MINIMAL DNA-SEQUENCES THAT CONTROL THE CELL LINEAGE-SPECIFIC EXPRESSION OF THE PRO-ALPHA-2(I) COLLAGEN PROMOTER IN TRANSGENIC MICE

The pattern of expression of the proalpha2(I) collagen gene is highly tissue specific in adult mice and shows its strongest expression in bones, tendons, and skin. Transgenic mice were generated harboring promoter fragments of the mouse proalpha2(I) collagen gene linked to the Escherichia coli beta-galactosidase or firefly luciferase genes to examine the activity of these promoters during development. A region of the mouse proalpha2(I) collagen promoter between -2,000 and +54 exhibited a pattern of beta-galactosidase activity during embryonic development that corresponded to the expression pattern of the endogenous proalpha2(I) collagen gene as determined by in situ hybridization. A similar pattern of activity was also observed with much smaller promoter fragments containing either 500 or 350 bp of upstream sequence relative to the start of transcription. Embryonic regions expressing high levels of beta-galactosidase activity included the bulbus arteriosus, valves of the developing heart, sclerotomes, meninges, limb buds, connective tissue fascia between muscle fibers, osteoblasts in newly formed bones, fibroblasts in tendons, periosteum, dermis, and peritoneal membranes. The pattern of beta-galactosidase activity was similar and included within the extracellular immunohistochemical localization pattern of transforming growth factor-beta1 (TGF-beta1). The -315--284 region of the proalpha2(I) collagen promoter was previously shown to mediate the stimulatory effects of TGF-beta1 on the proalpha2(I) collagen promoter in DNA transfection experiments with cultured fibroblasts. A construct containing this sequence tandemly repeated 5' to a very short alpha2(I) collagen promoter (-40-+54) showed preferential activity in tail and skin of 4-wk-old transgenic mice. Except for low expression of the transgene in bone, this pattern mimics the expression of the endogenous proalpha2(I) collagen gene. We propose the hypothesis that the tissue-specific expression of the proalpha2(I) collagen gene during embryogenesis is controlled by both TGF-beta1 and cell-specific transcription factors; one of these could interact directly or indirectly with either the -315--284 or the -40-+54 segment.