• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Aug 1996; 16(8): 4512–4523.
PMCID: PMC231450

An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

Abstract

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.

Full Text

The Full Text of this article is available as a PDF (670K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Cancedda R, Descalzi Cancedda F, Castagnola P. Chondrocyte differentiation. Int Rev Cytol. 1995;159:265–358. [PubMed]
  • Cheah KS, Lau ET, Au PK, Tam PP. Expression of the mouse alpha 1(II) collagen gene is not restricted to cartilage during development. Development. 1991 Apr;111(4):945–953. [PubMed]
  • Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. [PMC free article] [PubMed]
  • Dyer RB, Herzog NK. Isolation of intact nuclei for nuclear extract preparation from a fragile B-lymphocyte cell line. Biotechniques. 1995 Aug;19(2):192–195. [PubMed]
  • Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 1991 Sep;5(9):1513–1523. [PubMed]
  • Garofalo S, Vuorio E, Metsaranta M, Rosati R, Toman D, Vaughan J, Lozano G, Mayne R, Ellard J, Horton W, et al. Reduced amounts of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycine-to-cysteine mutation in the mouse type II procollagen alpha 1-chain gene. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9648–9652. [PMC free article] [PubMed]
  • Goldberg H, Helaakoski T, Garrett LA, Karsenty G, Pellegrino A, Lozano G, Maity S, de Crombrugghe B. Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells. J Biol Chem. 1992 Sep 25;267(27):19622–19630. [PubMed]
  • Herr W, Cleary MA. The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes Dev. 1995 Jul 15;9(14):1679–1693. [PubMed]
  • Hinkley C, Perry M. Histone H2B gene transcription during Xenopus early development requires functional cooperation between proteins bound to the CCAAT and octamer motifs. Mol Cell Biol. 1992 Oct;12(10):4400–4411. [PMC free article] [PubMed]
  • Horton W, Miyashita T, Kohno K, Hassell JR, Yamada Y. Identification of a phenotype-specific enhancer in the first intron of the rat collagen II gene. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8864–8868. [PMC free article] [PubMed]
  • Kadonaga JT, Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. [PMC free article] [PubMed]
  • Krebsbach PH, Nakata K, Bernier SM, Hatano O, Miyashita T, Rhodes CS, Yamada Y. Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene. J Biol Chem. 1996 Feb 23;271(8):4298–4303. [PubMed]
  • Lefebvre V, Garofalo S, Zhou G, Metsäranta M, Vuorio E, De Crombrugghe B. Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice. Matrix Biol. 1994 Aug;14(4):329–335. [PubMed]
  • Metsäranta M, Garofalo S, Smith C, Niederreither K, de Crombrugghe B, Vuorio E. Developmental expression of a type II collagen/beta-galactosidase fusion gene in transgenic mice. Dev Dyn. 1995 Oct;204(2):202–210. [PubMed]
  • Morrissey JH. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. [PubMed]
  • Mukhopadhyay K, Lefebvre V, Zhou G, Garofalo S, Kimura JH, de Crombrugghe B. Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene. J Biol Chem. 1995 Nov 17;270(46):27711–27719. [PubMed]
  • Rossert J, Eberspaecher H, de Crombrugghe B. Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice. J Cell Biol. 1995 Jun;129(5):1421–1432. [PMC free article] [PubMed]
  • Ryan MC, Sieraski M, Sandell LJ. The human type II procollagen gene: identification of an additional protein-coding domain and location of potential regulatory sequences in the promoter and first intron. Genomics. 1990 Sep;8(1):41–48. [PubMed]
  • Savagner P, Krebsbach PH, Hatano O, Miyashita T, Liebman J, Yamada Y. Collagen II promoter and enhancer interact synergistically through Sp1 and distinct nuclear factors. DNA Cell Biol. 1995 Jun;14(6):501–510. [PubMed]
  • Savagner P, Miyashita T, Yamada Y. Two silencers regulate the tissue-specific expression of the collagen II gene. J Biol Chem. 1990 Apr 25;265(12):6669–6674. [PubMed]
  • Schöler HR, Hatzopoulos AK, Balling R, Suzuki N, Gruss P. A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor. EMBO J. 1989 Sep;8(9):2543–2550. [PMC free article] [PubMed]
  • Vikkula M, Metsäranta M, Ala-Kokko L. Type II collagen mutations in rare and common cartilage diseases. Ann Med. 1994 Apr;26(2):107–114. [PubMed]
  • Wang LQ, Balakir R, Horton WE., Jr Identification of a cis-acting sequence in the collagen II enhancer required for chondrocyte expression and the binding of a chondrocyte nuclear factor. J Biol Chem. 1991 Oct 25;266(30):19878–19881. [PubMed]
  • Yamada Y, Miyashita T, Savagner P, Horton W, Brown KS, Abramczuk J, Xie HX, Kohno K, Bolander M, Bruggeman L. Regulation of the collagen II gene in vitro and in transgenic mice. Ann N Y Acad Sci. 1990;580:81–87. [PubMed]
  • Zhou G, Garofalo S, Mukhopadhyay K, Lefebvre V, Smith CN, Eberspaecher H, de Crombrugghe B. A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice. J Cell Sci. 1995 Dec;108(Pt 12):3677–3684. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...