Abstract
On a global scale, the incidence of cutaneous melanoma is increasing swiftly, outpacing the growth of other types of cancer. Metastasis, particularly the spread of melanoma to the brain, is the primary cause of death in melanoma patients. Therefore, it's imperative to deeply investigate how this metastatic process occurs and to explore new treatment alternatives.
Currently, there has been a promising development in understanding the role of epigenetic drivers in the progression of melanoma to metastasis. In various diseases, especially cancer, DNA hypermethylation at gene promoters has been well-established as a mechanism that typically reduces gene expression. However, recent findings from our lab and others have revealed a paradoxical phenomenon where high levels of gene expression coincide with high promoter methylation. This challenges the conventional dogma that promoter DNA methylation solely acts as a silencing mechanism and suggests the intriguing possibility that, for a specific set of genes, promoter hypermethylation might actually activate gene expression.
Conventional methods for manipulating DNA methylation usually involve the use of demethylating agents like 5-azacytidine, which has a global impact on methylation levels and depend on DNA replication. To investigate the precise role of promoter hypermethylation in gene activation, I adopted an innovative approach utilising Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-SunTag technology for methylation editing. This modified system employs a deactivated Cas9 (dCas9) protein, enabling the targeted manipulation of methylation at a specific genomic location without altering the DNA sequence.
In this study, I focused on targeted DNA methylation editing of a 58 bp target segment of the EBF3 (early B cell factor 3) gene promoter in melanoma cell lines, previously identified as a potential 'epigenetic driver' of melanoma metastasis. Interestingly, as melanoma progresses toward metastasis, EBF3 experiences significant DNA methylation at its promoter region, coinciding with an increase in gene expression. My objective is to establish an association between these specific methylation changes and gene expression, and consequently examine how these alterations affect the chromatin associations within the genome of unedited and edited cell line conditions. Subsequently, I aim to determine whether a reduction in promoter
DNA methylation leads to the repression of gene expression in this context, thereby initiating the exploration of the underlying mechanisms behind paradoxical gene activation.
Overall, this project challenges the long-standing notion of methylation as an exclusively gene silencing mechanism and aims to establish a new paradigm. The findings from this study may also help unveil epigenetic drivers of malignancy and metastasis.