Fig. 6: UTX-dependence of H3K27me3-mediated Igf2 transcriptional repression.

Selected H3K27me3-enriched GO terms in UtxKO chondrocytes (a). Gene set enrichment analysis of H3K27me3 marks revealed significant enhancement a specific pathway in UtxKO cells. Igf2 was selected as differentially regulated and earlier reported H3K27me3-mediated candidates controlling chondrocyte differentiation (enlarged section) (b). Gene track illustrating limited H3K27me3 chromatin occupation at regulator loci and promoter regions of Igf2 gene in UtxKO chondrocytes (red) as compared to WT cells (blue) (c). This confirmed highly significant differences in the enrichment of H3K27me3 by ChIP-PCR assay (d). Increased transcription of Igf2 in UtxKO cells (e) and evident Igf2 immunostaining (scale bar, 10 μm) in UtxKO cartilage (f). Antibody-mediated blockade of Igf2 suppressed Sox9, Col2a1, and Acan expression (g), as well as glycosaminoglycan production (h, i scale bar, 500 μm), in UtxKO chondrocytes. Cells were incubated in medium with Igf2 antibody or IgG. RT-PCR was conducted upon antibody treatment for 24 h. Micromass for Alcian blue staining was incubated for 7 days. Cell cultures were harvested from three mice and experiments were repeated three times. Data are expressed as mean ± standard errors. *P < 0.05; **P < 0.001.