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RNA-sequencing analysis of differentiating wildtype and Ezh2 deficient immature mouse chondrocytes

ABSTRACT: Objective: To evalute the role of Ezh2 in chondrocyte differentiation. Method: Ezh2 was inactivated in chondrocytes using the Col2a1-Cre driver in vivo. Immature mouse chondrocytes (IMC) were isolated from wildtype and Ezh2 deficient mice, and plated in micromass culture. Chondrocytes were differentiated and RNA was isolated at days 3, 7, and 14 of culture. Isolated RNA was subjected to RNA sequencing analysis. Results: Ezh2 deficient chondrocytes exhibit enhanced expression of osteogenic genes compared to wildtype cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE97118 | GEO | 2017/03/29

SECONDARY ACCESSION(S): PRJNA380751

REPOSITORIES: GEO

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Project description:Objective: Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skeleton and both Gdf5 and Erg are proposed as regulators of chondrocyte differentiation during and post interzone formation. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Design: Erg expression was identified in mouse knee joints at E13.5. Expression and microarray analyses were performed using micromass cultures of murine C3H10T1/2 mesenchymal cells undergoing induced chondrogenesis in the presence of absence of Gdf5 and Erg. Results: At E13.5, Erg expression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expression profile was altered by the addition of recombinant Gdf5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression identified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by Gdf5. Conclusion: Erg regulation by Gdf5 in mesenchymal cells in vitro is dependent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg expression causes downregulation of Col2a1 and Col10a1 expression and this effect is potentially mediated by Osr2, which is a known regulator of chondrocyte differentiation. The identification of Ube2b as a putative downstream target of Erg-010 suggests that it may contribute to the regulation of the ubiquitination pathway and thereby BMP2 signaling, which is essential for normal knee joint development. RNA from overexpression experiments was extracted as described above. Total RNA quality was confirmed using a Bioanalyzer 2100 (Agilent). Labelled targets were generated from total RNA and hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymatrix) (Genomics Centre, University of Manchester). The raw fluorescence intensity values were normalized using a Robust Multi-array Average approach using dChip (V2005)

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RNA-sequencing analysis of differentiating wildtype and Ezh2 deficient immature mouse chondrocytes

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