Investigations on the transcriptional regulation of chick aggrecan

Aggrecan is a large chondroitin sulfate proteoglycan whose expression is tissue-specific, developmentally-regulated and necessary for skeletal formation; alterations of message levels have been associated with arthritis and congenital chondrodystrophies. Cloning and sequencing of the 1.8 kilobase genomic 5' flanking sequence of the chick aggrecan gene reveal potential Sp1, NF-I, and polypyrimidine related sites, as well as several putative transcription factor binding sites, including the cartilage-associated silencers CSII1 and CSII2. Though lacking a classic TATA box, there are two instances in the 1.8 kb genomic fragment of TATA-like TCTAA sequences, and primer extension and S1 protection analyses reveal three major transcription start sites. 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 which are consistent with the sequence analysis and determined the minimal core sequence required to initiate RNA transcription. These findings identified a putative repressor region that spans 400 base pairs. DNase I footprinting experiments reveal that six regions of this genomic sequence bind to nuclear proteins in a tissue specific manner. To examine the functional significance of these protected sequences, mutated constructs of these protected sequences were transiently transfected into primary chondrocyte and fibroblast cultures, and four of the six protected sequences, which contained the sequence TCCTCC or TCCCCT, were shown to have repressor activities in transfected chick chondrocytes. Electromobility shift assays (EMSA) were conducted to examine the nature of DNA-protein interactions with the protected sequences with chick chondrocyte or fibroblast nuclear protein, in the context of normal development or with the addition of the exogenous agents, retinoic acid, ascorbic acid, glucose and hydrocortisone. Competition EMSA analysis reveal a novel consensus sequence, (Y)TCCCCT(A/C)RRC, that has the capacity to form two major protein-DNA complexes. HPLC analysis and affinity chromatography were used to purify a 21 kD protein identified by Southwestern analysis as one of the trans factors that binds to this novel consensus sequence. Mass spectral analysis and protein micro-sequencing reveal the putative identity of this trans factor as the yet-to-be cloned chick cold-inducible RNA-binding protein.