Altered promoter nucleosome positioning is an early event in gene silencing

Gene silencing in cancer frequently involves hypermethylation and dense nucleosome occupancy across promoter regions. How a promoter transitions to this silent state is unclear. Using colorectal adenomas, we investigated nucleosome positioning, DNA methylation, and gene expression in the early stages of gene silencing. Genome-wide gene expression correlated with highly positioned nucleosomes upstream and downstream of a nucleosome-depleted transcription start site (TSS). Hypermethylated promoters displayed increased nucleosome occupancy, specifically at the TSS. We investigated 2 genes, <i>CDH1</i> and <i>CDKN2B</i>, which were silenced in adenomas but lacked promoter hypermethylation. Instead, silencing correlated with loss of nucleosomes from the -2 position upstream of the TSS relative to normal mucosa. In contrast, permanent <i>CDH1</i> silencing in carcinoma cells was characterized by promoter hypermethylation and dense nucleosome occupancy. Our findings suggest that silenced genes transition through an intermediary stage involving altered promoter nucleosome positioning, before permanent silencing by hypermethylation and dense nucleosome occupancy.

癌症中的基因沉默（gene silencing）常涉及启动子区域的高甲基化（hypermethylation）与密集核小体占据（dense nucleosome occupancy）。目前，启动子如何转变为该沉默状态仍不明确。本研究以结直肠腺瘤（colorectal adenomas）为研究对象，在基因沉默的早期阶段，对核小体定位（nucleosome positioning）、DNA甲基化（DNA methylation）与基因表达（gene expression）展开了探究。全基因组水平的基因表达与核小体缺失（nucleosome-depleted）的转录起始位点（TSS, transcription start site）上下游的高度定位核小体显著相关。高甲基化的启动子区域表现出核小体占据水平升高的特征，且该升高特异性地集中于转录起始位点处。我们针对CDH1与CDKN2B两个基因展开了研究：这两个基因在腺瘤中发生沉默，但并未出现启动子高甲基化现象；相较正常黏膜（normal mucosa），其基因沉默与转录起始位点上游-2位的核小体丢失密切相关。与之形成鲜明对比的是，癌细胞（carcinoma cells）中CDH1的永久性沉默则以启动子高甲基化与密集核小体占据为典型特征。本研究结果表明，沉默基因在通过高甲基化与密集核小体占据实现永久性沉默之前，会先经历一个涉及启动子核小体定位改变的中间阶段（intermediary stage）。