cis elements that control the expression of chick aggrecan

Aggrecan is a large chondroitin sulfate proteoglycan whose expression is both cell-specific and developmentally regulated. Cloning and sequencing of the 1.8-kilobase genomic 5'-flanking sequence of the chick aggrecan gene revealed the presence of potential tissue-specific control elements including a consensus sequence found in the cartilage-associated silencers, CSIIS1 and CSIIS2, that were first characterized in the type II collagen promoter sequences, as well as numerous other cis elements. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively deleted portions of the chick aggrecan promoter and enhancer region demonstrated cell type-specific promoter activity and identified a 420-base pair region in the genomic 5-flanking region responsible for negative regulation of the aggrecan gene. In this report, three complementary methods, DNase I footprinting assays, transient transfections, and electrophoretic mobility shift assays (EMSA), provided an integral approach to better understand the regulation of the aggrecan gene. DNase I footprinting revealed that six regions of this genomic sequence bind to nuclear proteins in a tissue-specific manner. Transient transfection of reporter constructs bearing ablations of these protected sequences showed that four of the six protected sequences, which contain the sequence TCCTCC or TCCCCT, had repressor activities in transfected chick chondrocytes. Cross-competition EMSA using nuclear protein extracted from chondrocytes or fibroblasts explored the contributions of the different sequence elements in formation of DNA-protein complexes specific to cell type. This is the first parallel examination of the EMSA patterns for six functionally defined cis elements with highly similar sequences, using protein from primary cultured cells.