IDENTIFICATION OF NEGATIVE AND POSITIVE REGULATORY ELEMENTS IN THE RAT ALPHA-1(I) COLLAGEN GENE PROMOTER

Type I collagen is the main constituent of extracellular matrix found in various organs including the heart. Under some pathological conditi ons accumulation of excess type I collagen in the interstitium leads t o organ dysfunction. In order to identify the regulatory elements in t he rat alpha 1(I) collagen gene promoter, deletions were made in the p romoter region. Various plasmid constructs were transfected into diffe rent fibroblasts using LipofectAMINE. The results indicated a negative cis-element between nucleotides -310 to -440 in the rat alpha 1(I) co llagen gene promoter. Presence of this sequence significantly diminish ed the reporter gene activity. In addition we have observed that the s equence between -220 to -330 contained a positively acting cis-element , which is highly active in rat fibroblasts. Analysis of the nuclear f actors binding to the negative element by electrophoretic mobility shi ft assays indicated that similar or identical factors are present in d ifferent fibroblasts as well as human HeLa cells and that these factor s appear to bind to a composite sequence within -325 to -400. Competit ion with different oligonucleotides suggested that two distinct but co ntiguous sequence motifs may constitute the negative regulatory elemen t. Our results with the rat alpha 1(I) collagen promoter confirm the p resence of a negative cis-element previously described for the mouse p romoter and provided additional information on the bipartite nature of this element. (C) 1997 Elsevier Science Ltd.