Sixth International Conference on the Molecular Biology and Pathology of Matrix
chromatin in the region of this polypyrimidine sequence, but when this promoter is inactive, the DNase I hypersensitive site is absent. This polypyrimidine sequence is also known to contain an in vitro $1 nuclease-sensitive site, suggesting that it can form non canonical DNA structures. We have recently reported that several DNA-binding proteins present in nuclear extracts from fibroblasts and chondrocytes interact with this single-stranded pyrimidine sequence. By screening a chick embryo fibroblast cDNA expression library using a singlestranded oligonucleotide corresponding to this sequence, we have isolated several specific clones. One of them, designated CT-3, has a sequence that is not similar to any known gene. This sequence has a single open reading frame of 1211 bp that encodes a 286 aa protein. No commonly occurring motifs were found in this aa sequence. The cDNA has a relatively short 3'-untranslated region with a consensus polyadenylation signal (AAUAAA). There are several phosphorylation sites for casein kinase I I and protein kinase C. The affinity-purified protein was found to bind specifically to the singlestranded pyrimidine sequence but not to a mutated sequence. Because of its high binding affinity and specificity for this promoter sequence, this protein could be involved in the transcriptional regulation of the (z,?.(I) collagen gene.
Cold-Shock Proteins Interact with the S l Nuclease-Sensitive Site in the Promoter o f the Chick (z2(I) Collagen Gene. D. Bayarsaihan and L. N. Lukens Department of Molecular Biology and Bio-chemistry, Wesleyan University, Middletown, CT The sequence of the chicken (x2(I) collagen promoter from -712 to -85, relative to exon 1, has been shown to be important for transcriptional activity. Within this region a pyrimidine/purine asymmetrical element forms an in vitro $1 nucleasesensitive site. The pyrimidine strand of this element interacts specifically with single-stranded DNA binding proteins present in fibroblast nuclear extracts. To identify these proteins we performed expression screening of a chick embryo fibroblast cDNA library using a single-stranded polypyrimidine sequence derived from this element. One of the isolated clones was found to encode a member of the cold shock gene family, either chicken YB-1 or a highly homologous protein. These proteins are members of the Y-box family of proteins, which share an extremely conserved DNA-binding region, known
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as the cold-shock domain, from its homology to bacterial cold-shock proteins. This protein, as well as a sample of the chick YB-1 protein, were found to bind sequence-specifically to this pyrimidine element in the (z2(I) collagen promoter. The binding mechanism of these proteins could be based on the formation of a non-canonical triplex DNA structure (HDNA). Although members of this widespread and conserved protein family have been reported to modulate the expression of a number of genes, the findings reported here provide the first evidence for a possible role of cold-shock proteins in the regulation of the type I collagen genes.
An AP-1 Binding Sequence is Essential for Regulation of the Human (x2(I) Collagen (COLIA2) Promoter Activity by Transforming Growth Factor-~. Alain Mauviel, Kee-Yang Chung, and Jouni Uitto Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, PA Previous studies have shown that transforming growth factor-~ (TGF-I]) and tumor necrosis factor-e~ (TNF-e0 exert opposite modulation of type I collagen gene expression in fibroblasts. To fine-map the correponding response elements in the human o.2(I) collagen (COL1A2) promoter, we have generated a series of 5' deletion promoter/chloramphenicol acetyl transferase (CAT) reporter gene constructs. Cell transfection assays using human dermal and NIH 3T3 fibroblasts identified the region located between residues-265 and -241, containing both an AP-1 and an overlapping NF-KB binding sites, as critical for both TGF-J] response and the antagonistic effect of TNF-0~. Gel mobility shift assays indicate that nuclear factor binding is competed by AP-1, but not by NF-1 or NF-~;B, oligonucleotides. Site-directed mutagenesis indicates that the AP-1, but not the NF~B binding site, plays a significant role in the basal activity of the promoter. Furthermore, this AP-1 sequence is essential for TGF-~ response, and does not require the presence of the three Sp-l-binding sites located further upstream, between nucleotides -273 and -304. In addition, over-expression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs blocks the TGF-J3 response, further implicating APol in the regulation of COL1A2 gene expression. However, cotransfection experiments with expression vectors for various NF-k-'B sub-units together with COL1A2 promoter/CAT constructs indicate that 1, basal transcriptional activity is enhanced by p50 but not by p65, 2, over-expression of p105 subunit results in