Chromatin dynamics of gene activation and repression in response to interferon alpha (IFN(alpha)) reveal new roles for phosphorylated and unphosphorylated forms of the transcription factor STAT2

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.

FIGURE 1.

ISRE occupancy by STAT2 before and after IFNα treatment. A, STAT2-ChIPped DNAs from untreated and IFNα-treated Huh7 cells were labeled and hybridized to the STAT2/IFNα array as described under “Experimental Procedures.” Genes were divided into four categories depending on whether they: 1) already display STAT2 binding to their ISRE site before IFNα treatment and maintain it after the treatment (pos-pos; 53%) 2) acquire STAT2 binding after treatment (neg-pos; 29%) 3) STAT2 is bound prior to IFNα treatment and detaches after treatment (pos-neg; 9%) 4) display no STAT2 binding before and after IFNα treatment (neg-neg; 9%). B, dynamics of ISRE/STAT2 occupancy according to Gene Ontology categories (within brackets the number of genes belonging to each category). The figures represent the percentage of genes for each GO category displaying ISRE STAT2 binding before (pos-pos and pos-neg groups, as defined in A) and after IFNα stimulation (pos-pos or neg-pos groups), respectively. The schemes representing the ISGF3 include all the known components of the complex (STAT1, STAT2, and IRF9). However, because our ChIP-chip experiments investigated only STAT2 direct binding to the ISREs, STAT1, and IRF9 are depicted as shaded with dotted line (STAT1) and as only dotted line (IRF9) (see the Introduction section for a detailed discussion of IRF9 requirement for STAT2 activity).

FIGURE 2.

ISRE occupancy by P-STAT2 before and after IFNα treatment. A, cross-linked chromatin from untreated and IFNα-treated Huh7 cells was sequentially ChIPed first with α-STAT2 and then with α-P-STAT2, labeled and hybridized to the STAT2/IFNα array as described under “Experimental Procedures.” Genes were divided into 4 categories as in Fig. 1A: 1) [pos-pos]: 15%; 2) [neg-pos]: 43%; 3) [pos-neg]: 5%; 4) [neg-neg]: 37%. B, dynamics of ISRE/P-STAT2 occupancy according to Gene Ontology categories (within brackets the number of genes belonging to each category). The figures represent the percentage of genes for each GO category displaying ISRE P-Stat2 binding before (pos-pos or pos-neg groups, see Fig. 1A) and after IFNα stimulation (pos-pos or neg-pos groups), respectively. The ISGF3 cartoons are represented as in Fig. 1. Moreover, as a fraction of the promoters that are negative for P-STAT2 binding could be occupied by un-phosphorylated STAT2 in this categories the whole ISGF3 complex is drawn with dotted lines.

FIGURE 3.

Validation of STAT2/P-STAT2 occupancy of ISRE sites in selected array genes. Chromatin from untreated (K) and IFNα-treated (1000 UI/ml for 1 h) Huh7 cells was immunoprecipitated with either α-STAT2 or α-P-STAT2 (right column) antibodies and analyzed by real-time PCR with primers amplifying the regions spanning the oligonucleotides specifically enriched according to the ChIP-chip arrays data analysis (see supplemental Tables S1 and S4 for the complete list of primers). Results are expressed as specific enrichment (% of input values on selected regions divided for % of input values on the control (CTL) region), (as detailed in the “Experimental Procedures”). The figure shows mean values derived from three independent ChIPs experiments. Bars indicate standard deviation.

FIGURE 4.

ISGs expression profiles in Huh7 cells and PHH after IFNα treatment. A, custom real-time PCR liquid arrays (TLDAs, Applied Biosystems) designed to include 76 genes from the STAT2/IFNα ChIP-chip array (see “Experimental Procedures” for more details) were loaded with 200 ng of cDNA obtained from total RNAs extracted from Huh7 cells (upper panel) or PHH (lower panel) treated with 1000 IU/ml for the indicated time points. Results are plotted as log₁₀ enrichment over the basal level of expression in untreated cells. Genes that are not expressed in Huh7 cells, but are expressed and regulated in PHH by IFNα are shown in C. The complete list of expression values obtained from the average of three independent experiments is shown in supplemental Table S5. Standard deviation is always lower than 5% B, Venn representation of gene expression overlaps between Huh7 cells and PHH.

FIGURE 5.

Histone marks at ISRE sites and transcriptional modulation of selected array genes. Chromatin from untreated (K) and IFNα-treated (1000 UI/ml for 1 h) Huh7 cells was immunoprecipitated with α-H3K4me1, α-H3K4me3 or an anti-H3K27me3 antibodies and analyzed by real-time PCR with primers amplifying the regions corresponding to the oligonucleotide specifically enriched according to the ChIP-chip arrays data analysis (see supplemental Tables S1 and S4). Results are expressed as specific enrichment (% of input values on selected regions divided for % of input values on the control (CTL) region) (see “Experimental Procedures” for more details). The figure shows mean values derived from three independent ChIPs experiments. Bars indicate standard deviation.