Cutaneous melanoma is rapidly on the rise globally, surpassing the growth rate of other cancers, with metastasis being the primary cause of death in melanoma patients. Consequently, understanding the mechanisms behind this metastatic process and exploring innovative treatments is of paramount importance. Recent research has shown promise in unravelling the role of epigenetic factors in melanoma progression to metastasis. While DNA hypermethylation at gene promoters typically suppresses gene expression, we have contributed to establishing the newly understood mechanism of paradoxical activation of genes via DNA methylation, where high methylation coincides with increased gene activity. This mechanism challenges the conventional paradigm that promoter methylation solely silences genes, suggesting that, for specific genes, it might actually activate them. Traditionally, altering DNA methylation in vitro has involved using global demethylating agents, which is insufficient for studying the mechanism and testing the direct consequence of gene methylation changes. To investigate promoter hypermethylation and its association with gene activation, we employed a novel approach utilising a CRISPR-SunTag All-in-one system. Here, we focused on editing the DNA methylation of a specific gene promoter segment (EBF3) in melanoma cells using the All-in-one system. Using bisulfite sequencing and qPCR with RNA-Seq, we successfully demonstrated highly effective methylation and demethylation of theEBF3promoter, with subsequent gene expression changes, to establish and validate the paradoxical role of DNA methylation. Further, our study provides novel insights into the function of theEBF3gene, which remains largely unknown. Overall, this study challenges the conventional view of methylation as solely a gene-silencing mechanism and demonstrates a potential function ofEBF3in IFN pathway signalling, potentially uncovering new insights into epigenetic drivers of malignancy and metastasis.
皮肤黑色素瘤在全球范围内迅速增加，其增长速度超过其他癌症，而转移是黑色素瘤患者死亡的主要原因。因此，理解这一转移过程的机制并探索创新治疗方法至关重要。最近的研究在揭示表观遗传因素在黑色素瘤进展至转移中的作用方面显示出希望。虽然基因启动子区域的DNA高甲基化通常会抑制基因表达，但我们为建立新近理解的通过DNA甲基化反常激活基因的机制做出了贡献，即高甲基化与基因活性增加同时发生。这一机制挑战了启动子甲基化仅沉默基因的传统范式，表明对于特定基因，它实际上可能激活它们。传统上，在体外改变DNA甲基化涉及使用全局去甲基化剂，这对于研究机制和测试基因甲基化变化的直接后果是不够的。为了研究启动子高甲基化及其与基因激活的关联，我们采用了一种利用CRISPR-SunTag一体化系统的新方法。在此，我们专注于使用一体化系统编辑黑色素瘤细胞中特定基因启动子片段（EBF3）的DNA甲基化。通过亚硫酸氢盐测序和qPCR结合RNA-Seq，我们成功证明了EBF3启动子的高效甲基化和去甲基化，以及随后的基因表达变化，从而建立并验证了DNA甲基化的反常作用。此外，我们的研究为EBF3基因的功能提供了新的见解，该功能在很大程度上仍然未知。总体而言，这项研究挑战了甲基化仅作为基因沉默机制的传统观点，并证明了EBF3在IFN通路信号传导中的潜在功能，可能为恶性肿瘤和转移的表观遗传驱动因素提供新的见解。
肿瘤(癌症)患者之家