Structural and Functional Analysis of the Chick Chondroitin Sulfate Proteoglycan (Aggrecan) Promoter and Enhancer Region

Abstract

Aggrecan is a large chondroitin sulfate proteoglycan, the expression of which is both tissue-specific and developmentally regulated. Here we report the cloning and sequencing of the 1.8-kilobase genomic 5' flanking sequence of the chick aggrecan gene and provide a functional and structural characterization of its promoter and enhancer region. Sequence analysis reveals potential Sp1, AP2, and NF-I related sites, as well as several putative transcription factor binding sites, including the cartilage-associated silencers CIIS1 and CIIS2. A number of these transcription factor binding motifs are embedded in a sequence flanked by prominent inverted repeats. Although lacking a classic TATA box, there are two instances in the 1.8-kb genomic fragment of TATA-like TCTAA sequences, as have been defined previously in other promoter regions. Primer extension and S1 protection analyses reveal three major transcription start sites, also located between the inverted repeats. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively reduced portions of the aggrecan promoter region allowed mapping of chondrocyte-specific transcription enhancer and silencer elements that are consistent with the sequence analysis. These findings suggest the importance of this regulatory region in the tissue-specific expression of the chick aggrecan gene.

Transcriptional Regulation of the Endogenous Danger Signal Tenascin-C: A Novel Autocrine Loop in Inflammation

“…The activity of this basal promoter can be both positively and negatively modulated by noncoding sequences; the untranslated first exon contains two discrete regions that repress or enhance transcription of the reporter in human glioblastoma cells lines (44). In addition, as with many ECM genes, including collagen type I (45) and aggrecan (46), cell-specific transcription of tenascin-C is directed by the cooperation of intronic enhancer elements with the basal promoter; the first intron contributes to cell-type specific inducible expression in human melanoma cell lines (39). Our inspection of the tenascin-C genomic locus revealed that this gene spans a large area of chromosome 9, although the coding region made up a relatively low proportion of this area.…”

Cartilage proteoglycans

“…As yet there has been little examination of the transcriptional regulation of the aggrecan gene in either its tissue-specific distribution or its temporal expression during chondrogenesis, although numerous potentially active cis elements have been found in the 1.8 kb 5 flanking region. 26 SOX9 is a transactivating factor that stimulates the expression and maintenance of the chondrocyte phenotype. Recently it was reported that SOX9 enhanced the aggrecan gene promoter activity through a Sry/Sox consensus sequence.…”

APBP-1, a DNA/RNA-binding Protein, Interacts with the Chick Aggrecan Regulatory Region

“…Transient Transfection-Conditions of transfections conducted for luciferase and Northern blot experiments were as described by Pirok et al (1,3) using the calcium phosphate method. Triplicate plates containing ϳ5 ϫ 10 6 cells (day 12 chondrocytes)/100-mm tissue culture dishes (Falcon) received between 10 and 20 pmol of a given plasmid construct to be assayed.…”

“…We mapped a 400-bp region that actively mediates repression of aggrecan expression, removal of which increases aggrecan promoter activity by nearly 3-fold (3). Three DNase I-protected footprint sequences that mediate repressor activity in the context of this 400-bp sequence have been identified and characterized (1).…”