Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

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. In mammalian cells, DNA methylation is a major epigenetic modification, which regulates gene expression without alteration of the DNA sequence and thus plays a pivotal role in a myriad of physiological and pathological processes, including cell development and differentiation, genome imprinting, and tumorigenesis 1. We reported previously that the DNA methylation status of hepatic metabolic genes dynamically changes in early life, especially during the suckling period, thereby sequentially developing metabolic function in the liver to adapt to the drastic changes in the major nutrition source 2-4. Peroxisome proliferator-activated receptor-α (PPARα) is a nuclear receptor and a key regulator of hepatic lipid metabolism, which is activated by milk lipids as ligands at the onset of lactation. PPARα governs the transcription of major hepatic metabolism-related genes, and the activation of PPARα physiologically leads to DNA demethylation of fatty acid β-oxidation genes in the postnatal mouse liver 3,4. Of note, administration of a synthetic PPARα ligand to mouse dams during the perinatal period induced enhanced reduction of DNA methylation of PPARα target genes in the offspring liver, suggesting that the DNA methylation status of PPARα target genes can be modulated with ease during the perinatal period 3,4. 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 5-7 .

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

“…Several studies have proven that FGF21 stimulates the brown (BAT) and beige adipose tissue thermogenesis, which lowers body weight and improves insulin sensitivity in mice ( 73 , 74 ). It has also been reported that the Fgf21 promoter could be demethylated by CRISPR/dCas9-mediated epigenome editing ( 75 ). Therefore, we tried to activate FGF21 in mouse liver to regulate energy homeostasis.…”

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

“… Lau and Suh (2018) successfully demonstrated this by targeting a CGG expansion mutation in the 5'-untranslated region (5'-UTR) of the fragile X mental retardation 1 ( FMR1 ) gene in Fragile X syndrome (FXS) patients, which is the most common form of genetic intellectual disability in males. Several studies have shown CRISPR/dCas9-TET1 capable of removing up to 90% of targeted methylations, significantly increasing gene expression at the target sites ( Liu et al, 2016 ; Morita et al, 2016 ; Hanzawa et al, 2020 ; Horii et al, 2020 ). Horii et al (2020) also used this technology successfully to generate an epigenetic disease model in mice via targeted demethylation of the epigenome.…”

CRISPR-mediated promoter de/methylation technologies for gene regulation

“…Hanzawa et al used the dCas9-SunTag-scFv-sfGFP-TET1CD fusion vector for demethylation of the Fgf21 promoter (Table 2) (Hanzawa et al 2020). In this system, dCas9 was fused to SunTag epitopes for recruiting multiple copies of antibody-fused TET1 for enhanced demethylation activities at the target site (Fig.…”

“…The dCas9-SunTag-TET1 vector with a single-chain variable fragment (scFv) was used to target the Fgf21 promoter for demethylation in the adult mouse liver (Fig. 2b) (Hanzawa et al 2020). This study also showed that regulation of gene expression is achieved by a site-specific epigenetic tool without altering the DNA sequence in vivo.…”