite sequencing (RRBS), and performed RNA-seq to assess transcription in matched ESC lines. Overall design: Examination of mRNA profiles in Dnmt TKO-ESCs expressing wild-type/mutant Dnmt3a2.

组蛋白修饰与DNA甲基化是表观遗传调控体系中两种截然不同的模式，但二者的确切相互关系仍未明确。先前研究表明，组蛋白H3赖氨酸4三甲基化（histone H3 lysine 4 trimethylation, H3K4me3）可通过ATRX-DNMT3-DNMTL（ADD）结构域抑制从头DNA甲基转移酶（de novo DNA methyltransferases, Dnmt）的结合，从而使带有H3K4me3标记的CpG岛（CpG islands, CGI）免受DNA甲基化修饰。除H3K4me3外，本研究还鉴定出组蛋白H3苏氨酸3磷酸化（H3T3 phosphorylation, H3T3ph）与ADD结构域结合未修饰H3 N末端之间存在拮抗关系。为评估上述调控限制的生理相关性，我们对Dnmt3a的野生型ADD结构域（WT）进行工程改造，使其能够额外结合H3K4me3（WWD）或H3T3ph（R）；随后利用PiggyBac转座子系统，将带有FLAG标签的全长野生型或突变型Dnmt3a2稳定导入至完全缺失所有Dnmt的胚胎干细胞（embryonic stem cells, ESCs）中，该细胞系为Dnmt1、Dnmt3a与Dnmt3b三基因敲除细胞系（triple knock-out, TKO）。针对WT-Dnmt3a2、WWD-Dnmt3a2与R-Dnmt3a2三种亚型，我们分别构建了混合群体与单克隆来源的ESC细胞系。随后我们采用染色质免疫共沉淀测序（chromatin immunoprecipitation followed by high-throughput DNA sequencing, ChIP-seq）技术，检测全长WT-Dnmt3a2、WWD-Dnmt3a2及R-Dnmt3a2的基因组分布情况，并分析H3K4me3的分布特征。同时，我们采用优化版简化亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）技术，在碱基分辨率水平上定量检测全基因组范围内的CpG（胞嘧啶，cytosine）甲基化水平；并通过RNA测序（RNA-seq）分析匹配ESC细胞系的转录水平。实验整体设计：对表达野生型/突变型Dnmt3a2的Dnmt三基因敲除ESC细胞系的mRNA表达谱进行检测分析。