Li Shiyong, Hu Guanghui, Chen Yulu, Sang Ye, Tang Qin, Liu Rengyun
Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan Second Road, Guangzhou, Guangdong, 510080, China.
Cancer Cell Int. 2024 Aug 3;24(1):271. doi: 10.1186/s12935-024-03459-2.
DNA hypermethylation and hotspot mutations were frequently observed in the upstream and core promoter of telomerase reverse transcriptase (TERT), respectively, and they were associated with increased TERT expression and adverse clinical outcomes in thyroid cancer. In TERT promoter mutant cancer cells, the hypomethylated TERT mutant allele was active and the hypermethylated TERT wild-type allele was silenced. However, whether and how the upstream promoter methylation regulates TERT expression in TERT mutation-negative cells were largely unknown.
DNA demethylating agents 5-azacytidine and decitabine and a genomic locus-specific demethylation system based on dCas9-TET1 were used to assess the effects of TERT upstream promoter methylation on TERT expression, cell growth and apoptosis of thyroid cancer cells. Regulatory proteins binding to TERT promoter were identified by CRISPR affinity purification in situ of regulatory elements (CAPTURE) combined with mass spectrometry. The enrichments of selected regulatory proteins and histone modifications were evaluated by chromatin immunoprecipitation.
The level of DNA methylation at TERT upstream promoter and expression of TERT were significantly decreased after treatment with 5-azacytidine or decitabine in TERT promoter wild-type thyroid cancer cells. Genomic locus-specific demethylation of TERT upstream promoter induced TERT downregulation, along with cell apoptosis and growth inhibition. Consistently, demethylating agents sharply inhibited the growth of thyroid cancer cells harboring hypermethylated TERT but had little effect on cells with TERT hypomethylation. Moreover, we identified that the chromatin remodeling protein CHD4 binds to methylated TERT upstream promoter and promotes its transcription by suppressing the enrichment of H3K9me3 and H3K27me3 at TERT promoter.
This study uncovered the mechanism of promoter methylation mediated TERT activation in TERT promoter mutation-negative thyroid cancer cells and indicated TERT upstream promoter methylation as a therapeutic target for thyroid cancer.
端粒酶逆转录酶（TERT）上游和核心启动子区域分别频繁出现DNA高甲基化和热点突变，且它们与甲状腺癌中TERT表达增加及不良临床预后相关。在TERT启动子突变的癌细胞中，低甲基化的TERT突变等位基因具有活性，而高甲基化的TERT野生型等位基因则被沉默。然而，上游启动子甲基化是否以及如何调节TERT突变阴性细胞中的TERT表达，在很大程度上尚不清楚。
使用DNA去甲基化剂5-氮杂胞苷和地西他滨以及基于dCas9-TET1的基因组位点特异性去甲基化系统，评估TERT上游启动子甲基化对甲状腺癌细胞TERT表达、细胞生长和凋亡的影响。通过CRISPR原位调控元件亲和纯化（CAPTURE）结合质谱法鉴定与TERT启动子结合的调控蛋白。通过染色质免疫沉淀评估选定调控蛋白和组蛋白修饰的富集情况。
在TERT启动子野生型甲状腺癌细胞中，用5-氮杂胞苷或地西他滨处理后，TERT上游启动子的DNA甲基化水平和TERT表达显著降低。TERT上游启动子的基因组位点特异性去甲基化诱导TERT下调，同时伴有细胞凋亡和生长抑制。一致地，去甲基化剂显著抑制了TERT高甲基化的甲状腺癌细胞的生长，但对TERT低甲基化的细胞影响很小。此外，我们鉴定出染色质重塑蛋白CHD4与甲基化的TERT上游启动子结合，并通过抑制TERT启动子处H3K9me3和H3K27me3的富集来促进其转录。
本研究揭示了TERT启动子突变阴性甲状腺癌细胞中启动子甲基化介导TERT激活的机制，并表明TERT上游启动子甲基化可作为甲状腺癌的治疗靶点。
