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Cited by 101Osteoarthritis Treatment and MechanismsGenomics and Chromatin DynamicsProteoglycans and glycosaminoglycans research

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SOX9 Regulates Multiple Genes in Chondrocytes, Including Genes Encoding ECM Proteins, ECM Modification Enzymes, Receptors, and Transporters

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Oh, Chun-do, Lu, Yue, Liang, Shoudan, Mori-Akiyama, Yuko, Chen, Di, de Crombrugghe, Benoit, Yasuda, Hideyo·2014·PLoS ONE
DOI 10.1371/journal.pone.0107577PMC PMC4168005
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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.

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Figure 1 Decrease of Sox9 expression in mouse Sox9flox/flox primary rib chondrocytes infected with Ad-CMV-Cre. Primary rib chondrocytes obtained from 4-day-old Sox9flox/flox mice were cultured for 24 hours and then infected with Ad-CMV-Cre or control vector Ad-CMV-Null at 200 moi. In these cells, exon 2 through exon 3 of the Sox9 gene was flanked with loxP sites. Forty-eight hours after infection, the Sox9 mRNA levels in cells infected with Ad-CMV-Cre, measured by RT-qPCR, were decreased by more than 8-fold compared to the levels in cells infected with Ad-CMV-Null.
Figure 1 Decrease of Sox9 expression in mouse Sox9flox/flox primary rib chondrocytes infected with Ad-CMV-Cre. Primary rib chondrocytes obtained from 4-day-old Sox9flox/flox mice were cultured for 24 hours and then infected with Ad-CMV-Cre or control vector Ad-CMV-Null at 200 moi. In these cells, exon 2 through exon 3 of the Sox9 gene was flanked with loxP sites. Forty-eight hours after infection, the Sox9 mRNA levels in cells infected with Ad-CMV-Cre, measured by RT-qPCR, were decreased by more than 8-fold compared to the levels in cells infected with Ad-CMV-Null.CC BY · PubMed Central
Figure 2 Classification of genes whose expression was decreased after removal of SOX9. Genes whose expression was decreased by more than 4-, 6-, or 8-fold by the removal of SOX9 were classified into functional categories. The percentage of proteins in each category is shown. The number of corresponding proteins in each category is shown in Table S1. Some proteins were assigned to more than one category. Genes whose expression was decreased by more than 6-fold by the removal of SOX9 were classified into 20 functional groups; the percentages in each group of more than five genes containing a SOX9-interaction site revealed by ChIP-Seq are underlined in red on the left: the first percentage is for mouse rib chondrocytes and the second for RCS cells. Among the genes whose expression was decreased by more than 6- or 8-fold, the ECM and transferase groups had the highest percentages of SOX9-interaction sites, and these percentages were higher than for the same groups of genes whose expression was decreased by only 4-fold after SOX9 removal.
Figure 2 Classification of genes whose expression was decreased after removal of SOX9. Genes whose expression was decreased by more than 4-, 6-, or 8-fold by the removal of SOX9 were classified into functional categories. The percentage of proteins in each category is shown. The number of corresponding proteins in each category is shown in Table S1. Some proteins were assigned to more than one category. Genes whose expression was decreased by more than 6-fold by the removal of SOX9 were classified into 20 functional groups; the percentages in each group of more than five genes containing a SOX9-interaction site revealed by ChIP-Seq are underlined in red on the left: the first percentage is for mouse rib chondrocytes and the second for RCS cells. Among the genes whose expression was decreased by more than 6- or 8-fold, the ECM and transferase groups had the highest percentages of SOX9-interaction sites, and these percentages were higher than for the same groups of genes whose expression was decreased by only 4-fold after SOX9 removal.CC BY · PubMed Central
Figure 3 Distribution of SOX9-interacting peak locations. The locations are defined as follows: upstream: −50 to −5 Kb from transcription start site (TSS); promoter: −5 to +0.5 Kb from TSS; termination site (TES): −0.5 to +5 Kb from TES; downstream: +5 to +50 Kb from TES; distant: localized outside the boundaries already defined. Both mouse and rat genes were aligned by using the database of mouse genome sequences because the annotation and the assignment of genes to the rat genome is not yet complete.
Figure 3 Distribution of SOX9-interacting peak locations. The locations are defined as follows: upstream: −50 to −5 Kb from transcription start site (TSS); promoter: −5 to +0.5 Kb from TSS; termination site (TES): −0.5 to +5 Kb from TES; downstream: +5 to +50 Kb from TES; distant: localized outside the boundaries already defined. Both mouse and rat genes were aligned by using the database of mouse genome sequences because the annotation and the assignment of genes to the rat genome is not yet complete.CC BY · PubMed Central
Figure 4 Examples of ChIP-Seq peaks of mouse primary rib chondrocytes and RCS cells. The SOX9-interacting peaks of the Col9a2 (collagen IX, alpha-2 polypeptide) and Acan (aggrecan) genes are shown in A, B) and C, D), respectively. In the bottoms of B) and D), the sequence conservation peaks among 6 vertebrates are shown. The peaks detected by our criteria are indicated by *. A) The peaks in the mouse Col9a2 gene from −5 Kb from the TSS to +5 Kb from the TSE are shown. In the right of this graph, the portion surrounded by dotted square is shown. The solid squares in Col9a2 gene show the exons including non-coding ones. B) The peaks in the rat Col9a2 gene are shown. In this region two additional genes, Zmpste24 and Smap2, exist in addition to Col9a2. C) The peaks in the mouse Acan gene from −50 Kb from the TSS to +50 Kb from the TSE are shown. Below in this graph, the portion surrounded by dotted square in C) is shown. D) The peaks in the rat Acan gene are shown. The solid squares in Acan gene show the exons including non-coding ones. In this region, two additional genes in addition to Acan, Hapln3 and Mfge8, are localized in the reverse direction to Acan. Three sharp peaks were detected in mouse and rat Acan genes. The locations of two of the three peaks are similar in genes of mouse chondrocytes and rat RCS cells. The red arrows show the peaks that have been shown to have SOX9/SOX5/SOX6-dependent enhancer activity [33].
