Genome-wide characterization of cytosine-specific 5-hydroxymethylation in normal breast tissue

Despite recent evidence that 5-hydroxymethylcytosine (5hmC) possesses roles in gene regulation distinct from 5-methylcytosine (5mC), relatively little is known regarding the functions of 5hmC in mammalian tissues. To address this issue, we utilized an approach combining both paired bisulfite (BS) and oxidative bisulfite (oxBS) DNA treatment, to resolve genome-wide patterns of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC was differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity, such as heterochromatin and repressed Polycomb regions, were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. Furthermore, genes containing abundant 5hmC were enriched among those actively transcribed in normal breast tissue. Finally, in independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Primarily, our findings identify genomic loci containing abundant 5hmC in breast tissues and provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue. Additionally, these data suggest 5hmC may participate in gene regulatory programs that are dysregulated during breast-related carcinogenesis.

尽管近期研究表明，5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)具有区别于5-甲基胞嘧啶(5-methylcytosine, 5mC)的基因调控功能，但目前对哺乳动物组织中5hmC的功能仍知之甚少。为解决这一问题，本研究采用亚硫酸氢盐(BS)与氧化亚硫酸氢盐(oxBS) DNA联合处理的策略，解析了无病女性正常乳腺组织中5hmC与5mC的全基因组分布模式。尽管5hmC的丰度低于5mC，但其分布具有差异性，且在乳腺特异性增强子与转录活性染色质中持续富集；与之相反，与转录沉默相关的调控区域（如异染色质及受抑制的多梳蛋白(Polycomb)结合区域）中，5hmC的丰度相对较低。携带高丰度5hmC的基因区域显著富集于乳酸氧化、免疫细胞功能及催乳素信号通路；此外，这类基因在正常乳腺组织的活跃转录基因集合中显著富集。最后，在独立数据集的分析中，正常乳腺组织的5hmC在浸润前乳腺癌及浸润性乳腺肿瘤中被证实存在甲基化改变的CpG位点中显著富集。本研究首要成果为鉴定了乳腺组织中高丰度5hmC的基因组位点，并构建了正常乳腺组织中核苷酸分辨率的全基因组5hmC图谱；此外，本研究数据提示5hmC可能参与了乳腺相关癌变过程中失调的基因调控程序。