Epigenetic regulation of ZNF687 by miR-142a-3p and DNA methylation during osteoblast differentiation and mice bone development and aging

Domingos Varela, Débora Cristina; Domingos Varela, Tatiana da Conceição; Conceição, Natércia; Cancela, M. Leonor

http://hdl.handle.net/10400.1/26968

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Authors

Domingos Varela, Débora Cristina

Domingos Varela, Tatiana da Conceição

Conceição, Natércia

Cancela, M. Leonor

Abstract(s)

Zinc finger protein 687 (ZNF687), a transcription factor implicated in osteoblast/osteoclast differentiation and linked to Paget's disease of bone, has unclear mechanisms in bone metabolism. Epigenetic disruptions can affect bone cell activity and contribute to bone-related diseases. This work aimed to elucidate the regulatory role of epigenetics in modulating Zfp687 expression throughout osteoblast differentiation and bone growth/aging in mice. Differentiation of the mouse-derived osteoblast precursor cell line (MC3T3-E1) showed increased expression of osteogenic markers and decreased Zfp687 expression. In the hindlimb bones of C57BL/6J mice, the expression of most bone-forming genes decreased from youth to adulthood, while Zfp687 and Runx2 expression was maintained, being only significantly reduced in old mice in comparison to young mice. Bisulfite sequencing revealed hypomethylation of the Zfp687 promoter during MC3T3-E1 differentiation and bone growth/aging. Bioinformatics predicted miR-142a-3p, miR-122b-5p, and miR-124-3p binding sites in Zfp687 3 ' UTR, and RT-qPCR analysis showed higher expression of these miRNAs in mature osteoblasts. Transfection of a miR-142-3p mimic reduced luciferase activity in the wildtype Zfp687 3 ' UTR but not the mutant 3 ' UTR and downregulated the Zfp687 gene and protein levels. In conclusion, miR-142a-3p directly targets the Zfp687 3 ' UTR, promoting its downregulation during osteoblastogenesis. Furthermore, DNA methylation does not appear to regulate Zfp687 during osteoblast differentiation or bone development in mice.