Promoter Occupancy of STAT1 in Interferon Responses Is Regulated by Processive Transcription

Abstract

Interferons regulate immunity by inducing DNA binding of the transcription factor STAT1 through Y701 phosphorylation. Transcription by STAT1 needs to be restricted to minimize the adverse effects of prolonged immune responses. It remains unclear how STAT1 inactivation is regulated such that the transcription output is adequate. Here we show that efficient STAT1 inactivation in macrophages is coupled with processive transcription. Ongoing transcription feeds back to reduce the promoter occupancy of STAT1 and, consequently, the transcriptional output. Once released from the promoter, STAT1 is ultimately inactivated by Y701 dephosphorylation. We observe similar regulation for STAT2 and STAT3, suggesting a conserved inactivation mechanism among STATs. These findings reveal that STAT1 promoter occupancy in macrophages is regulated such that it decreases only after initiation of the transcription cycle. This feedback control ensures the fidelity of cytokine responses and provides options for pharmacological intervention. Cytokines perform their functions as key regulators of immune responses through activation of the JAK-STAT signaling pathway (1). Inadequate (either low or exacerbated) cytokine signaling may result in diseases such as immunodeficiency, autoimmunity, or cancer (2, 3). The strength of cytokine responses is regulated by various positive and negative feedback mechanisms that act at all steps of the signaling pathway: the cytokine receptors, JAKs, and STAT transcription factors. Yet it remains unclear how the process of cytokine-induced transcription is controlled once the transcription machinery has been turned on by activated STAT.STAT1 is indispensable for the biological function of interferons (IFNs), which are crucial cytokines for antiviral and antibacterial immunity. STAT1 nuclear translocation and DNA binding are activated by JAK-mediated phosphorylation of Y701. Other modifications that tune STAT1 function in IFN signaling include CDK8-mediated S727 phosphorylation, IB kinase ε (IKKε)-mediated S708 phosphorylation, and K703 sumoylation (4-6). Y701-phosphorylated STAT1 binds to target gene promoters in two major forms: (i) STAT1 homodimers induced by type I, II, and III IFNs bind to gamma interferon-activated sequence (GAS) elements, and (ii) the trimeric interferon-stimulated gene factor 3 (ISGF3; composed of STAT1, STAT2, and IRF9) induced by type I and III IFNs binds to interferon-stimulated response elements (ISRE) (1, 7). The principal mechanism of STAT1 inactivation is Y701 dephosphorylation, which causes both STAT1 homodimers and ISGF3 to lose their DNA-binding activity and to relocate to the cytoplasm. The nuclear T-cell protein tyrosine phosphatase (TC-PTP) is the major Y701-directed phosphatase (8). STAT1 acetylation was reported to facilitate dephosphorylation by TC-PTP (9), but this issue has been controversial (10). The access of phosphatase to phosphorylated Y701 appears to be restricted, since DNA-bound STAT1 is protected from Y701 dephosphorylation (11,12). For type II IFN (IFN-␥) ...

A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

“…4a, b). Furthermore, we observed a weak association between STAT2 and STAT1 that, in line with earlier observations, did not increase after IFN-I treatment 22,23 . STAT1 and IRF9 could not be coprecipitated, despite earlier studies assigning transcriptional activity to STAT1–IRF9 complexes 25,26 .…”

The C-Terminal Transactivation Domain of STAT1 Has a Gene-Specific Role in Transactivation and Cofactor Recruitment

“…The STAT1 C-terminal TAD is also required for the recruitment of CDK8, a component of the Mediator kinase module, which has been implicated in multiple aspects of the transcriptional cycle, including transcriptional re-initiation (3). The recent finding that STAT1 requires processive transcription for its dephosphorylation and promoter dissociation (69) prompts the hypothesis that transcriptionally compromised STAT1β homodimers accumulate at the promoters and prevent transcriptional re-initiation. However, it is also possible that the time-dependent effects observed relate to the heterogeneity of the cell population and reflect an increase in the number of cells responding to IFNγ over time.…”

Toxoplasma gondiistabilises tetrameric complexes of tyrosine-phosphorylated signal transducer and activator of transcription-1 and leads to its sustained and promiscuous DNA binding

“…Thirdly, nuclear STAT1 in infected cells appeared to be fully activated to a large extent, as revealed by the high levels of phosphorylation at Tyr 701 and Ser 727 . DNA‐bound STAT1 is indeed protected from dephosphorylation at Tyr 701 (Mertens et al, ; Meyer et al, ; Wiesauer, Gaumannmuller, Steinparzer, Strobl, & Kovarik, ) and has been reported to be required for phosphorylation at Ser 727 (Sadzak et al, ). Thus, high levels of phosphorylated STAT1 in the nuclei of T. gondii ‐infected cells clearly argue for a DNA‐bound state, thereby hindering nuclear export.…”

“…Thirdly, nuclear STAT1 in infected cells appeared to be fully activated to a large extent, as revealed by the high levels of phosphorylation at Tyr 701 and Ser 727 . DNA-bound STAT1 is indeed protected from dephosphorylation at Tyr 701 (Mertens et al, 2006;Meyer et al, 2003;Wiesauer, Gaumannmuller, Steinparzer, Strobl, & Kovarik, 2015) and has been reported to be required for phosphorylation at Ser 727 (Sadzak et al, 2008 (Köster & Hauser, 1999) was cultured in DMEM supplemented with 10% FCS, 1-mM sodium pyruvate, non-essential amino acids, antibiotics as above, and 2.5 μg/ml puromycin. Immortilised primary murine fibroblasts expressing either wild-type STAT1 (STAT1 WT ) or mutant STAT1 with a Phe 77 -to-Ala substitution (STAT1 F77A ;Begitt et al, 2014;Meyer et al, 2004) were cultured in DMEM medium as specified above but without puromycin.…”