Figure 4.
Chondrogenic genes exhibit normal expression upon loss of Ezh2 and H3K27me3. A, IMCs were isolated from WT (Ezh2f/f; Col2a1-Cre−/−) and cKOCol2 mice and plated in micromass culture for 14 days (D) in the presence of chondrogenic mixture (Chondro mix). B, abundance of Ezh2 and H3K27me3 was reduced in cKOCol2 micromasses compared with the WT. The numbers on the right indicate locations of molecular weight markers (kilodalton). C, histological staining of WT (Ezh2f/f; Col2a1-Cre−/−) and cKOCol2 micromasses show a reduced Alcian blue extracellular matrix in cKOCol2 chondrocytes. D, RNA-Seq analysis was performed on WT (Ezh2f/f; Col2a1-Cre−/−) and cKOCol2 IMCs on day 3, day 7, and day 14 of culture. Gene expression data were filtered to exclude genes that were not expressed in any of the samples (0 RPKM, 19,641 of 23,359 genes were visualized). Hierarchical clustering of the filtered RNA-Seq identified three clusters that grouped together WT and cKOCol2 samples taken at similar time points, indicating that the majority of expressed genes were similar between samples. E, chondrogenic genes associated with immature and proliferating chondrocytes (Col2a1, Acan, and Sox9) are highly expressed on day 3, whereas the hypertrophic markers Col10a1 and Vegfa are up-regulated on day 14. F, validation of RNA-Seq data shows good concordance with quantitative PCR.