Our day-to-day experiences provide a clear understanding of cause-and-effect; a lab mate’s birthday means cake for lunch, and cake for lunch means hours of hard work in the gym! While this cake-centric concept might be simple and easy to follow, the seemingly straightforward causal relationship between DNA demethylation and the upregulation of gene expression, much like the movie Primer, requires some additional consideration.
In their recent article, Daniel M. Sapozhnikov and Moshe Szyf (McGill University, Montreal, Canada) describe how experimental “confounds” complicate our understanding of the causal relationship between DNA methylation and gene expression when using flexibly tethered enzyme-based approaches (e.g., dcas9-TET-based epigenetic editors). As a solution, the authors examined what happened when dCas9 physically blocks (aka sterically hinders) DNA methylation at specific CpGs to cast off confounding factors and “crack” this epigenetic case of cause and effect.
Let’s hear how this cool new tool helped Sapozhnikov and Szyf consider the causality of DNA methylation and gene expression:
- Specific guide RNAs direct an enzyme-free dCas9 to target CpGs, where it sterically-hinders DNA methyltransferase activity (a previously unrecognized ability) over a narrow region
- An optimized approach induces near-complete DNA demethylation in dividing cells without off-target effects (e.g., alterations to adjacent CpG methylation) to allow the exploration of the causal role of DNA methylation dynamics in altering gene expression
- Using this approach, the authors reveal that DNA demethylation causes context-dependent alterations to gene expression, which may facilitate responses to additional factors
- Targeted DNA demethylation at an inducible promoter (Il33-002 gene) induces a small effect; therefore, DNA methylation may prevent leaky expression/transcriptional noise in the absence of the primary expression-inducing signal in this context
- DNA demethylation of the large, highly-methylated FMR1 repeat region in Fragile X syndrome patient fibroblasts prompts a highly significant increase in gene expression, suggesting that repeat element methylation plays a prominent role in gene silencing
The authors report that their cool new causality-confirming tool, which casts off confounding factors quicker than cake disappears in a lab, represents a straightforward and scalable approach to distinguish “causational instances of DNA methylation” and provide a more robust base to the understanding of DNA methylation. Indeed, comparisons with results obtained using general DNA methylation inhibitors suggest that confounding factors may have overestimated the effect of DNA methylation at specific sites.
First author Daniel M. Sapozhnikov shares, “Any DNA regions of interest in cells or in vitro can be completely demethylated by nuclease dead Cas9 (dCas9) without any tethered enzyme, simply through the physical interference with DNA methyltransferases. This brings a new, robust, unconfounded, and simple ability for researchers to ask whether the DNA methylation they are studying has causal consequences on gene expression.”
See what confounds have been hindering our understanding of DNA demethylation over at Nature Communications, September 2021.