Abstract
BackgroundOur previous work has provided strong evidence that the transcription factor SOX9 is completely needed for chondrogenic differentiation and cartilage formation acting as a “master switch” in this differentiation. Heterozygous mutations in SOX9 cause campomelic dysplasia, a severe skeletal dysmorphology syndrome in humans characterized by a generalized hypoplasia of endochondral bones. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies.Methodology/Principal FindingsThe ChIP DNA was hybridized to a microarray, which covered 80 genes, many of which are involved in chondrocyte differentiation. Hybridization peaks were detected in a series of cartilage extracellular matrix (ECM) genes including Col2a1, Col11a2, Aggrecan and Cdrap as well as in genes for specific transcription factors and signaling molecules. Our results also showed SOX9 interaction sites in genes that code for proteins that enhance the transcriptional activity of SOX9. Interestingly, a strong SOX9 signal was also observed in genes such as Col1a1 and Osx, whose expression is strongly down regulated in chondrocytes but is high in osteoblasts. In the Col2a1 gene, in addition to an interaction site on a previously identified enhancer in intron 1, another strong interaction site was seen in intron 6. This site is free of nucleosomes specifically in chondrocytes suggesting an important role of this site on Col2a1 transcription regulation by SOX9.Conclusions/SignificanceOur results provide a broad understanding of the strategies used by a “master” transcription factor of differentiation in control of the genetic program of chondrocytes.
Highlights
The transcription factor SOX9 plays a critical role in cell fate decisions of a discrete number of cell types [1,2,3,4]
Other genes of group 1 include the chains of type I collagen, which are not expressed in chondrocytes but are prominent in both SOX9-expressing mesenchymal precursors and in osteoblasts [24]
The profile of the negative controls using non-specific IgGs showed essentially no hybridization peaks. Overall these results showed that the chromatin immunoprecipitation (ChIP)-on-chip approach was very efficient in identifying highly specific SOX9 interaction sites
Summary
The transcription factor SOX9 plays a critical role in cell fate decisions of a discrete number of cell types [1,2,3,4]. Heterozygous mutations in Sox cause Campomelic Dysplasia (CD), a generalized disease of cartilage characterized by hypoplasia of endochondral bones [5,6]. Chondrogenesis is associated with activation of a repertoire of cartilage-specific ECM genes. In several of these genes, chondrocyte-specific enhancers have been identified. In order to determine whether genes involved in cartilage function and regulation are direct targets of SOX9 in the genome of chondrocytes, and to examine patterns of SOX9 interactions with the chromatin of these genes in these cells, we have used a chromatin immunoprecipitation (ChIP)-on-chip approach [18]. Our study, which identified many new direct targets of SOX9 as well as potential binding sites for SOX9 in these genes, provides new insights in the strategies used by SOX9 in the control of chondrogenesis. To obtain insights into the logic used by SOX9 to control a network of target genes in chondrocytes, we performed a ChIP-on-chip experiment using SOX9 antibodies
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