DNMT and HDAC inhibitors globally induce cryptic TSSs encoded in long terminal repeats. Homo sapiens

By mapping global transcription start site (TSS) and chromatin dynamics, we observed the activation of thousands of cryptic, currently non-annotated TSSs (TINATS) following DNMTi and/or HDACi treatment. The resulting transcripts encode truncated or chimeric open reading frames that can be translated into products with predicted abnormal functions or immunogenic potential. TINAT activation after DNMTi coincided with DNA hypomethylation and gain in H3K4me3, H3K9ac, and H3K27ac histone marks. In contrast, HDACi induced only canonical TSSs in association with histone acetylation, but TINATs via a yet unknown mechanism. Nevertheless, both inhibitors convergently induced unidirectional transcription from identical sites since TINATs are encoded in solitary long-terminal repeats of the endogenous retrovirus-9 family, epigenetically repressed in virtually all normal cells. Overall design: CAGE-, ChIP-, and WGB-sequencing of NCI-H1299 EGFP-NEO reporter cells after treatment with DMSO, DAC, SB939, or DAC+SB

通过对全局转录起始位点（transcription start site, TSS）与染色质动态特征进行图谱绘制，本研究观察到在DNA甲基转移酶抑制剂（DNA methyltransferase inhibitor, DNMTi）和/或组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor, HDACi）处理后，数千个隐秘且目前未被注释的转录起始位点（cryptic non-annotated TSSs, TINATS）被激活。由此产生的转录本编码截短型或嵌合型开放阅读框，此类阅读框可被翻译为功能异常或具备免疫原性潜能的预测产物。DNMTi处理后TINAT的激活与DNA低甲基化，以及H3K4me3、H3K9ac、H3K27ac组蛋白修饰标记的富集同步发生。与之相反，HDACi仅诱导经典转录起始位点的激活，该过程与组蛋白乙酰化相关，但其激活TINAT的具体机制仍未明确。尽管如此，两种抑制剂均可从相同位点诱导单向转录，这是因为TINAT编码于内源性逆转录病毒9家族的孤立长末端重复序列中，该序列在几乎所有正常细胞中均处于表观遗传沉默状态。
整体实验设计：对经二甲基亚砜（dimethyl sulfoxide, DMSO）、DAC、SB939或DAC+SB处理后的NCI-H1299 EGFP-NEO报告细胞系，开展CAGE测序（Cap Analysis of Gene Expression sequencing, CAGE-seq）、染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）以及全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing, WGB-seq）。