Identification and characterization of CCAAT enhancer-binding protein (C/EBP) as a transcriptional activator for Epstein-Barr virus oncogene latent membrane protein 1

Chieko Noda, Takayuki Murata, Teru Kanda, Hironori Yoshiyama, Atsuko Sugimoto, Daisuke Kawashima, Shinichi Saito, Hiroki Isomura, Tatsuya Tsurumi

Research output: Contribution to journalArticle

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Abstract

Epstein-Barr virus LMP1, a major oncoprotein expressed in latent infection, is critical for primary B cell transformation, functioning as a TNFR family member by aggregation in the plasma membrane resulting in constitutive activation of cellular signals, such as NF-κB, MAPK, JAK/STAT, and AKT. Although transcription of LMP1 in latent type III cells is generally under the control of the viral coactivator EBNA2, little is known about EBNA2-independent LMP1 expression in type II latency. We thus screened a cDNA library for factors that can activate the LMP1 promoter in an EBNA2-independent manner, using a reporter assay system. So far, we have screened >20,000 clones, and here identified C/EBPε as a new transcriptional activator. Exogenous expression of C/EBPα, -β, or -ε efficiently augmented LMP1 mRNA and protein levels in EBV-positive cell lines, whereas other members of the C/EBP family exhibited modest or little activity. It has been demonstrated that LMP1 gene transcription depends on two promoter regions: proximal (ED-L1) and distal (TR-L1). Interestingly, although we first used the proximal promoter for screening, we found that C/EBP increased transcription from both promoters in latent EBV-positive cells. Mutagenesis in reporter assays and EMSA identified only one functional C/EBP binding site, through which activation of both proximal and distal promoters is mediated. Introduction of point mutations into the identified C/EBP site in EBV-BAC caused reduced LMP1 transcription from both LMP1 promoters in epithelial cells. In conclusion, C/EBP is a newly identified transcriptional activator of the LMP1 gene, independent of the EBNA2 coactivator.

Original languageEnglish
Pages (from-to)42524-42533
Number of pages10
JournalJournal of Biological Chemistry
Volume286
Issue number49
DOIs
Publication statusPublished - 09-12-2011

Fingerprint

CCAAT-Enhancer-Binding Proteins
Viruses
Oncogenes
Membrane Proteins
Transcription
Human Herpesvirus 4
Cells
Assays
Genes
Chemical activation
Binding Sites
Mutagenesis
Oncogene Proteins
Cell membranes
Epstein-Barr virus EBV-associated membrane antigen
Gene Library
Point Mutation
Genetic Promoter Regions
Protein Binding
Screening

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Noda, Chieko ; Murata, Takayuki ; Kanda, Teru ; Yoshiyama, Hironori ; Sugimoto, Atsuko ; Kawashima, Daisuke ; Saito, Shinichi ; Isomura, Hiroki ; Tsurumi, Tatsuya. / Identification and characterization of CCAAT enhancer-binding protein (C/EBP) as a transcriptional activator for Epstein-Barr virus oncogene latent membrane protein 1. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 49. pp. 42524-42533.
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Identification and characterization of CCAAT enhancer-binding protein (C/EBP) as a transcriptional activator for Epstein-Barr virus oncogene latent membrane protein 1. / Noda, Chieko; Murata, Takayuki; Kanda, Teru; Yoshiyama, Hironori; Sugimoto, Atsuko; Kawashima, Daisuke; Saito, Shinichi; Isomura, Hiroki; Tsurumi, Tatsuya.

In: Journal of Biological Chemistry, Vol. 286, No. 49, 09.12.2011, p. 42524-42533.

Research output: Contribution to journalArticle

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T1 - Identification and characterization of CCAAT enhancer-binding protein (C/EBP) as a transcriptional activator for Epstein-Barr virus oncogene latent membrane protein 1

AU - Noda, Chieko

AU - Murata, Takayuki

AU - Kanda, Teru

AU - Yoshiyama, Hironori

AU - Sugimoto, Atsuko

AU - Kawashima, Daisuke

AU - Saito, Shinichi

AU - Isomura, Hiroki

AU - Tsurumi, Tatsuya

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AB - Epstein-Barr virus LMP1, a major oncoprotein expressed in latent infection, is critical for primary B cell transformation, functioning as a TNFR family member by aggregation in the plasma membrane resulting in constitutive activation of cellular signals, such as NF-κB, MAPK, JAK/STAT, and AKT. Although transcription of LMP1 in latent type III cells is generally under the control of the viral coactivator EBNA2, little is known about EBNA2-independent LMP1 expression in type II latency. We thus screened a cDNA library for factors that can activate the LMP1 promoter in an EBNA2-independent manner, using a reporter assay system. So far, we have screened >20,000 clones, and here identified C/EBPε as a new transcriptional activator. Exogenous expression of C/EBPα, -β, or -ε efficiently augmented LMP1 mRNA and protein levels in EBV-positive cell lines, whereas other members of the C/EBP family exhibited modest or little activity. It has been demonstrated that LMP1 gene transcription depends on two promoter regions: proximal (ED-L1) and distal (TR-L1). Interestingly, although we first used the proximal promoter for screening, we found that C/EBP increased transcription from both promoters in latent EBV-positive cells. Mutagenesis in reporter assays and EMSA identified only one functional C/EBP binding site, through which activation of both proximal and distal promoters is mediated. Introduction of point mutations into the identified C/EBP site in EBV-BAC caused reduced LMP1 transcription from both LMP1 promoters in epithelial cells. In conclusion, C/EBP is a newly identified transcriptional activator of the LMP1 gene, independent of the EBNA2 coactivator.

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