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首页> 外文期刊>Molecular and Cellular Biology >A Krüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene
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A Krüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

机译：Krüppel相关的框锌指蛋白NT2抑制α2（XI）胶原基因的细胞类型特异性启动子活性

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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.
机译：XI型胶原蛋白由三条链α1（XI），α2（XI）和α3（XI）组成，在软骨胶原纤维的形成和骨骼形态发生中起关键作用。以前有报道说，α2（XI）胶原基因（ Col11a2 ）的？530 bp启动子片段足以实现软骨特异性表达，并且该片段的24 bp序列能够将该序列置于异源神经丝轻基因（ NFL ）启动子中时，将启动子活性从转基因小鼠的神经组织转移到软骨。为了鉴定与 Col11a2 启动子的24 bp序列结合的蛋白质因子，我们在酵母单杂交筛选系统中筛选了小鼠肢芽cDNA表达文库，并获得了cDNA克隆NT2。序列分析显示，NT2是由Krüppel相关盒（KRAB）组成的锌指蛋白，是人FPM315的同源物，以前是通过随机克隆和测序分离的。尽管已经鉴定出含KRAB的锌指蛋白的靶基因很少，但已在许多锌指蛋白中发现了KRAB域，并暗示其为转录抑制域。在这里，我们证明NT2充当 Col11a2 的负调节剂。对发育中的小鼠软骨的原位杂交分析表明， NT2 mRNA在肥大的软骨细胞中高表达，而在静息和增殖的软骨细胞中最低表达，与 Col11a2的表达模式呈负相关。 em>。凝胶位移分析表明NT2结合了 Col11a2 启动子的24 bp位点内的特定序列。我们发现NT2表达载体在软骨肉瘤细胞系RSC细胞中的转染抑制了 Col11a2 启动子的活性。缺少KRAB结构域的突变体NT2的表达载体未能抑制 Col11a2 启动子活性。这些结果表明KRAB-锌指蛋白NT2抑制其生理靶基因的转录，表明 Col11a2 的软骨特异性表达的新调节机制。

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