Permissive Transcriptional Activity at the Centromere through Pockets of DNA Hypomethylation

DNA methylation is a hallmark of transcriptional silencing, yet transcription has been reported at the centromere. To address this apparent paradox, we employed a fully sequence-defined ectopic human centromere (or neocentromere) to investigate the relationship between DNA methylation and transcription. We used sodium bisulfite PCR and sequencing to determine the methylation status of 2,041 CpG dinucleotides distributed across a 6.76-Mbp chromosomal region containing a neocentromere. These CpG dinucleotides were associated with conventional and nonconventional CpG islands. We found an overall hypermethylation of the neocentric DNA at nonconventional CpG islands that we designated as CpG islets and CpG orphans. The observed hypermethylation was consistent with the presence of a presumed transcriptionally silent chromatin state at the neocentromere. Within this neocentric chromatin, specific sites of active transcription and the centromeric chromatin boundary are defined by DNA hypomethylation. Our data demonstrate, for the first time to our knowledge, a correlation between DNA methylation and centromere formation in mammals, and that transcription and ��chromatin-boundary activity�� are permissible at the centromere through the selective hypomethylation of pockets of sequences without compromising the overall silent chromatin state and function of the centromere.

DNA甲基化（DNA methylation）是转录沉默的标志性特征，但已有研究报道着丝粒区域存在转录现象。为解析这一表观矛盾，我们采用序列完全确定的异位人类着丝粒（neocentromere，又称新着丝粒），探究DNA甲基化与转录之间的关联。我们通过亚硫酸氢盐PCR测序（sodium bisulfite PCR and sequencing）技术，对包含新着丝粒的6.76 Mb染色体区域内分布的2041个CpG二核苷酸（CpG dinucleotides）的甲基化状态进行检测。这些CpG二核苷酸分别与常规CpG岛（CpG islands）及非常规CpG序列相关联。我们发现，被命名为CpG小岛（CpG islets）与CpG孤儿序列（CpG orphans）的非常规CpG区域内的新着丝粒DNA整体呈现高甲基化状态。这一结果与新着丝粒处推测的转录沉默染色质状态相符。在该新着丝粒染色质区域内，活跃转录的特定位点与着丝粒染色质边界（centromeric chromatin boundary）由DNA低甲基化（hypomethylation）所定义。据我们所知，本研究首次在哺乳动物中证实了DNA甲基化与着丝粒形成之间存在相关性；同时证明，通过对局部序列区域进行选择性低甲基化，着丝粒处可允许转录与染色质边界活性的存在，且不会破坏整体的沉默染色质状态与着丝粒功能。