Elsevier

Matrix Biology

Volume 20, Issue 7, November 2001, Pages 451-462
Matrix Biology

The hcKrox gene family regulates multiple extracellular matrix genes

https://doi.org/10.1016/S0945-053X(01)00167-6Get rights and content

Abstract

The transcription factor cKrox was originally identified as a protein that bound to a negative transcription regulatory element in the murine α1(I) collagen promoter. We recently reported the cloning and characterization of human cKrox (hcKrox). Overexpression of hcKrox in NIH3T3 fibroblasts efficiently repressed the promoters of the fibronectin and α1(I) collagen genes (70–90%) in transient transfection assays and suppressed the endogenous genes in hcKrox expressing permanent cell lines. We have now isolated genomic clones and cDNAs encoding two novel transcription factors related to hcKrox termed hcKrox-β and hcKrox–γ (the original clone is now referred to as hcKrox-α). Both contain three kruppel-like zinc-finger DNA binding motifs that are 71–78% identical to those of hcKrox-α. The NH2-terminus 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 expressed in foreskin and dermal fibroblasts. Transient transfection studies in NIH3T3 fibroblasts demonstrate that hcKrox-α -β and -γ, as well as the murine cKrox-β homologue, LRF, suppress transcription driven by promoters for the α1(I) and α2(I) collagen, fibronectin and elastin genes. Electrophoretic mobility shift assays and coimmunoprecipitation studies suggest that homo- and heterodimerization occurs between cKrox family members. Dimer formation is influenced by amino acids in the NH2-terminal POZ domain and the Zn+2-finger region. Immunoprecipitation studies indicate that cKrox can form heterodimers in solution in the absence of DNA. Thus, a multi-gene family exists that can coordinately regulate several extracellular matrix genes and has the potential to form many heterodimeric transcription factors.

Introduction

Extracellular matrix genes are regulated by a combination of positive and negative trans-acting transcription factors which act together to achieve appropriate levels of expression. The transcription factor cKrox was identified due to its binding to a GC-rich negative control element in the murine α1(I) collagen promoter. Multiple binding sites for cKrox have been identified in the murine α1 and α2 type I collagen gene promoters (Galera et al., 1996) and in the human α1(I) and α2(I) collagen promoters (Ghayor et al., 2000, Widom et al., 1997). Many binding sites for positive regulatory factors in these promoters have also been described, including Sp-1, NF-1, and CBF (Jimenez et al., 1994, Li et al., 1995, Maity et al., 1988, Nehls et al., 1993, Nehls et al., 1991). It is thus likely that both positive and negative trans-acting factors play important roles in regulating collagen and other matrix genes. Although extracellular matrix genes are often considered ‘housekeeping’ genes requiring little regulation, upregulation is seen in normal wound repair and the consequences of sustained overexpression can be severe, as seen in many fibrotic diseases.

In the fibrotic disease, scleroderma, fibroblasts cultured from lesional skin of patients overexpress the α1(I), α2(I) and α1(III) collagen genes, as well as the fibronectin gene when compared to fibroblasts derived from normal skin (Jimenez et al., 1986, Varga et al., 1990). This phenomenon suggests that these different matrix genes can be coordinately regulated. This could be achieved by a mechanism using common positive and negative transcription factors that bind to the promoters of all of these genes. The promoter DNA of matrix genes tends to be GC-rich, making it likely that binding sites for factors like Sp-1 and cKrox are present.

Many transcription factors exist as members of closely related families of proteins which share a high degree of homology, especially in their DNA binding domains. Such factors often bind to identical or very similar DNA sequences, yet may have opposite transcriptional effects, such as Sp-1 and Sp-3 (Hagen et al., 1994, Majello et al., 1994). Other factors can form heterodimers with family members to give new DNA binding properties and activation potential, such as nuclear steroid receptors (Ijpenberg et al., 1997, Schulman et al., 1998, Thompson et al., 1998). The existence of families of related factors expands the ability of a cell to precisely regulate gene expression and in some cases it also provides a redundancy of function which could be important if a particular factor is mutated or otherwise inactivated.

In the current study, we have identified two novel human transcription factors related to hcKrox termed hcKrox-β and hcKrox–γ (the original gene is now referred to as hcKrox-α). All contain three kruppel-like zinc-finger DNA binding motifs that are 71–83% identical, while the NH2-terminus of all three proteins contains a POZ1 domain. Messenger RNA for all three hcKrox family members can be detected in both foreskin and adult dermal fibroblasts. hcKrox family members mediate transcriptional suppression of the promoters for the α1(I) collagen, α2(I) collagen, fibronectin and elastin genes. Homo- and heterodimerization between family members occurs, the formation of which is influenced by amino acids in the Zn+2-finger region and the NH2-terminal POZ domain. The POZ domain also plays a role in DNA binding affinity and transcriptional repression.

Section snippets

hcKrox is a member of a multigene family

Several families of Zn+2-finger containing transcription factors exist which share very similar DNA binding domains, yet diverge in other parts of the protein; both the EGR family and the nuclear receptors display this property. In order to determine whether hcKrox belongs to such a family, cDNA and genomic libraries were screened at reduced stringency using the hcKrox Zn+2-finger region as a probe. Several cDNA clones from fibroblast and placenta were isolated and sequenced, revealing two

Discussion

We have now identified a multigene family of transcription factors that can suppress the activity of multiple extracellular matrix gene promoters. The cKrox family members are highly homologous (>70% identity) in their NH2-terminal POZ domain and Zn+2-finger DNA binding domains, yet are <60% similar in other regions of the proteins. The degree of homology in the DNA binding domains suggests that these proteins will bind to similar recognition sequences. However, only hcKrox-α shows strong

Library screening, DNA purification and sequencing

A human placenta cDNA library (#HL1008b, Clontech, Palo Alto, CA) and human genomic library (Clontech # HL1006d) were screened using standard methods (Sambrook et al., 1989). Approximately 5×105 and 1×106 clones were screened for the placenta library and for the genomic library, respectively. The probe was a 310bp fragment of hcKrox spanning the Zn+2-finger DNA binding domain (nt 1209–1518) (Widom et al., 1997). Hybridization at reduced stringency was carried out for 12 h at 37°C in 33%

Acknowledgements

The authors wish to thank Dr Maria Trojanowska for the gift of the α2(1) promoter constructs, Dr Judith Foster for the elastin promoter constructs, Dr E. Schwarz for APM-1 and Dr S. Bourgeois for the human fibronectin promoter DNA. We are indebted to Dr Arthur Zelent for providing the LRF and LRFΔPOZ expression vectors and for sharing unpublished data. This work was supported by grants from the Arthritis Foundation (RLW), the Scleroderma Foundation (RLW), the Scleroderma Research Fund of Boston

References (29)

  • R.L. Widom et al.

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

    Gene

    (1997)
  • B.L. Wolfe et al.

    Insulin-like growth factor-I regulates transcription of the elastin gene

    J. Biol. Chem.

    (1993)
  • V.J. Bardwell et al.

    The POZ domain: A conserved protein-protein interaction motif

    Genes. Dev.

    (1994)
  • J.M. Davies et al.

    Novel BTB/POZ domain zinc-finger protein, LRF, is a potential target of the LAZ-3/BCL-6 oncogene

    Oncogene

    (1999)
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