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. 2022 Feb;36(2):e22153.
doi: 10.1096/fj.202101402R.

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation

Chirada Dusadeemeelap  1   2 , Thira Rojasawasthien  1 , Takuma Matsubara  1 , Shoichiro Kokabu  1 , William N Addison  1
Affiliations

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation

Chirada Dusadeemeelap et al. FASEB J. 2022 Feb.

Abstract

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.

Keywords: DNA demethylation; DNA methylation; SP7; TET; TET inhibitors; epigenetics; osteoblast.

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References

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