The hcKrox gene family regulates multiple extracellular matrix genes

The transcription factor cKrox was originally identified as a protein that bound to a negative transcription regulatory element in the murine alpha1(I ) collagen promoter. We recently reported the cloning and characterization of human cKrox (hcKrox). Overexpression of hcKrox in NIH3T3 fibroblasts eff iciently repressed the promoters of the fibronectin and alpha1(I) collagen genes (70-90%) in transient transfection assays and suppressed the endogeno us, genes in hcKrox expressing permanent cell lines. We have now isolated g enomic clones and cDNAs encoding two novel transcription factors related to hcKrox termed hcKrox-beta and hcKrox-gamma (the original clone is now refe rred to as hcKrox-alpha). Both contain three kruppel-like zinc-finger DNA b inding motifs that are 71-78% identical to those of hcKrox-alpha. The NH2-t erminus of all three proteins contains a POZ domain, a conserved 120 amino acid motif involved in transcriptional repression and protein dimerization. RT-PCR experiments demonstrate that all three hcKrox family members are ex pressed in foreskin and dermal fibroblasts. Transient transfection studies in NIH3T3 fibroblasts demonstrate that hcKrox-alpha -beta and -gamma, as we ll as the murine cKrox-beta homologue, LRF, suppress transcription driven b y promoters for the alpha1(I) and alpha2(I) collagen, fibronectin and elast in genes. Electrophoretic mobility shift assays and coimmunoprecipitation s tudies suggest that homo- and heterodimerization occurs between cKrox famil y members. Dimer formation is influenced by amino acids in the NH2-terminal POZ domain and the Zn+2-finger region. Immunoprecipitation studies indicat e that cKrox can form heterodimers in solution in the absence of DNA. Thus, a multi-gene family exists that can coordinately regulate several extracel lular matrix genes and has the potential to form many heterodimeric transcr iption factors. (C) 2001 Elsevier Science B.V./International Society of Mat rix Biology. All rights reserved.