Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells

doi:10.1083/jcb.116.1.227

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Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells

D Pavlin, AC Lichtler, A Bedalov, BE Kream, JR Harrison, HF Thomas, GA Gronowicz, SH Clark, CO Woody and DW Rowe
Department of Pediatrics, University of Connecticut Health Center, Farmington 06032.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30- fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.
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