High throughput bisulfite sequencing of the MLH1 promoter in human hematiopoietic progenitor cell clones. Homo sapiens

Using single molecule ultra-deep bisulfite sequencing we characterized the CpG methylation landscape from –938 to –337 bp upstream of the MLH1 transcriptional start site position 0, from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we are able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as previously described for many tumor cell types, MLH1 promoter methylation in hematopoietic stem cell clones correlates with the loss of MLH1 expression, a critically important gene in the mismatch repair pathway. Overall design: Examination of CpG methylation landscape upstream of the MLH1 transcriptional start site in human hematiopoietic progenitor cell clones

本研究采用单分子超深度亚硫酸氢盐测序（single molecule ultra-deep bisulfite sequencing），针对30株分别表达或不表达MLH1的造血集落形成细胞克隆（hematopoietic colony forming cell clones, CFC），对MLH1转录起始位点（位置0）上游-938 bp至-337 bp区域的CpG甲基化图谱进行了表征。我们明确了MLH1启动子甲基化与MLH1表达缺失之间的相关性。此外，依托本研究获取的CpG位点甲基化频率数据，我们构建了可有效区分表达与不表达MLH1的CFC的分类算法。综上，正如此前针对多种肿瘤细胞类型的研究报道，造血干细胞克隆中的MLH1启动子甲基化与MLH1表达缺失显著相关——MLH1是错配修复通路中的关键功能基因。整体实验设计：检测人类造血祖细胞克隆中MLH1转录起始位点上游区域的CpG甲基化图谱。