Abstract
The regulation of Col2a1, which encodes type II collagen, likely results from a balance of both positive and negative proteins. Here we present evidence that the transcription factor deltaEF1 participates in the negative regulation of Col2a1 transcription. A deletion analysis suggested that a region between -100 and -307 of the rat Col2a1 gene was required for activity in differentiating chick limb bud mesenchymal cells; however, mutation of a conserved E2 box site in this region actually increased promoter activity. Supershift analysis demonstrated that deltaEF1, a known transcriptional repressor, bound to the E2 box in a sequence-dependent manner. Chick limb bud mesenchymal cells, which do not express type II collagen, expressed abundant deltaEF1 mRNA, but, following differentiation in micromass culture, deltaEF1 mRNA expression was lost. Primary embryonic chick sternal chondrocytes, which express abundant type II collagen, displayed minimal levels of deltaEF1 mRNA. The inhibition of Col2a1 transcription following treatment of chick sternal chondrocytes with growth factors was accompanied by increased deltaEF1 expression. Overexpression of deltaEF1 in differentiated chondrocytes resulted in decreased expression of a reporter construct containing a collagen II promoter/enhancer insert; however, this negative regulation was not dependent on the proximal E2 box. This is the first report of a specific transcription factor involved in the negative regulation of Col2a1.
Highlights
The regulation of Col2a1, which encodes type II collagen, likely results from a balance of both positive and negative proteins
A construct containing only 100 bp of 5Ј-flanking sequence averaged only 35% of the activity observed with the 307-bp construct (Fig. 1B). These results suggested that the information contained in the 307-bp Col2a1 promoter construct was sufficient to direct enhancer-mediated transcriptional activity in differentiating chick limb bud (CLB) mesenchymal cells and that specific sequences between position Ϫ100 and Ϫ307 were required for enhancer-mediated promoter activity in these cells
Collective evidence shows that the control of type II collagen expression in chondrocytes involves transcription factors that operate through cis-acting elements found in both the promoter and first intron enhancer [10, 11, 13,14,15,16,17, 19, 34]
Summary
Vol 275, No 5, Issue of February 4, pp. 3610 –3618, 2000 Printed in U.S.A. The Transcription Factor ␦EF1 Is Inversely Expressed with Type II Collagen mRNA and Can Repress Col2a1 Promoter Activity in Transfected Chondrocytes*. A recent study determined that ␦EF1 was expressed at a high level in the early limb mesenchyme and that its expression was lost in these cells after condensation and the initiation of chondrogenesis [27] This pattern of expression suggests that ␦EF1 is involved in skeletal patterning during limb development and may function as a negative regulator of chondrocyte-specific genes. We present evidence that ␦EF1 binds to a CACCT sequence contained within an E2 box motif that is conserved in position and sequence in the proximal promoter region of the Col2a1 gene across several species Mutation of this sequence increased transcriptional activity of the rat Col2a1 promoter in differentiating chick limb bud mesenchymal cells. ␦EF1 expression inhibited the activity of a co-transfected Col2a1 reporter construct, the conserved proximal E2 box was not required to mediate this suppression These results suggest that ␦EF1 either directly or indirectly participates in the negative regulation of Col2a1 transcription. This is the first report to identify a specific transcription factor involved in the negative regulation of type II collagen gene expression
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