Investigating the Direct Role of Promoter DNA Methylation in Gene Expression using a Targeted TET1CD-dCas9 Demethylase Tool

Abstract

DNA hypermethylation of gene promoter regions has long been associated with transcriptional repression. Furthermore, the establishment of dense promoter methylation within tumour- suppressor genes has been widely-characterised as a prominent hallmark of oncogenesis. The presence of dense DNA methylation, concentrated to promoter region CpG islands, is known to recruit methyl-CpG binding domain proteins (MBDs) and transcriptional repressors which manipulate the local chromatin environment to downregulate transcriptional activity. However, there is an accumulating body of evidence which now suggests, in some specific contexts, that promoter hypermethylation is also associated with high or increased levels of active gene expression. Recent research has uncovered this phenomenon in the context of cutaneous melanoma. Early B-cell factor 3 (EBF3) has been identified as a putative ‘epigenetic driver’ of metastasis in melanoma, associated with an increase in aggressive phenotypic behaviour. Interestingly, EBF3 acquires substantial promoter DNA methylation during the transition to metastasis, which is associated with a corresponding increase in gene expression. Moreover, when treated with a global demethylating agent, EBF3 expression was subsequently reduced. Unfortunately, investigations using global demethylation inhibitors do not conclusively demonstrate that promoter-specific methylation gain is responsible for a change in gene expression. However, with the recent emergence of Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenetic editing technologies, there is now scope for more targeted investigation into this relationship. Here, I have constructed a CRISPR-based targeting system to investigate the EBF3 promoter region in more detail. The CRISPR construct is fused to the SUperNova Tagging (SunTag) protein scaffold, which allows for the recruitment of effector proteins to induce active demethylation at the target locus. In this case, the effector protein is a catalytic domain derived from the human ten-eleven translocation 1 (TET1) dioxygenase. Directed by locus-specific guide-RNA (gRNA) molecules, this system was used to induce site- specific active demethylation within the target promoter region of EBF3, in human melanoma cell lines. Methylation changes across this target locus have been evaluated using Combined Bisulfite Restriction Assay (CoBRA) and methylation-specific Illumina MiSeq sequencing. Variable levels of targeted demethylation have been observed, of up to 58.8% absolute methylation difference between edited and control samples. In generating a modifiable, RNA-guided construct for site-specific DNA methylation editing, this work has laid a strong platform for further investigation into the direct relationship between DNA methylation and gene expression with regards to both EBF3, and a limitless range of additional contexts in future.