DNA methylation represses IFN-γ-induced and signal transducer and activator of transcription 1-mediated IFN regulatory factor 8 activation in colon carcinoma cells

Jon M. McGough, Dafeng Yang, Shuang Huang, David Georgi, Stephen M. Hewitt, Christoph Röcken, Marc Tänzer, Matthias P.A. Ebert, Kebin Liu

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

IFN regulatory factor 8 (IRF8) is both constitutively expressed and IFN-γ inducible in hematopoietic and nonhematopoietic cells. We have shown that IRF8 expression is silenced by DNA methylation in human colon carcinoma cells, but the molecular mechanism underlying methylation-dependent IRF8 silencing remains elusive. In this study, we observed that IRF8 protein level is inversely correlated with the methylation status of the IRF8 promoter and the metastatic phenotype in human colorectal carcinoma specimens in vivo. Demethylation treatment or knocking down DNMT1 and DNMT3b expression rendered the tumor cells responsive to IFN-γ to activate IRF8 transcription in vitro. Bisulfite genomic DNA sequencing revealed that the entire CpG island of the IRF8 promoter is methylated. Electrophoresis mobility shift assay revealed that DNA methylation does not directly inhibit IFN-γ-activated phosphorylated signal transducer and activator of transcription 1 (pSTAT1) binding to the IFN-γ activation site element in the IRF8 promoter in vitro. Chromatin immunoprecipitation assay revealed that pSTAT1 is associated with the IFN-γ activation site element of the IRF8 promoter in vivo regardless of the methylation status of the IRF8 promoter. However, DNA methylation results in preferential association of PIAS1, a potent inhibitor of pSTAT1, with pSTAT1 in the methylated IRF8 promoter region. Silencing methyl-CpG binding domain protein 1 (MBD1) expression resulted in IRF8 activation by IFN-γ in human colon carcinoma cells with methylated IRF8 promoter. Our data thus suggest that human colon carcinoma cells silence IFN-γ-activated IRF8 expression through MBD1-dependent and PIAS1-mediated inhibition of pSTAT1 function at the methylated IRF8 promoter.

Original languageEnglish (US)
Pages (from-to)1841-1851
Number of pages11
JournalMolecular Cancer Research
Volume6
Issue number12
DOIs
StatePublished - Dec 1 2008

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STAT1 Transcription Factor
DNA Methylation
Colon
Carcinoma
Methylation
Carrier Proteins
CpG Islands
Chromatin Immunoprecipitation
Electrophoretic Mobility Shift Assay
DNA Sequence Analysis
Genetic Promoter Regions
Electrophoresis
Colorectal Neoplasms
Phenotype
Neoplasms
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Oncology
  • Cancer Research

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DNA methylation represses IFN-γ-induced and signal transducer and activator of transcription 1-mediated IFN regulatory factor 8 activation in colon carcinoma cells. / McGough, Jon M.; Yang, Dafeng; Huang, Shuang; Georgi, David; Hewitt, Stephen M.; Röcken, Christoph; Tänzer, Marc; Ebert, Matthias P.A.; Liu, Kebin.

In: Molecular Cancer Research, Vol. 6, No. 12, 01.12.2008, p. 1841-1851.

Research output: Contribution to journalArticle

McGough, Jon M. ; Yang, Dafeng ; Huang, Shuang ; Georgi, David ; Hewitt, Stephen M. ; Röcken, Christoph ; Tänzer, Marc ; Ebert, Matthias P.A. ; Liu, Kebin. / DNA methylation represses IFN-γ-induced and signal transducer and activator of transcription 1-mediated IFN regulatory factor 8 activation in colon carcinoma cells. In: Molecular Cancer Research. 2008 ; Vol. 6, No. 12. pp. 1841-1851.
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AU - Georgi, David

AU - Hewitt, Stephen M.

AU - Röcken, Christoph

AU - Tänzer, Marc

AU - Ebert, Matthias P.A.

AU - Liu, Kebin

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AB - IFN regulatory factor 8 (IRF8) is both constitutively expressed and IFN-γ inducible in hematopoietic and nonhematopoietic cells. We have shown that IRF8 expression is silenced by DNA methylation in human colon carcinoma cells, but the molecular mechanism underlying methylation-dependent IRF8 silencing remains elusive. In this study, we observed that IRF8 protein level is inversely correlated with the methylation status of the IRF8 promoter and the metastatic phenotype in human colorectal carcinoma specimens in vivo. Demethylation treatment or knocking down DNMT1 and DNMT3b expression rendered the tumor cells responsive to IFN-γ to activate IRF8 transcription in vitro. Bisulfite genomic DNA sequencing revealed that the entire CpG island of the IRF8 promoter is methylated. Electrophoresis mobility shift assay revealed that DNA methylation does not directly inhibit IFN-γ-activated phosphorylated signal transducer and activator of transcription 1 (pSTAT1) binding to the IFN-γ activation site element in the IRF8 promoter in vitro. Chromatin immunoprecipitation assay revealed that pSTAT1 is associated with the IFN-γ activation site element of the IRF8 promoter in vivo regardless of the methylation status of the IRF8 promoter. However, DNA methylation results in preferential association of PIAS1, a potent inhibitor of pSTAT1, with pSTAT1 in the methylated IRF8 promoter region. Silencing methyl-CpG binding domain protein 1 (MBD1) expression resulted in IRF8 activation by IFN-γ in human colon carcinoma cells with methylated IRF8 promoter. Our data thus suggest that human colon carcinoma cells silence IFN-γ-activated IRF8 expression through MBD1-dependent and PIAS1-mediated inhibition of pSTAT1 function at the methylated IRF8 promoter.

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