Uhrf1-mediated DNA methylation governs chondrocyte differentiation.

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Uhrf1-mediated DNA methylation governs chondrocyte differentiation. Uhrf1-mediated DNA methylation governs chondrocyte differentiation. (A) The amount of MBD2-mediated enrichment in methylated DNA from 1 μg of total DNA obtained from control and cKO chondrocytes (n=3). (B) Distribution of Uhrf1-mediated methylated DNA that was annotated with the given intervals and scores with genome features by CEAS (cis-regulatory element annotation system). All differences were significant. (C) Meta-gene analysis of Uhrf1-mediated methylated DNA peaks. Peak signals on ‘all genes’ (black), ‘down’ (red) and ‘up’ (purple) regulated genes by Uhrf1 evaluated by RNA-seq results are described. (D) Venn diagram for the 324 upregulated genes in cKO (blue), 190 genes having Uhrf1-mediated methylated DNA peaks within the Refseq gene body ±10 kb (light green) and 28 genes with peaks in the promoter (3 kb) region (green), as listed in Table S2. (E) Real-time RT-PCR results for Hspb1 expression in primary chondrocytes (n=3). (F) Methylated DNA signals around Hspb1 genes (about 6 kb) illustrated by Igv (integrative genome viewer). (G) Immunohistochemistry for Hspb1 (green) and DAPI (blue) expressed at postnatal day 7 in the proximal humeral growth plate of control and cKO mice. Representative data from at least three individual mice are shown. Scale bars: 50 µm. (H) In vitro BrdU incorporation assay for primary cultured chondrocytes of control, cKO and cKO treated with siRNA for each gene (n=3). (I) Real-time RT-PCR results for cKO primary chondrocytes treated with rhBMP-2 for 3 days after siRNA for Hspb1 treatment (n=3). All data are mean±s.d. *P<0.05 (Student's t-test, Mann–Whitney U-test or ANOVA). Michiko Yamashita et al. Development 2018;145:dev157412 © 2018. Published by The Company of Biologists Ltd