Figure 4 Examples of ChIP-Seq peaks of mouse primary rib chondrocytes and RCS cells. The SOX9-interacting peaks of the Col9a2 (collagen IX, alpha-2 polypeptide) and Acan (aggrecan) genes are shown in A, B) and C, D), respectively. In the bottoms of B) and D), the sequence conservation peaks among 6 vertebrates are shown. The peaks detected by our criteria are indicated by *. A) The peaks in the mouse Col9a2 gene from −5 Kb from the TSS to +5 Kb from the TSE are shown. In the right of this graph, the portion surrounded by dotted square is shown. The solid squares in Col9a2 gene show the exons including non-coding ones. B) The peaks in the rat Col9a2 gene are shown. In this region two additional genes, Zmpste24 and Smap2, exist in addition to Col9a2. C) The peaks in the mouse Acan gene from −50 Kb from the TSS to +50 Kb from the TSE are shown. Below in this graph, the portion surrounded by dotted square in C) is shown. D) The peaks in the rat Acan gene are shown. The solid squares in Acan gene show the exons including non-coding ones. In this region, two additional genes in addition to Acan, Hapln3 and Mfge8, are localized in the reverse direction to Acan. Three sharp peaks were detected in mouse and rat Acan genes. The locations of two of the three peaks are similar in genes of mouse chondrocytes and rat RCS cells. The red arrows show the peaks that have been shown to have SOX9/SOX5/SOX6-dependent enhancer activity [33].CC BY · PubMed Central
Figure 5 The SOX9-dependent enhancer activity of the SOX9-interacting peak at −80 Kb in the Sox9 gene. A) The peaks in the Sox9 gene from −300 Kb from the TSS to +50 Kb from the TSE are shown. In this region, no additional coding genes exist except for a mouse non-coding RNA in the reverse direction to Sox9. One peak (blue arrow) was detected in the mouse Sox9 gene and two sharp peaks (blue and red arrows) were observed in the rat Sox9 gene. B) The SOX9-dependent enhancer activity of the −80 Kb peak in the rat gene (red arrow) is demonstrated. A 750-bp DNA fragment surrounding the SOX9-interacting site at −80 Kb from the TSS was inserted 5′ to a 1-Kb Sox9 promoter plus exon1 fragment followed by an internal ribosome entry site (IRES) and a luciferase reporter as illustrated at the bottom of the figure. 293T cells were transfected with either a control vector or this construct in the presence or absence of a Sox9-expressing plasmid, and the luciferase activity was measured 48 hours after transfection. The reporter activity, which was normalized with renilla luciferase activity, was markedly increased in the presence of SOX9.
Figure 5 The SOX9-dependent enhancer activity of the SOX9-interacting peak at −80 Kb in the Sox9 gene. A) The peaks in the Sox9 gene from −300 Kb from the TSS to +50 Kb from the TSE are shown. In this region, no additional coding genes exist except for a mouse non-coding RNA in the reverse direction to Sox9. One peak (blue arrow) was detected in the mouse Sox9 gene and two sharp peaks (blue and red arrows) were observed in the rat Sox9 gene. B) The SOX9-dependent enhancer activity of the −80 Kb peak in the rat gene (red arrow) is demonstrated. A 750-bp DNA fragment surrounding the SOX9-interacting site at −80 Kb from the TSS was inserted 5′ to a 1-Kb Sox9 promoter plus exon1 fragment followed by an internal ribosome entry site (IRES) and a luciferase reporter as illustrated at the bottom of the figure. 293T cells were transfected with either a control vector or this construct in the presence or absence of a Sox9-expressing plasmid, and the luciferase activity was measured 48 hours after transfection. The reporter activity, which was normalized with renilla luciferase activity, was markedly increased in the presence of SOX9.CC BY · PubMed Central
Figure 6 Consensus binding motif of SOX9-interacting sites detected in chondrocytes. A) The MEME program was used to search for a common motif among 500 random peaks identified by ChIP-Seq experiments (p value <10−8) in RCS cells. The motif was found within 100 bp on either side of the top of the peak in 371 of the genomic SOX9-interaction sites. The motif is an inverted repeat of a canonical motif of six bases on each side with an interval of four bases. B) A search for the same motif within 100 bp on either side of the top of 800 of the strongest binding peaks (p value ≤1e-5, fold ≤2) identified the motif in rib chondrocytes. C) A secondary motif was found in the same binding peaks shown in B). D) In RCS cells, a third motif was found to be similar to a RUNX1 consensus binding site.
Figure 6 Consensus binding motif of SOX9-interacting sites detected in chondrocytes. A) The MEME program was used to search for a common motif among 500 random peaks identified by ChIP-Seq experiments (p value <10−8) in RCS cells. The motif was found within 100 bp on either side of the top of the peak in 371 of the genomic SOX9-interaction sites. The motif is an inverted repeat of a canonical motif of six bases on each side with an interval of four bases. B) A search for the same motif within 100 bp on either side of the top of 800 of the strongest binding peaks (p value ≤1e-5, fold ≤2) identified the motif in rib chondrocytes. C) A secondary motif was found in the same binding peaks shown in B). D) In RCS cells, a third motif was found to be similar to a RUNX1 consensus binding site.CC BY · PubMed Central

