Epigenetic landscape of 5-hydroxymethylcytosine and associations with gene expression in placenta

5-hydroxymethylcystosine (5hmC), is an intermediate product in the DNA demethylation pathway, but may act as a functional epigenetic modification. We have conducted the largest study of site-specific 5hmC in placenta to date using parallel bisulphite and oxidative bisulphite modification with array-based assessment. Incorporating parallel RNA-sequencing data allowed us to assess associations between 5hmC and gene expression, using expression quantitative trait hydroxymethylation (eQTHM) analysis. We identified ~ 47,000 loci with consistently elevated (systematic) 5hmC proportions. Systematic 5hmC was significantly depleted (<i>p</i> &lt; 0.0001) at CpG islands (CGI), and enriched (<i>p</i> &lt; 0.0001) in ‘open sea’ regions (CpG &gt;4 kb from CGI). 5hmC was most and least abundant at CpGs in enhancers and active transcription start sites (TSS), respectively (<i>p</i> &lt; 0.05). We identified 499 significant (empirical-p &lt;0.05) eQTHMs within 1 MB of the assayed gene. At most (75.4%) eQTHMs, the proportion of 5hmC was positively correlated with transcript abundance. eQTHMs were significantly enriched among enhancer CpGs and depleted among CpGs in active TSS (<i>p</i> &lt; 0.05 for both). Finally, we identified 107 differentially hydroxymethylated regions (DHMRs, <i>p</i> &lt; 0.05) across 100 genes. Our study provides insight into placental distribution of 5hmC, and sheds light on the functional capacity of this epigenetic modification in placenta.

5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC）是DNA去甲基化通路中的中间产物，同时也可作为一种功能性表观遗传修饰。本研究开展了迄今为止规模最大的胎盘位点特异性5hmC分析，采用平行亚硫酸氢盐与氧化亚硫酸氢盐修饰结合阵列检测的方法。结合平行RNA测序数据，我们通过表达数量性状羟甲基化（expression quantitative trait hydroxymethylation，eQTHM）分析，评估了5hmC与基因表达之间的关联。我们鉴定出约47000个5hmC比例持续升高的系统性位点。系统性5hmC在CpG岛（CpG islands，CGI）中显著缺失（*p* < 0.0001），而在“开放海域”区域（距CGI大于4kb的CpG位点）中显著富集（*p* < 0.0001）。5hmC在增强子区域的CpG位点中丰度最高，在活跃转录起始位点（transcription start sites，TSS）的CpG位点中丰度最低（*p* < 0.05）。我们在检测基因的1Mb范围内鉴定出499个具有统计学意义的eQTHMs（经验*p* < 0.05）。在大多数（75.4%）eQTHMs中，5hmC的比例与转录本丰度呈正相关。eQTHMs在增强子CpG位点中显著富集，而在活跃TSS的CpG位点中显著缺失（两者*p*均<0.05）。最后，我们在100个基因中鉴定出107个差异羟甲基化区域（differentially hydroxymethylated regions，DHMRs，*p* < 0.05）。本研究揭示了5hmC在胎盘中的分布特征，并阐明了该表观遗传修饰在胎盘中的功能意义。