Chromatin Dynamics of Gene Activation and Repression in Response to Interferon α (IFNα) Reveal New Roles for Phosphorylated and Unphosphorylated Forms of the Transcription Factor STAT2

Sabine Gerbal-Chaloin,Christine Völlenkle,Massimo Levrero,Francesca Guerrieri,Giovanni Blandino,Barbara Testoni

doi:10.1074/jbc.m111.231068

Abstract

Signal transducer and activator of transcription 2 (STAT2), the critical component of type I interferons signaling, is a prototype latent cytoplasmic signal-dependent transcription factor. Activated tyrosine-phosphorylated STAT2 associates with STAT1 and IRF9 to bind the ISRE elements in the promoters of a subset of IFN-inducible genes (ISGs). In addition to activate hundreds of ISGs, IFNα also represses numerous target genes but the mechanistic basis for this dual effect and transcriptional repression is largely unknown. We investigated by ChIP-chip the binding dynamics of STAT2 and "active" phospho(P)-STAT2 on 113 putative IFNα direct target promoters before and after IFNα induction in Huh7 cells and primary human hepatocytes (PHH). STAT2 is already bound to 62% of our target promoters, including most "classical" ISGs, before IFNα treatment. 31% of STAT2 basally bound promoters also show P-STAT2 positivity. By correlating in vivo promoter occupancy with gene expression and changes in histone methylation marks we found that: 1) STAT2 plays a role in regulating ISGs expression, independently from its phosphorylation; 2) P-STAT2 is involved in ISGs repression; 3) "activated" ISGs are marked by H3K4me1 and H3K4me3 before IFNα; 4) "repressed" genes are marked by H3K27me3 and histone methylation plays a dominant role in driving IFNα-mediated ISGs repression.

Highlights

Interferons are pleiotropic cytokines induced upon virus infection and other stimuli to modulate host immune response and are classified as type I interferon ␣ and ␤ (IFN␣ and IFN␤),3 type II (IFN␥), and the recently discovered type III (IFN␭) [1,2,3])
Both the enrichment of STAT2 Cy5 channel over the NoAb Cy5 channel hybridized in parallel and of Cy5-ChIPed chromatin over the corresponding Cy3-Input chromatin were considered for data analysis
In this work we investigated by Chromatin Immunoprecipitation (ChIP)-chip the binding dynamics of STAT2 and its phosphorylated form phosphorylated STAT2 (P-STAT2) to the ISRE sequences present on a large repertoire of IFN␣/␤ specific target genes before and after IFN␣ induction in hepatocellular carcinoma Huh7 cells and in primary human hepatocytes (PHH)

Summary

The abbreviations used are

IFN␣, interferon ␣; ISRE, interferon-stimulated responsive element; ISG, interferon-stimulated gene; STAT, signal transducer and activator of transcription; PHH, primary human hepatocytes. Expression profiling studies have shown that, in addition to activate hundreds of genes, IFN␣ is able to repress numerous target genes [21, 25,26,27]. The basis for this dual effect, and in particular for transcriptional repression, is still unclear as the composition of STAT containing complexes in genes activated or repressed in response to IFNs is largely unknown. To generate further mechanistic knowledge on ISREs-bound protein complexes, we investigated the binding dynamics of STAT2 and its “active” phospho-STAT2 form to a large panel of putative IFN␣ direct target promoters by ChIP-chip on a custom oligonucleotide promoter array. “repressed” genes are consistently marked by H3K27me, suggesting a dominant role of histone methylation in driving IFN␣-mediated ISGs repression

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION