Elsevier

Gene

Volume 258, Issues 1–2, 27 November 2000, Pages 63-69
Gene

Modulation of early growth response (EGR) transcription factor-dependent gene expression by using recombinant adenovirus

https://doi.org/10.1016/S0378-1119(00)00445-5Get rights and content

Abstract

Early growth response (EGR) transcription factors link initial cytoplasmic events to long-term alterations of cellular gene expression and are induced by various stimuli. To test their roles in cell physiology, we constructed adenoviral recombinants encoding NGFI-A binding protein 2 (NAB2, a repressor of EGR1, EGR2, and EGR3), EGR1, NAB-insensitive EGR1(I293F) (EGR1), EGR2, and the NAB-binding, repressive domain 1 (R1) of EGR1. These viruses regulated EGR-dependent expression of GFP and luciferase reporter genes in heterologous expression assays. Infection of a myoblast cell line with EGR1 and EGR1 adenovirus induced the endogenous gene for platelet-derived growth factor A chain (PDGF-A). In addition, in neuroblastoma cells, the two novel EGR1 target genes EGR3 and NAB2 were identified by using adenoviral transfer of EGR1 and EGR1. Our results demonstrate that recombinant adenovirus is useful to regulate heterologous and endogenous EGR target gene expression, and suggest that EGR transcription factors can autoregulate themselves.

Introduction

Early growth response (EGR) transcription factors are cellular immediate early genes that become rapidly induced in response to stimulation of cells, e.g. by growth factors. They play a key role in coordinating subsequent waves of gene expression that underlie the long-term changes in a variety of cell biological processes, including proliferation, apoptosis, and differentiation. The EGR family is composed of EGR1 (also termed NGFI-A, Krox-24, Zif268, and TIS8), EGR2 (Krox-20), EGR3 (PILOT), and EGR4 (NGFI-C). All EGR proteins contain a DNA-binding domain of three zinc fingers that recognize a GC-rich 9 bp sequence (the ‘GSG motif’) in the promoter region of EGR target genes, and transactivation by all EGR members through the GSG motif has been shown (Beckmann and Wilce, 1998, O'Donovan et al., 1999).

The activity of EGR1, EGR2, and EGR3 is selectively inhibited by NAB1 (Russo et al., 1995) and NAB2/Mader (Kirsch et al., 1996, Svaren et al., 1996), nuclear corepressor proteins that bind to the regulatory R1 domain found in EGR1, EGR2, and EGR3, but not EGR4 (O'Donovan et al., 1999). NAB1 is constitutively expressed in many tissues (Russo et al., 1995), whereas NAB2 is mostly expressed in brain and thymus and induced by the same stimuli that upregulate EGR1 (Svaren et al., 1996). Comparison of the expression patterns suggests that NAB2 rather than NAB1 is more closely linked to EGR1 function. After induction, NAB2 expression lags behind that of EGR1; thus, NAB2 may constitute a negative feedback signal to inhibit EGR1 activity (Svaren et al., 1996).

In order to study the roles of EGR transcription factors in cellular function, we propose to use recombinant adenovirus to either overexpress or suppress the activity of EGR family members. Adenovirus contains double-stranded DNA as a genome and infects a wide range of cell types, usually by initial attachment to the coxsackievirus and adenovirus receptor (Bergelson et al., 1997) and to αv integrins (Nemerow et al., 1994). Upon infection, the DNA is transported to the nucleus, where it exists as a linear extrachromosomal molecule. Adenoviral vectors lack the viral E1A and E1B genes that normally deregulate cell cycle control, induce apoptosis, and inhibit host gene expression (Shenk, 1996). Recombinant, E1-deficient adenovirus does not prevent host gene transcription and translation and, therefore, has been successfully used for functional tests of certain transcription factors (e.g., Wang et al., 1999). We show here that adenoviral vectors are valuable to modulate EGR-dependent expression of heterologous reporter genes and of the endogenous PDGF-A gene in a myoblast cell line. In addition, by using adenoviral gene transfer, we determined EGR3 and NAB2 as previously unknown EGR1 target genes in neuroblastoma cells.

Section snippets

Adenoviral recombinants and reporter constructs

As described previously (Ehrengruber et al., 1998), NAB-insensitive EGR1 (Russo et al., 1993), NAB2 (Svaren et al., 1996), and the R1 domain (aa 269–304) of EGR1 (Russo et al., 1993) fused to aa 1–147 of the Gal4 DNA-binding domain were inserted into the E1 region of adenovirus Ad5PacIGFP (Qu et al., 1998) to obtain AdGFP EGR1, AdGFP NAB2, and AdGFP R1 respectively. In addition, wild-type EGR1 (Milbrandt, 1987), EGR1, and EGR2 (Chavrier et al., 1988) were inserted into the E1 regions of

Overexpression of EGR1 and EGR2

As a tool to suppress and overexpress EGR1-type activity, we constructed adenoviral recombinants carrying the genes for the following proteins in the viral E1 region: (i) NAB2 (a corepressor of EGR1, EGR2, and EGR3); (ii) EGR1; (iii) NAB-insensitive EGR1; (iv) EGR2; (v) the repressive R1 domain of EGR1; (vi) R1, which does not bind NAB proteins (Fig. 1). Transcriptional regulation by these viruses was tested in CHO cells transiently transfected with GFP and luciferase reporter plasmids. Both

Acknowledgements

We thank Dr Jeffrey Milbrandt and Dr John Svaren for providing A2ProLuc and the cDNAs for EGR1, EGR1, EGR2, NAB2, R1, and R1, Dr Barbara J. Wold and Dr Jeong Kyo Yoon for the sGFP cDNA, Anita Buchli for the Neuro-2a cells, Susanne Erb for the C2C12 cells, Dr Hanns Möhler and Laura Huopaniemi for supporting the TaqMan PCR, and Dr John Svaren and Dr Sondra Schlesinger for helpful discussions and comments on the manuscript. This work was supported by the Swiss National Science Foundation (grant

References (28)

  • M.U. Ehrengruber et al.

    Activation of heteromeric G protein-gated inward rectifier K⊞ channels overexpressed by adenovirus gene transfer inhibits the excitability of hippocampal neurons

    Proc. Natl. Acad. Sci

    (1997)
  • R. Heim et al.

    Improved green fluorescence

    Nature

    (1996)
  • K.H. Kirsch et al.

    Mader: a novel protein over expressed in human melanomas

    Oncogene

    (1996)
  • J. Milbrandt

    A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor

    Science

    (1987)
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