Abstract
Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.
Highlights
In mammalian cells, DNA methylation is a major epigenetic modification, which regulates gene expression without alteration of the DNA sequence and plays a pivotal role in a myriad of physiological and pathological processes, including cell development and differentiation, genome imprinting, and tumorigenesis[1]
We found that the DNA methylation status of the mouse fibroblast growth factor 21 (FGF21) gene (Fgf21) promoter, which is established during the suckling period, is maintained into adulthood[4]
Even though we found no significant difference in Fgf[21] mRNA levels after K-877 administration between gRNA1 and gRNA1+2, a correlation plot showed a negative correlation between the degree of DNA methylation (%DNA methylation) and the induction of gene expression (Fig. 5b)
Summary
DNA methylation is a major epigenetic modification, which regulates gene expression without alteration of the DNA sequence and plays a pivotal role in a myriad of physiological and pathological processes, including cell development and differentiation, genome imprinting, and tumorigenesis[1]. A genome-wide analysis of DNA methylation revealed that a few PPARα target genes undergo ligand-activated, PPARα-dependent DNA demethylation during the perinatal period, and the DNA hypomethylation status of these persists into adulthood. Among these genes, which may be referred to as “epigenetic memory genes,” we focused on fibroblast growth factor 21 (FGF21), which is a metabolic hormone derived from the liver and a master regulator of glucose and lipid metabolism[5,6,7]. Single guide RNA (sgRNA)-mediated DNA targeting, followed by Cas[9] endonuclease-mediated DNA cleavage enables site-specific genome editing[10]
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