Variants of DNMT3A cause transcript-specific DNA methylation changes

The de novo DNA methyltransferase 3A (DNMT3A) plays a pivotal role in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing of DNMT3A has characteristic epigenetic and functional sequels. Various transcripts of DNMT3A were either knocked down or overexpressed in human hematopoietic stem and progenitor cells resulting in complementary and transcript-specific DNA methylation (DNAm) and gene expression changes. Our results demonstrate that different splice variants of DNMT3A have distinct epigenetic and functional sequels. Six biological replicas of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs; #1-6) were isolated for the knockdown (KD; #1-3) and the overexpression (OE; #4-6) of DNMT3A transcripts. HSPCs were lentivirally infected with either shRNA or overexpression vectors 1 day after isolation. Control HSPCs were infected with a scrambled or empty vector. Genomic DNA was isolated at day 12 after infection, bisulfite converted and hybridized to the Illumina Infinium 450k Human Methylation Beadchip.

从头DNA甲基转移酶3A（de novo DNA methyltransferase 3A，DNMT3A）在造血分化过程中发挥关键作用。本研究基于如下假说：DNMT3A的可变剪接具有特征性的表观遗传与功能效应。我们在人类造血干祖细胞中对DNMT3A的多种转录本分别进行敲低或过表达，由此引发了转录本特异性且具有互补性的DNA甲基化（DNA methylation，DNAm）与基因表达变化。本研究结果表明，DNMT3A的不同剪接变体具有截然不同的表观遗传与功能效应。我们分离得到6份脐带血来源的CD34+造血干祖细胞（hematopoietic stem and progenitor cells，HSPCs；编号1-6）生物学重复样本，分别用于DNMT3A转录本的敲低（knockdown，KD；编号1-3）与过表达（overexpression，OE；编号4-6）实验。细胞于分离后1天，分别通过慢病毒感染携带短发夹RNA（short hairpin RNA，shRNA）的载体或过表达载体。对照组造血干祖细胞则使用乱序对照载体或空载体进行感染。于感染后第12天提取基因组DNA，经亚硫酸氢盐转化处理后，与Illumina Infinium 450k Human Methylation Beadchip进行杂交。