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Metadata

Authors
Oh, C.-D., Lu, Y., Liang, S., Mori-Akiyama, Y., Chen, D., de Crombrugghe, B., & Yasuda, H.
Affiliations
  • The University of Texas MD Anderson Cancer Center (US)
  • Rush University Medical Center (US)
Corresponding author
Yasuda, Hideyo
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2014-09-17
Volume
9
Issue
9
Pages
e107577
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0107577
PMC
PMC4168005
OpenAlex
W2040278668
Cited by
101
References
54
Citations by year
2026: 2 · 2025: 6 · 2024: 7 · 2023: 4 · 2022: 9 · 2021: 9 · 2020: 17 · 2019: 10 · 2018: 6 · 2017: 6 · 2016: 11 · 2015: 14
FWCI
8.59
Type
journal article
Language
en
Update notices
correction (2015-11-12)
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health (CA-16 672)
  • University of Texas MD Anderson Cancer Center (#CA16672)
  • National Cancer Institute (CA-16672, P30CA016672)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR309854, R01AR054465)
Topics
  • Health Sciences › Medicine › Rheumatology › Osteoarthritis Treatment and Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Proteoglycans and glycosaminoglycans research
Keywords
SOX9, chondrocytes, transcriptional regulation, extracellular matrix genes, transcription factor binding sites, gene expression profiling, Cre-loxP conditional knockout, chondrogenesis, cell surface receptors, transporters
MeSH terms
Animals, Binding Sites, Gene Expression Regulation, Humans, Protein Binding, Gene Expression, Extracellular Matrix Proteins, Mice, Knockout, Chondrocytes, Gene Expression Profiling, Protein Transport, Chromatin Immunoprecipitation, Mice, SOX9 Transcription Factor, Position-Specific Scoring Matrices, High-Throughput Nucleotide Sequencing, Nucleotide Motifs

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