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

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, IFN␣ 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 IFN␣ direct target promoters before and after IFN␣ induction in Huh7 cells and primary human hepatocytes (PHH). STAT2 is already bound to 62% of our target promoters, including most "classical" ISGs, before IFN␣ 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 IFN␣; 4) "repressed" genes are marked by H3K27me3 and histone methylation plays a dominant role in driving IFN␣-mediated ISGs repression.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) (1-3)). Interferons exert their function by phosphorylating latent transcription factors belonging to the signal transducer and activator of transcription (STAT) family after a signaling cascade, which begins with the binding of interferon to their membrane receptors and involves Janus kinases (JAKs). Receptor dimerization or oligomerization leads to Jak apposition and the trans-phosphorylation on tyrosine residues that releases their intrinsic catalytic activity. Tyrosine-phosphorylated cytokine-receptor cytoplasmic domains then provides binding sites for the Src homology-2 (SH2) domain of the STAT proteins, which are recruited to the JAKs and phosphorylated on a single tyrosine residue (Tyr-689 in the case of STAT2). The interaction between phosphorylated-SH2 domains on STAT proteins leads to homo-or hetero-dimerization and nuclear translocation (4 -6). STAT2 Tyr-689 and STAT1 Tyr-701 can also be phosphorylated by non-receptor TKs, including SRC and ABL in the absence of ligand-induced receptor signaling (7). STAT dimers directly activate genes containing the IFN␥ activation site (GAS) element, while the association of STATs with the DNA-binding protein interferon regulatory factor (IRF) 9 expands the range of DNA response elements that can be targeted by the JAK-STAT pathway to interferon-stimulate response element (ISRE) and IRF response element (IRE) (8). Un-phosphorylated STAT2 binds IRF9 and constitutively shuttles i...

Molecular Pathways: Interferon/Stat1 Pathway: Role in the Tumor Resistance to Genotoxic Stress and Aggressive Growth

“…Importantly, U-STAT1 occupies only a fraction of the promoters that bind phosphorylated STAT1 (P-STAT1) after IFN-g stimulation (47). Unphosphorylated Stat2 and Stat3 activated transcription of subsets of downstream genes, only partially overlapping with genes activated by phosphorylated Stats (46,48). If prosurvival ISGs are preferentially activated by U-Stat1, then the equilibrium between P-STAT1 and U-STAT1 can be of critical importance to affect the prosurvival functions of STAT1.…”

The Tumor Suppressive Effects of HPP1 Are Mediated Through JAK-STAT-Interferon Signaling Pathways

“…However, recent evidence suggests that unphosphorylated STAT1 and STAT2 are able to shuttle in and out of the nucleus (Banninger and Reich, 2004;Cheon and Stark, 2009). Moreover, unphosphorylated STAT1 and STAT2 have been shown to play a role in regulating the expression of IFN-inducible genes (Cheon and Stark, 2009;Testoni et al, 2011a), including some typical IFN-inducible genes such as RIG-G (Lou et al, 2009). Interestingly, Testoni et al (2011a) also demonstrated recruitment of unphosphorylated STAT2 to the promoter region of OAS1 with IFN-a treatment.…”

“…Similarly, about one-quarter of the IFN-inducible genes found to be repressed following IFN-a treatment recruited both STAT2 and pSTAT2 to the promoter regions. Moreover, half of the genes repressed by IFN-a treatment demonstrated STAT2 binding before treatment and pSTAT2 binding afterward (Testoni et al, 2011a). We focused our investigation of HPP1-mediated tumor suppression on three typical STAT phosphorylation is mediated by kinases of the Janus Family (Schindler and Darnell, 1995).…”

“…Type II IFNs (including IFN-g) are known to elicit STAT1 homodimer formation, while type I IFNs (including IFN-a) are known to result in STAT1:STAT2 heterodimer formation. Interestingly, STAT2 is absolutely required for a fully effective response to IFN-a (Testoni et al, 2011a). We have previously observed that STAT2 is upregulated and activated in response to HPP1 overexpression and consequently, we hypothesize that HPP1s tumor-suppressive effects are likely mediated by elements of the IFN-a pathway.…”

Responses to Cytokines and Interferons that Depend upon JAKs and STATs

“…Differently from U-STAT5, U-ISGF3 and phosphorylated ISGF3 bind to similar ISRE elements in the promoters of ISGs that are induced by both transcription factors . Using a ChIP-on-chip analysis, Testoni et al (2011) showed that U-STAT2, possibly as a component of U-ISGF3, binds to more than half of the ISG promoters investigated before IFN-a treatment, but the effect on the expression of ISGs is not clear. Although U-ISGF3 itself increases the expression of some ISGs in the absence of IFN treatment, we do not yet know whether or not U-ISGF3 inhibits the expression of other ISGs in response to IFN.…”