MZF-1 and DbpA interact with DNase I hypersensitive sites that correlate with expression of the human MUC1 mucin gene

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Abstract

The MUC1 mucin is a large membrane-tethered glycoprotein that shows differential expression in many adenocarcinomas, where it contributes to their invasive and metastatic properties. We previously identified DNase I hypersensitive sites at −750 and −250 bp in the human MUC1 gene promoter and showed concordance between the −250 site and MUC1 mRNA levels in vivo. Transient expression assays using promoter constructs, in which the core DHS was deleted, to drive reporter gene expression revealed in vivo evidence for their activity. DNase I footprinting using nuclear extracts from HPAF human pancreatic carcinoma cells and MCF7 breast carcinoma cells identified three protein-binding elements in these regions (−250FP1, FP2 and −750FP). Electrophoretic mobility shift assays detected several complexes between HPAF nuclear proteins and labeled FP DNA probes. Southwestern blots and UV cross-linking experiments identified myeloid zinc finger-1 (MZF-1) as a candidate transcription factor among proteins binding to the −250FP1 and FP2 sequences. Another candidate that was identified by screening an HPAF cDNA expression library with the −250FP1 probe is DNA binding protein A (DbpA). Exogenous DbpA expression in COS-7 cells was accompanied by upregulation of MUC1 promoter activity via the −250 DHS, suggesting that DbpA binding to the −250 DHS can influence human MUC1 gene expression.

Introduction

The apical surfaces of mammalian epithelial cells carry many membrane-tethered, O-glycosylated proteins, which play an important role in epithelial protection and in signaling from the extracellular milieu to the inside of the cell [1]. The gene encoding one such glycoprotein, the human MUC1 mucin, was cloned as a tumor-associated cell membrane antigen. MUC1 has been shown to influence adhesion and anti-adhesion processes during invasion and metastasis [2] and to engage in intracellular signaling [3].

Human MUC1 is expressed in diverse epithelial tissues and is differentially expressed (mRNA and protein) in many adenocarcinomas, as compared to normal cell counterparts [1]. Hence, elucidation of mechanisms that regulate MUC1 expression is important to further our understanding of the biology of normal epithelia and its transformation to a malignant condition. Regulation of expression of the MUC1 mucin gene has been evaluated extensively in vitro, which led to the identification of several potentially important cis-elements and trans-acting factors [4], [5], [6], [7], [8], [9]. However, the regulatory mechanisms that control overexpression of MUC1 in tumors have not been established. Activation of Signal Transducer and Activator of Transcription (STATs) proteins has been implicated through a functional STAT3/1 element in the MUC1 promoter that is responsive to IL-6 and γ-interferon in reporter gene assays [10]. In addition, c-ErbB2 and ras signaling pathways regulate MUC1 expression in human mammary cell lines [11].

In contrast to the in vitro studies, there have been few reports on regions of the MUC1 gene that control basal or tissue-specific expression of MUC1 in vivo. We previously identified two DNase I hypersensitive sites (DHS) in the promoter region of MUC1, which may be important to gene expression in vivo [12], since DHS are often associated with regulatory elements. We have now characterized the cis-elements within the DHS at −750 and −250 bp in the human MUC1 gene promoter region and have identified MZF-1 and DbpA as nuclear proteins that bind to the DHS and may regulate MUC1 expression.

Section snippets

Cell culture

The following cell lines were used: HPAF [13], Caco-2 [14], MCF7 [15] and COS-7 were cultured in Dulbecco's modified Eagle's medium (DMEM).

