Groves I J, Knight E L A, Ang Q Y, Scarpini C G, Coleman N
Department of Pathology, University of Cambridge, Cambridge, UK.
Oncogene. 2016 Sep 8;35(36):4773-86. doi: 10.1038/onc.2016.8. Epub 2016 Feb 15.
In cervical squamous cell carcinomas, high-risk human papillomavirus (HRHPV) DNA is usually integrated into host chromosomes. Multiple integration events are thought to be present within the cells of a polyclonal premalignant lesion and the features that underpin clonal selection of one particular integrant remain poorly understood. We previously used the W12 model system to generate a panel of cervical keratinocyte clones, derived from cells of a low-grade premalignant lesion naturally infected with the major HRHPV type, HPV16. The cells were isolated regardless of their selective advantage and differed only by the site of HPV16 integration into the host genome. We used this resource to test the hypothesis that levels of HPV16 E6/E7 oncogene expression in premalignant cells are regulated epigenetically. We performed a comprehensive analysis of the epigenetic landscape of the integrated HPV16 DNA in selected clones, in which levels of virus oncogene expression per DNA template varied ~6.6-fold. Across the cells examined, higher levels of virus expression per template were associated with more open chromatin at the HPV16 long control region, together with greater loading of chromatin remodelling enzymes and lower nucleosome occupancy. There were higher levels of histone post-translational modification hallmarks of transcriptionally active chromatin and lower levels of repressive hallmarks. There was greater abundance of the active/elongating form of the RNA polymerase-II enzyme (RNAPII-Ser2P), together with CDK9, the component of positive transcription elongation factor b complex responsible for Ser2 phosphorylation. The changes observed were functionally significant, as cells with higher HPV16 expression per template showed greater sensitivity to depletion and/or inhibition of histone acetyltransferases and CDK9 and less sensitivity to histone deacetylase inhibition. We conclude that virus gene expression per template following HPV16 integration is determined through multiple layers of epigenetic regulation, which are likely to contribute to selection of individual cells during cervical carcinogenesis.
在宫颈鳞状细胞癌中，高危型人乳头瘤病毒（HRHPV）DNA通常整合到宿主染色体中。多克隆癌前病变细胞内被认为存在多个整合事件，而支持特定整合体克隆选择的特征仍知之甚少。我们之前使用W12模型系统生成了一组宫颈角质形成细胞克隆，这些克隆源自自然感染主要HRHPV类型HPV16的低级别癌前病变细胞。这些细胞是随机分离的，仅因HPV16整合到宿主基因组的位点不同而有所差异。我们利用这一资源来检验癌前细胞中HPV16 E6/E7癌基因表达水平受表观遗传调控的假说。我们对选定克隆中整合的HPV16 DNA的表观遗传格局进行了全面分析，其中每个DNA模板的病毒癌基因表达水平相差约6.6倍。在所检测的细胞中，每个模板较高的病毒表达水平与HPV16长控制区更开放的染色质相关，同时染色质重塑酶的负载量更大，核小体占有率更低。转录活性染色质的组蛋白翻译后修饰标记水平较高，而抑制性标记水平较低。RNA聚合酶II酶（RNAPII-Ser2P）的活性/延伸形式以及负责Ser2磷酸化的正转录延伸因子b复合物的成分CDK9的丰度更高。观察到的这些变化具有功能意义，因为每个模板HPV16表达较高的细胞对组蛋白乙酰转移酶和CDK9的缺失和/或抑制表现出更高的敏感性，而对组蛋白脱乙酰酶抑制的敏感性较低。我们得出结论，HPV16整合后每个模板的病毒基因表达是通过多层表观遗传调控决定的，这可能在宫颈癌发生过程中有助于单个细胞的选择。
