STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE HUMAN DECORIN GENE PROMOTER - A HOMOPURINE-HOMOPYRIMIDINE S1 NUCLEASE-SENSITIVE REGION ISINVOLVED IN TRANSCRIPTIONAL CONTROL

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan w hich binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternativel y spliced leader exons, designated exon Ia and Ib, in the 5'-untransla ted region. Initial analysis of the sequences upstream to these two ex ons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the tran scription start site. To determine if these 5'-flanking sequences exhi bited promoter activity, chimeric chloramphenicol acetyltransferase ex pression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The result s showed that only the region flanking exon Ib was functional. In vitr o transcription assay generated two transcripts of 92 and 82 base pair s (bp) indicating that both TATA boxes could be used. Using stepwise 5 ' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT an d the two TATA boxes, exhibited strong promoter activity. When a large r construct containing an additional 800 bp of upstream region was tes ted, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappaB) and for transf orming growth factor-beta and a 150-bp homopurine/homopyrimidine eleme nt with several mirror repeats. When contained in a supercoiled plasmi d, this sequence exhibited sensitivity to endonuclease S1, an enzyme t hat preferentially digests single-stranded DNA. Precise S1 mapping, ob tained by direct sequencing of nine distinct Sl-generated clones, reve aled that in all cases the borders of the sensitive sequence resided w ithin the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and m ay play a role in the chromatin organization at the decorin gene locus . In addition, this region was able to up-regulate a minimal heterolog ous promoter in transient transfection assays. The results show that t he structure of the decorin gene promoter is different from that of an y other proteoglycan promoter characterized so far and indicate that t he pur/pyr segment plays a role in the regulation of gene transcriptio n.