A Krüppel-associated box-zinc finger protein, NT2, represses cell-type-specific promoter activity of the α2(XI) collagen gene

Kazuhiro Tanaka, Noriyuki Tsumaki, Christine A. Kozak, Yoshihiro Matsumoto, Fumihiko Nakatani, Yukihide Iwamoto, Yoshihiko Yamada

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Type XI collagen is composed of three chains, α1(XI), α2(XI), and α3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the α2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB- zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

Original languageEnglish
Pages (from-to)4256-4267
Number of pages12
JournalMolecular and cellular biology
Volume22
Issue number12
DOIs
Publication statusPublished - Jun 5 2002

Fingerprint

Zinc Fingers
Collagen
Cartilage
Genes
Chondrocytes
Proteins
Collagen Type XI
Limb Buds
Chondrosarcoma
Intermediate Filaments
Gene Library
Morphogenesis
Transgenic Mice
In Situ Hybridization
Transfection
Sequence Analysis
Organism Cloning
Complementary DNA
Clone Cells
Yeasts

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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A Krüppel-associated box-zinc finger protein, NT2, represses cell-type-specific promoter activity of the α2(XI) collagen gene. / Tanaka, Kazuhiro; Tsumaki, Noriyuki; Kozak, Christine A.; Matsumoto, Yoshihiro; Nakatani, Fumihiko; Iwamoto, Yukihide; Yamada, Yoshihiko.

In: Molecular and cellular biology, Vol. 22, No. 12, 05.06.2002, p. 4256-4267.

Research output: Contribution to journalArticle

Tanaka, Kazuhiro ; Tsumaki, Noriyuki ; Kozak, Christine A. ; Matsumoto, Yoshihiro ; Nakatani, Fumihiko ; Iwamoto, Yukihide ; Yamada, Yoshihiko. / A Krüppel-associated box-zinc finger protein, NT2, represses cell-type-specific promoter activity of the α2(XI) collagen gene. In: Molecular and cellular biology. 2002 ; Vol. 22, No. 12. pp. 4256-4267.
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abstract = "Type XI collagen is composed of three chains, α1(XI), α2(XI), and α3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the α2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Kr{\"u}ppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB- zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.",
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AU - Tanaka, Kazuhiro

AU - Tsumaki, Noriyuki

AU - Kozak, Christine A.

AU - Matsumoto, Yoshihiro

AU - Nakatani, Fumihiko

AU - Iwamoto, Yukihide

AU - Yamada, Yoshihiko

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