pubmed.ncbi.nlm.nih.gov

Phosphorylation-induced dimerization of interferon regulatory factor 7 unmasks DNA binding and a bipartite transactivation domain - PubMed

Phosphorylation-induced dimerization of interferon regulatory factor 7 unmasks DNA binding and a bipartite transactivation domain

I Marié et al. Mol Cell Biol. 2000 Dec.

Abstract

Interferon regulatory factor 7 (IRF7) is an interferon (IFN)-inducible transcription factor required for activation of a subset of IFN-alpha genes that are expressed with delayed kinetics following viral infection. IRF7 is synthesized as a latent protein and is posttranslationally modified by protein phosphorylation in infected cells. Phosphorylation required a carboxyl-terminal regulatory domain that controlled the retention of the active protein exclusively in the nucleus, as well as its binding to specific DNA target sequences, multimerization, and ability to induce target gene expression. Transcriptional activation by IRF7 mapped to two distinct regions, both of which were required for full activity, while all functions were masked in latent IRF7 by an autoinhibitory domain mapping to an internal region. A conditionally active form of IRF7 was constructed by fusing IRF7 with the ligand-binding and dimerization domain of estrogen receptor (ER). Hormone-dependent dimerization of chimeric IRF7-ER stimulated DNA binding and transcriptional transactivation of endogenous target genes. These studies demonstrate the regulation of IRF7 activity by phosphorylation-dependent allosteric changes that result in dimerization and that facilitate nuclear retention, derepress transactivation, and allow specific DNA binding.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1

Phosphorylated IRF7 accumulates in the nucleus and binds DNA. (A) Isoelectric-focusing analysis of NDV-activated IRF7. 293T cells were transfected with an IRF7-HA expression plasmid. At 16 h post-transfection, cells were mock infected (lane 1) or NDV infected (lane 2) for 7 h and cell extracts were immunoprecipitated and analyzed by native isoelectric focusing and Western blotting using anti-HA antibodies. The mobilities of IRF7 and phosphorylated IRF7 (IRF7-P) are indicated. (B) Phosphorylated IRF7 is exclusively nuclear. 293T cells were transfected with an IRF7 expression plasmid, and nuclear (N) and cytoplasmic (C) extracts prepared from mock- or NDV-infected cells 9 h postinfection were analyzed by Western blotting using anti-IRF7 antibodies raised against the carboxyl terminus. The mobilities of IRF7 and phosphorylated IRF7 (IRF7-P) are indicated. (C) IRF7 binds DNA in response to viral infection. EMSA was performed on nuclear (lanes 1 to 5) and cytoplasmic (lanes 6 and 7) extracts derived from vector-transfected 293T cells (lanes 1 and 2) or cells expressing IRF7-Flag (lanes 3 to 7) that had been mock or NDV infected for 9 h, as indicated. Extracts were incubated with an ISRE probe from the ISG15 gene. Anti-Flag M2 antibodies were added to the reaction mixture (lane 5) to confirm the identity of the complex.

FIG. 2
FIG. 2

Transactivation of the IFN-α6 promoter by IRF7. (A) COS cells were transfected with the diagrammed IRF7 splice variant and truncation mutant expression constructs along with a luciferase reporter driven by the IFN-α6 promoter. At 24 h after transfection, cells were mock infected (hatched bars) or infected with NDV for 12 h (solid bars) before being assayed for luciferase activity. The values are expressed as fold induction relative to cells transfected with empty vector after normalization to cotransfected β-galactosidase. Mean values from a single representative experiment performed in duplicate are shown. Each construct was tested in at least three separate experiments, and variation between experiments was less than 10%. (B) IRF7Δ238–410 lacking the autoinhibitory domain does not respond to viral infection. COS cells were transfected and treated as in panel A, except that fivefold less IRF7Δ238–410 DNA was transfected relative to wild-type IRF7. (C) IRF7Δ238–410 does not require phosphorylation regulatory sequences for constitutive transcriptional activity. Cells were transfected with IRF7Δ238–410 or with IRF7Δ238–410 in which the two serine residues required for phosphorylation of the regulatory domain were converted to alanines (AA). Data are expressed as fold activation of the IFN-α6-luc reporter relative to its activation by virus-activated wild-type IRF7.

FIG. 3
FIG. 3

Structure-function mapping of IRF7 transactivation and autoinhibitory domains using Gal4 chimeras. Gal4-IRF7 fusion protein constructs are diagrammed on the left, indicating the Gal4 DBD and the exon structure of IRF7. Distinct functional regions of IRF7 are shaded. Fold activation of a Gal4 upstream activation site luciferase reporter cotransfected in COS cells with the indicated Gal4-IRF7 chimeric expression plasmids is shown on the right. The values are expressed as fold induction relative to basal activation by Gal4-DBD alone after normalization to cotransfected β-galactosidase activity. The graph represents the mean value from a single experiment performed in duplicate and is representative of at least three trials for each construct. Overall experimental variation was consistently less than 15%. Note that the results for constructs 1 to 9 and 10 to 11 are plotted on different scales. (B) Diagram of IRF7 functional domains, indicated by different shading patterns and labeled underneath, summarizing the data derived from transfection experiments. Exons are numbered for identification.

