Dataset Information

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

ABSTRACT: Recently, we reported PPAR?-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPAR? 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 PPAR?-deficient mice, with increased gene expression response to PPAR? 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.

SUBMITTER: Hanzawa N

PROVIDER: S-EPMC7083849 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Hanzawa Nozomi N Hashimoto Koshi K Yuan Xunmei X Kawahori Kenichi K Tsujimoto Kazutaka K Hamaguchi Miho M Tanaka Toshiya T Nagaoka Yuya Y Nishina Hiroshi H Morita Sumiyo S Hatada Izuho I Yamada Tetsuya T Ogawa Yoshihiro Y

Scientific reports 20200320 1

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 vi ...[more]

PMID: 32198422

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Project description:RationaleDSP (desmoplakin), the most abundant component of desmosomes, which maintain the mechanical integrity of epithelium, is a genome-wide association study-identified genetic risk locus in human idiopathic pulmonary fibrosis (IPF). Subjects with IPF express a significantly higher level of DSP than control subjects.ObjectivesDetermine potential mechanisms by which DSP is regulated in lung fibrosis.MethodsMatrigel-coated soft and stiff polyacrylamide gels were made to simulate the stiffness of normal and fibrotic lungs. Quantitative chromatin immunoprecipitation and electrophoretic mobility shift assay were used to evaluate transcription factor binding to the DSP promoter. Targeted DNA methylation was achieved by CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 (deactivated CRISPR-associated protein-9 nuclease)-mediated Dnmt3A (DNA methyltransferase 3A) expression under the guidance of sequence-specific single guide RNAs.Measurements and main resultsStiff matrix promotes DSP gene expression in both human and rodent lung epithelial cells as compared with soft matrix. A conserved region in the proximal DSP promoter is hypermethylated under soft matrix conditions and becomes hypomethylated/demethylated under stiff matrix conditions. Demethylation of this conserved DSP promoter region is associated with transactivation of transcription factor EGR1 (early growth response protein 1), resulting in EGR1-dependent DSP overexpression. Targeted DNA methylation by CRISPR/dCas9/Dnmt3A-mediated epigenome editing blocks EGR1 binding to the DSP promoter and inhibits stiff matrix-induced DSP overexpression.ConclusionsDSP is a matrix stiffness-regulated mechanosensitive gene. CRISPR/dCas9-Dnmt3A-mediated epigenome editing reverses DSP overexpression by reestablishment of the epigenetic control of DSP under the mechanically homeostatic environment. It provides a useful tool for investigations of the functional role of DSP in the pathogenesis of lung fibrosis.

Dataset Information

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

Publications

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

Similar Datasets

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