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- Title
Targeted DNA Methylation Editing Using an All-in-One System Establishes Paradoxical Activation of EBF3.
- Authors
Banerjee, Rakesh; Ajithkumar, Priyadarshana; Keestra, Nicholas; Smith, Jim; Gimenez, Gregory; Rodger, Euan J.; Eccles, Michael R.; Antony, Jisha; Weeks, Robert J.; Chatterjee, Aniruddha
- Abstract
Simple Summary: Contrary to the conventional understanding of DNA hypermethylation suppressing gene expression, a new mechanism emerges, suggesting that high methylation can paradoxically activate genes. This challenges the traditional notion that promoter methylation solely silences genes. The study employs a CRISPR-SunTag All-in-one system to manipulate the DNA methylation of the EBF3 gene promoter segment in melanoma cells. Successful methylation and demethylation demonstrate the paradoxical role of DNA methylation, offering insights into the EBF3 gene's function, particularly in the IFN pathway signaling. This research challenges conventional views on methylation, providing potential insights into epigenetic drivers of malignancy and metastasis. 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 the EBF3 promoter, 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 the EBF3 gene, which remains largely unknown. Overall, this study challenges the conventional view of methylation as solely a gene-silencing mechanism and demonstrates a potential function of EBF3 in IFN pathway signalling, potentially uncovering new insights into epigenetic drivers of malignancy and metastasis.
- Subjects
RNA analysis; IN vitro studies; METHYLATION; RESEARCH funding; EPIGENOMICS; POLYMERASE chain reaction; PROMOTERS (Genetics); CAUSES of death; CELLULAR signal transduction; DNA methylation; CELL lines; GENE expression; GENES; PARADIGMS (Social sciences); CRISPRS; GENOME editing; CUTANEOUS malignant melanoma; DEMETHYLATION; SEQUENCE analysis
- Publication
Cancers, 2024, Vol 16, Issue 5, p898
- ISSN
2072-6694
- Publication type
Article
- DOI
10.3390/cancers16050898