FIG. 4
FIG. 4

Induction of endogenous IFN-α gene expression by IRF7 isoforms. (A) Stat1−/− fibroblasts were transiently transfected with expression plasmids encoding IRF7 splice variants and truncation mutants, as indicated. After 36 h, cells were mock or NDV infected for 9 h and levels of non-IFN-α4 and GAPDH mRNA were monitored by RT-PCR, as indicated. (B) IRF7 accumulates in both the cytoplasm and nucleus. Expression levels of each IRF7 splice variant or truncation mutant in extracts from transiently transfected cells were monitored by Western blotting. The faster-migrating forms observed in lanes 1, 3, 7, and 8 (indicated by ∗) are most probably proteolytic breakdown products. The mobilities of molecular mass markers are indicated on the right in kilodaltons.

FIG. 5
FIG. 5

IRF7γ and IRF7ΔN102 competitively repress IRF7-mediated IFN-α6 induction. COS cells were cotransfected with expression constructs encoding IRF7α or IRF7β (50 ng), as indicated, and increasing amounts (0, 250, 1,000, and 2,000 ng) of either IRF7γ (upper panels) or IRF7ΔN102 (lower panels), along with a luciferase reporter construct driven by the IFN-α6 promoter. At 24 h after transfection, cells were infected with NDV for 12 h and extracts were assayed for luciferase activity. The values are expressed as a percentage of the activity without competitor after normalization to cotransfected β-galactosidase activity and represent the average of duplicate measurements.

FIG. 6
FIG. 6

Phosphorylated IRF7 displays an increased native molecular size. Nuclear extracts harvested from uninfected control (Ctl) (upper panel) or NDV-infected (lower panel) 293T cells that had been transfected with IRF7 were fractionated by glycerol gradient sedimentation. Individual fractions, as indicated, were assayed for IRF7 by immunoblotting following SDS-PAGE. The fractionation of molecular mass standards in a parallel gradient is indicated at the top, and the electrophoretic mobilities of phosphorylated and unphosphorylated IRF7 are indicated at the right.

FIG. 7
FIG. 7

Hormone-dependent dimerization of IRF7-ER induces specific DNA binding and IFN-α gene expression. (A) Diagram of the IRF7-ER chimeric proteins. The DBD, transactivation domain (TA), autoinhibitory domain (Inhib.), regulatory domain (Reg), and estrogen LBD are indicated for IRF7α (upper) and the IRF7γ splice variant (lower). (B) 293T cells were cotransfected with IFN-α6-luc plus vector, IRF7α-ER, or IRF7γ-ER, as indicated, and then treated for 16 h with 4-HT or left untreated (Ctl) before being assayed for luciferase activity. Data are shown as fold induction over untreated, vector-transfected cells and represent the mean and standard error of duplicate measurements. (C) Cells cotransfected with IFN-α6-luc plus vector, wild-type IRF7α, IRF7α-ER, or IRF7α(AA)-ER were left untreated or treated for 16 h with 4-HT before being assayed for luciferase activity. Data are shown as percent maximal activity obtained in NDV-infected cells transfected with wild-type IRF7 (results not shown) and represent the mean and standard error of triplicate determinations. (D) IRF7α (lane 1), IRF7α-ER (lane 2), and IRF7γ-ER (lane 3) protein levels were measured in extracts from transfected 293T cells by immunoblotting. (E) Extracts of cells transfected with IRF7α-ER (α-ER) or IRF7γ-ER (γ-ER), as indicated, that had been left untreated (lanes 1 and 3) or treated for 4 h with 4-HT (lanes 2 and 4) were analyzed by EMSA. The positions of the IRF7-ER protein-DNA complexes are indicated. (F) Stat1−/− fibroblasts were transfected with IRF7α-ER (lanes 1 and 2) or IRF7γ-ER (lanes 3 and 4) before being treated with 4-HT for 6 h (even-numbered lanes). RNA was analyzed for expression of the non-IFN-α4 subset or for GAPDH, as indicated.

Similar articles

Cited by

References

    1. Au W C, Moore P A, LaFleur D W, Tombal B, Pitha P M. Characterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon A genes. J Biol Chem. 1998;273:29210–29217. - PubMed
    1. Berry M, Metzger D, Chambon P. Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J. 1990;9:2811–2818. - PMC - PubMed
    1. Bluyssen H A R, Muzaffar R, Vlieststra R J, van der Made A C J, Leung S, Stark G R, Kerr I M, Trapman J, Levy D E. Combinatorial association and abundance of interferon-stimulated gene factor 3 components dictate the selectivity of interferon responses. Proc Natl Acad Sci USA. 1995;92:5645–5649. - PMC - PubMed
    1. Brandt M E, Vickery L E. Cooperativity and dimerization of recombinant human estrogen receptor hormone-binding domain. J Biol Chem. 1997;272:4843–4849. - PubMed
    1. Brass A L, Kehrli E, Eisenbeis C F, Storb U, Singh H. Pip, a lymphoid-restricted IRF, contains a regulatory domain that is important for autoinhibition and ternary complex formation with the Ets factor PU.1. Genes Dev. 1996;10:2335–2347. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources