title: A Novel Regulatory Mechanism of Type II Collagen Expression via a SOX9-dependent Enhancer in Intron 6.
abstract: Type II collagen α1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1. However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5-10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. Furthermore, SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, although both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1.
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Konkuk University, Neungdong-Ro, Gwangjin-gu, Seoul 05029, Republic of Korea, h-yasuda@yk9.so-net.ne.jp.|the Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, and.
authors.forename: Hideyo
authors.initials: H
authors.lastname: Yasuda
authors.affiliation: the Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, and.|the Department of Biochemistry, Rush University Medical Center, Chicago, Illinois 60612-3823.
authors.forename: Chun-do
authors.initials: CD
authors.lastname: Oh
authors.affiliation: the Department of Biochemistry, Rush University Medical Center, Chicago, Illinois 60612-3823.
authors.forename: Di
authors.initials: D
authors.lastname: Chen
authors.affiliation: the Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, and.
authors.forename: Benoit
authors.initials: B
authors.lastname: de Crombrugghe
authors.affiliation: From the Department of Stem Cell and Regenerative Biology, Konkuk University, Neungdong-Ro, Gwangjin-gu, Seoul 05029, Republic of Korea, jhkim541@konkuk.ac.kr.
authors.forename: Jin-Hoi
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D024043:Collagen Type II / Q000096:biosynthesis* / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007438:Introns / Q000502:physiology*; D051379:Mice; D051381:Rats; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D055755:SOXD Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology
chemical_list: C499177:COL2A1 protein, human; C511513:COL2A1 protein, rat; C447413:Col2a1 protein, mouse; D024043:Collagen Type II; C490301:SOX5 protein, human; C486270:SOX6 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D055755:SOXD Transcription Factors; C089186:Sox5 protein, mouse; C095939:Sox6 protein, mouse; C527933:Sox9 protein, mouse
keywords: ChIP-sequencing (ChIP-seq); SOX5/SOX6; SOX9; chondrogenesis; extracellular matrix protein; gene knockout; transcription enhancer; type II collagen