Cloning and characterization of hcKrox, a transcriptional regulator of extracellular matrix gene expression

Abstract

cKrox is a novel zinc finger-containing transcription factor that binds to the α1(I) and α2(I) collagen gene promoters. The gene coding for cKrox is a new member of a family of early growth response genes, that play important roles in development. In the mouse, cKrox is expressed, beginning at 9.5 days of gestation and at 10.5 days in regions destined to become skin. In adult animals, expression is predominantly in skin, one of the two major organs where type I collagen is expressed. We have isolated cDNA clones for human cKrox. Theoretical translation of the nucleic acid sequence reveals 90% conservation of amino acids between the mouse and human proteins; however, the human gene product contains a 117-amino-acid N-terminal extension. The amino acid sequences of the zinc-finger DNA binding domains of mouse and human cKrox are identical. RT-PCR analysis of human fibroblasts indicates constitutive low-level expression of cKrox which can be transiently elevated by treatment with retinoic acid. Transient transfection assays indicate that hcKrox represses transcription of the α1(I) procollagen promoter, and electrophoretic mobility shift assays demonstrate that hcKrox binds to both the human and murine promoter DNA. Deletion derivatives of hcKrox demonstrate transcription-activating potential that is promoter-dependent. NIH3T3 cells permanently expressing hcKrox demonstrate a threefold and 10-fold decrease in α1(I) procollagen and fibronectin mRNA levels, respectively, compared to control cells. Consistent with this finding, a fibronectin promoter reporter construct is repressed more than 80% by hcKrox. These data suggest that hcKrox represses collagen transcription directly, and it may function as a repressor of fibronectin and possibly other matrix genes.