Abstract
The transcription factor SOX9 plays an essential role in determining the fate of several cell types and is a master factor in regulation of chondrocyte development. Our aim was to determine which genes in the genome of chondrocytes are either directly or indirectly controlled by SOX9. We used RNA-Seq to identify genes whose expression levels were affected by SOX9 and used SOX9 ChIP-Seq to identify those genes that harbor SOX9-interaction sites. For RNA-Seq, the RNA expression profile of primary Sox9flox/flox mouse chondrocytes infected with Ad-CMV-Cre was compared with that of the same cells infected with a control adenovirus. Analysis of RNA-Seq data indicated that, when the levels of Sox9 mRNA were decreased more than 8-fold by infection with Ad-CMV-Cre, 196 genes showed a decrease in expression of at least 4-fold. These included many cartilage extracellular matrix (ECM) genes and a number of genes for ECM modification enzymes (transferases), membrane receptors, transporters, and others. In ChIP-Seq, 75% of the SOX9-interaction sites had a canonical inverted repeat motif within 100 bp of the top of the peak. SOX9-interaction sites were found in 55% of the genes whose expression was decreased more than 8-fold in SOX9-depleted cells and in somewhat fewer of the genes whose expression was reduced more than 4-fold, suggesting that these are direct targets of SOX9. The combination of RNA-Seq and ChIP-Seq has provided a fuller understanding of the SOX9-controlled genetic program of chondrocytes.
Highlights
The multistep differentiation process of chondrogenesis has an essential role in the development of the endochondral skeleton
Genes regulated by SOX9 in chondrocytes Rib chondrocytes were isolated from 4-day-old Sox9flox/flox mice, in which the DNA segment containing exons 2 and 3 of the Sox9 gene is flanked by loxP sites [2]
Forty-eight hours after infection, the cells were harvested and the expression of Sox9 in total RNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR)
Summary
The multistep differentiation process of chondrogenesis has an essential role in the development of the endochondral skeleton. Mesenchymal progenitor cells first form chondrogenic condensations and differentiate into overt chondrocytes characterized by a high level of expression of a number of typical cartilage extracellular matrix (ECM) genes. These cells sustain additional changes: first, a largely unidirectional proliferation step, followed by exit from the cell cycle and further differentiation into prehypertrophic and hypertrophic chondrocytes. These steps result in formation of the characteristic features of the so-called growth plate of endochondral bones. Conditional inactivation of the Sox gene at various times during mouse limb development demonstrated that SOX9 is necessary for mesenchymal condensations and for the commitment of the cells in these condensations to the chondrocytic cell fate
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