cis elements that control the expression of chick aggrecan

Aggrecan is a large chondroitin sulfate proteoglycan whose expression is bo th cell-specific and developmentally regulated. Cloning and sequencing of t he 1.8-kilo-base genomic 5'-flanking sequence of the chick aggrecan gene re vealed the presence of potential tissue-specific control elements including a consensus sequence found in the cartilage-associated silencers, CSIIS1 a nd CSIIS2, that were first characterized in the type II collagen promoter s equences, as well as numerous other cis elements. Transient transfections o f 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 Fi-flanking region responsible for nega tive regulation of the aggrecan gene. In this report, three complementary m ethods, DNase I footprinting assays, transient transfections, and electroph oretic mobility shift assays (EMSA), provided an integral approach to bette r understand the regulation of the aggrecan gene. DNase I footprinting reve aled that six regions of this genomic sequence bind to nuclear proteins in a tissue-specific manner. Transient transfection of reporter constructs bea ring ablations of these protected sequences showed that four of the six pro tected sequences, which contain the sequence TCCTCC or TCCCCT, had represso r activities in transfected chick chondrocytes, Cross-competition EMSA usin g nuclear protein extracted from chondrocytes or fibroblasts explored the c ontributions 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 si milar sequences, using protein from primary cultured cells.