Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.
Hum Mol Genet. 2011 Nov 15;20(22):4299-310. doi: 10.1093/hmg/ddr356. Epub 2011 Aug 11.
Despite the fact that 45% of all human gene promoters do not contain CpG islands, the role of DNA methylation in control of non-CpG island promoters is controversial and its relevance in normal and pathological processes is poorly understood. Among the few studies which investigate the correlation between DNA methylation and expression of genes with non-CpG island promoters, the majority do not support the view that DNA methylation directly leads to transcription silencing of these genes. Our reporter assays and gene reactivation by 5-aza-2'-deoxycytidine, a DNA demethylating agent, show that DNA methylation occurring at CpG poor LAMB3 promoter and RUNX3 promoter 1(RUNX3 P1) can directly lead to transcriptional silencing in cells competent to express these genes in vitro. Using Nucleosome Occupancy Methylome- Sequencing, NOMe-Seq, a single-molecule, high-resolution nucleosome positioning assay, we demonstrate that active, but not inactive, non-CpG island promoters display a nucleosome-depleted region (NDR) immediately upstream of the transcription start site (TSS). Furthermore, using NOMe-Seq and clonal analysis, we show that in RUNX3 expressing 623 melanoma cells, RUNX3 P1 has two distinct chromatin configurations: one is unmethylated with an NDR upstream of the TSS; another is methylated and nucleosome occupied, indicating that RUNX3 P1 is monoallelically methylated. Together, these results demonstrate that the epigenetic signatures comprising DNA methylation, histone marks and nucleosome occupancy of non-CpG island promoters are almost identical to CpG island promoters, suggesting that aberrant methylation patterns of non-CpG island promoters may also contribute to tumorigenesis and should therefore be included in analyses of cancer epigenetics.
尽管所有人类基因启动子中只有 45%不含 CpG 岛，但 DNA 甲基化在非 CpG 岛启动子控制中的作用仍存在争议，其在正常和病理过程中的相关性也知之甚少。在少数研究 DNA 甲基化与非 CpG 岛启动子基因表达之间相关性的研究中，大多数研究并不支持 DNA 甲基化直接导致这些基因转录沉默的观点。我们的报告基因实验和 5-氮杂-2′-脱氧胞苷（一种 DNA 去甲基化剂）对基因的重新激活表明，在具有 LAMB3 启动子和 RUNX3 启动子 1（RUNX3 P1）的 CpG 贫瘠区发生的 DNA 甲基化可直接导致体外有能力表达这些基因的细胞中基因转录沉默。我们利用核小体占据甲基化组测序（NOMe-Seq），一种单分子、高分辨率核小体定位检测方法，证明了活跃但非失活的非 CpG 岛启动子在转录起始位点（TSS）上游显示出核小体缺失区域（NDR）。此外，我们利用 NOMe-Seq 和克隆分析表明，在表达 RUNX3 的 623 号黑色素瘤细胞中，RUNX3 P1 具有两种不同的染色质构型：一种是 TSS 上游无甲基化的 NDR；另一种是甲基化和核小体占据的，表明 RUNX3 P1 是单等位基因甲基化的。综上所述，这些结果表明非 CpG 岛启动子的表观遗传特征包括 DNA 甲基化、组蛋白标记和核小体占据，与 CpG 岛启动子几乎相同，提示非 CpG 岛启动子的异常甲基化模式也可能有助于肿瘤发生，因此应包括在癌症表观遗传学分析中。