Promoter-luciferase constructs for transient transfection assays

The pCATE0.9 construct, containing −790 bp (M61170:2081) to + 33 (M61170:2903) relative to the transcription start site of the human MUC1 gene [16], was used as the parental construct (named WT). Unique restriction enzyme sites in the MUC1 promoter region were used to prepare −750 and/or −250 DHS-deleted clones: Δ−750 lacks the region between BstEII and SacI

Transient promoter assays

In HPAF cells, which express high levels of MUC1 mRNA, deletion of the −250 DHS (pGL3B Δ−250) resulted in a marked decrease in luciferase activity in comparison to the wild type promoter (WT) (P ≤ 0.0148). Furthermore, the deletion of both DHS −750 and −250 (FP1 and FP2) (Δ−750/−250) had an additive effect in that luciferase activity was lower than in Δ−250 alone (P ≤  0.003) (Fig. 1) with respect to WT and Δ−250. In contrast, the −750DHS deletion (Δ−750) alone caused no significant effect on

Discussion

We previously identified DHS at −750 and −250 bp with respect to the transcriptional start site of the human MUC1 gene and showed that the intensity of the −250 DHS correlated with MUC1 mRNA levels in vivo [12]. All cell types evaluated showed the −750 DHS. Only cells expressing high levels of MUC1, such as HPAF and MCF-7, showed the −250 DHS, and those with low levels of MUC1 such as lymphoblastoid cells did not. In transgenic mice carrying a genomic copy of human MUC1, the −750 and −250 DHS

Acknowledgments

We thank Dr. Jennifer Morris (Medical College of Wisconsin, USA) for anti-human MZF-1 antiserum (α-ZF), Dr. Timothy J. Ley (Washington University, USA) for anti-mouse DbpA antiserum, Dr. Alexey Pshezhetsky (University of Montreal, Canada) for anti-human PPCA antiserum and T. Caffrey for assistance. This work was funded by NIH grant CA79580, Wellcome Trust Advanced Training Fellowship (Grant No. 047002 and 059897) (JPW) and a Wellcome Trust Biomedical collaboration grant.

References (42)

  • L.S. Coles et al.

    An ordered array of cold shock domain repressor elements across tumor necrosis factor-responsive elements of the granulocyte-macrophage colony-stimulating factor promoter

    J. Biol. Chem.

    (2000)
  • P. Diamond et al.

    Cold shock domain factors activate the granulocyte-macrophage colony-stimulating factor promoter in stimulated Jurkat T cells

    J. Biol. Chem.

    (2001)
  • M.A. Hollingsworth et al.

    Mucins in cancer: protection and control of the cell surface

    Nat. Rev., Cancer

    (2004)
  • K.M. McDermott et al.

    Overexpression of MUC1 reconfigures the binding properties of tumor cells

    Int. J. Cancer

    (2001)
  • M. Abe et al.

    Characterization of cis-acting elements regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene

    Proc. Natl. Acad. Sci. U. S. A.

    (1993)
  • T. Irimura et al.

    Soluble factor in normal tissues that stimulates high-molecular-weight sialoglycoprotein production by human colon carcinoma cells

    Cancer Res.

    (1990)
  • K. Shirotani et al.

    Purification of nuclear proteins that potentially regulate transcription of the MUC1 mucin gene induced by a soluble factor

    J. Biochem. Tokyo

    (1998)
  • I.J. Lee et al.

    Transcriptional regulation of the hamster Muc1 gene: identification of a putative negative regulatory element

    Am. J. Physiol.: Lung Cell. Mol. Physiol.

    (2003)
  • A.G. Scibetta et al.

    Regulation of MUC1 expression in human mammary cell lines by the c-ErbB2 and ras signaling pathways

    DNA Cell Biol.

    (2001)
  • T. Shiraga et al.

    Identification of two novel elements involved in human MUCI gene expression in vivo

    Mol. Med.

    (2002)
  • Y.W. Kim et al.

    Characterization of clones of a human pancreatic adenocarcinoma cell line representing different stages of differentiation

    Pancreas

    (1989)

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    Current address: Division of Tumor Biochemistry, Department of Biochemistry, Miyazaki Medical College, University of Miyazaki, 5200 Kiyotake, Miyazaki 889-1692, Japan.

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