Octamer-binding factor 6 (Oct-6/Pou3f1) is induced by interferon and contributes to dsRNA-mediated transcriptional responses

Abstract

Background: Octamer-binding factor 6 (Oct-6, Pou3f1, SCIP, Tst-1) is a transcription factor of the Pit-Oct-Unc (POU) family. POU proteins regulate key developmental processes and have been identified from a diverse range of species. Oct-6 expression is described to be confined to the developing brain, Schwann cells, oligodendrocyte precursors, testes, and skin. Its function is primarily characterised in Schwann cells, where it is required for correctly timed transition to the myelinating state. In the present study, we report that Oct-6 is an interferon (IFN)-inducible protein and show for the first time expression in murine fibroblasts and macrophages.

Results: Oct-6 was induced by type I and type II IFN, but not by interleukin-6. Induction of Oct-6 after IFNbeta treatment was mainly dependent on signal transducer and activator of transcription 1 (Stat1) and partially on tyrosine kinase 2 (Tyk2). Chromatin immunopreciptitation experiments revealed binding of Stat1 to the Oct-6 promoter in a region around 500 bp upstream of the transcription start site, a region different from the downstream regulatory element involved in Schwann cell-specific Oct-6 expression. Oct-6 was also induced by dsRNA treatment and during viral infections, in both cases via autocrine/paracrine actions of IFNalpha/beta. Using microarray and RT-qPCR, we furthermore show that Oct-6 is involved in the regulation of transcriptional responses to dsRNA, in particular in the gene regulation of serine/threonine protein kinase 40 (Stk40) and U7 snRNA-associated Sm-like protein Lsm10 (Lsm10).

Conclusion: Our data show that Oct-6 expression is not as restricted as previously assumed. Induction of Oct-6 by IFNs and viruses in at least two different cell types, and involvement of Oct-6 in gene regulation after dsRNA treatment, suggest novel functions of Oct-6 in innate immune responses.

Figure 1

Oct-6 mRNA and protein is induced by IFNβ in primary fibroblasts (pMEFs) in a Tyk2-dependent manner. WT and Tyk2^-/- (A, B) pMEFs were treated with IFNβ for 6 h or incubated with medium alone (Ctrl). (A) Oct-6 and (B) IP-10 mRNA levels were determined by RT-qPCR using Ube2d2 as endogenous control and calculated relative to untreated WT cells. Mean values ± SD of two independent experiments are shown. (C) WT and Oct-6^-/- pMEFs were treated with IFNβ for 6 h or incubated with medium alone (0 h). Oct-6 was immunoprecipitated from whole cell extracts; panERK was used as an input control. (D) WT and Tyk2^-/- pMEFs were treated with IFNβ for the times indicated. Whole cell extracts were analysed by EMSA with an octamer motif containing oligonucleotide. (E) Oct6^-/-, Oct6^+/- and WT pMEFs were treated with IFNβ for the times indicated. Whole cell extracts were analysed by EMSA as described in (D). (C - E) Representatives of at least two independent experiments are shown.

Figure 2

IFNβ induces expression of Oct-6 in the murine Schwann cell line SW10. Cells were treated with IFNβ for the times indicated or incubated with medium alone (0 h). (A) Oct-6 and (B) IP-10 mRNA levels were determined by RT-qPCR as described in the legend to Figure 1B. Data are depicted relative to cells incubated without IFNβ from one of the experiments. Mean values ± SD of two independent experiments are shown. (C) Whole cell extracts were analysed by EMSA as described in the legend of Figure 1 D. A representative of two independent experiments is shown.

Figure 3

Oct-6 is induced by IFNβ and IFNγ in macrophages. WT BMMs were treated with IFNβ, IFNγ or IL-6 for the times indicated. (A, C) Whole cell extracts were analysed by EMSA as described in the legend of Figure 1 D. (A) DNA-binding complexes were identified by supershift of WT_8 h lysates with the respective antibodies (+AB), and by Oct-6 overexpression (ox) in a MEF cell line. (B) Oct-6 mRNA levels were determined by RT-qPCR as described in the legend of Figure 1A. Mean values ± SD of two experiments are shown. (D) Oct-6 was immunoprecipitated from whole cell extracts, panERK was used as an input control. (A, C, D) Representatives of at least two independent experiments are shown.

Figure 4

Oct-6 induction by IFNβ depends on Stat1 and partially on Tyk2. (A) WT, Stat1^-/- , Tyk2^-/- and Ifnar1^-/- BMMs were treated with IFNβ for the times indicated. Whole cell extracts were analysed by EMSA as described in the legend to Figure 1 D. A representative of at least two independent experiments per genotype is shown. (B) WT and Irf1^-/- BMMs were treated with IFNβ for the times indicated and Oct-6 mRNA expression was determined by RT-qPCR as described in the legend to Figure 1A. Mean values ± SD of two independent experiments are shown.

Figure 5

Oct-6 is induced by poly(I:C) and during MCMV infections via autocrine/paracrine actions of IFNα/β. (A, B) WT BMMs or (C) BMMs derived from mice of the indicated genotype were (A, C) treated with poly(I:C) or (B) infected with MCMV (MOI = 1) for the times indicated. Whole cell extracts were analysed by EMSA as described in the legend to Figure 1 D. Representatives of (A, B) three independent experiments and (C) at least two independent experiments per genotype are shown.

Figure 6

Stat1 binds to a conserved region in the Oct-6 promoter containing predicted GAS and ISRE sites. (A) Alignment of the selected conserved region upstream of the Oct-6 transcription start site (base counts correlate to the murine sequence). Potential GAS (black, bold) and ISRE (grey, bold) motifs are indicated. Position of primers used for the PCR reaction of the ChIP analysis are indicated by arrows. For the two PCRs, short (S) and long (L), the same forward primer was used, the reverse primer for the long PCR (not shown) is located 150 bp further downstream. (B) WT BMMs were treated with IFNβ (500 U/ml), IFNγ (200 U/ml) or were left untreated (Ctrl) for 1 h and 3 h. ChIP for Stat1 (α-Stat1 AB; nonspecific rabbit serum: Ctrl AB) was performed, followed by the two different PCRs for the Oct-6 promoter (Oct-6_S and Oct-6_L), and a PCR for the Irf1 promoter (Irf1) as a control. Representatives of two independent experiments are shown.

Figure 7

Oct-6 localises to the nucleus in response to IFNβ or poly (I:C). WT BMMs were grown on glass slides, treated with IFNβ (middle panels) or poly(I:C) (lowest panels) for 6 h or left untreated (upper panels). Oct-6 was detected by indirect immunofluorescence (right panels), nuclei were stained with DAPI (left panels). Representatives of two independent experiments are shown.

Figure 8

Absence of Oct-6 in macrophages does not influence poly(I:C) induced IFNβ and IFNα mRNA expression and has no impact on MCMV replication. (A - E) WT and Oct-6^-/- FLMs were treated with poly(I:C) or incubated with medium alone (0 h) for the times indicated. (A) Oct-6 was immunoprecipitated from whole cell extracts; panERK was used as an input control. (B - E) Expression levels were determined by RT-qPCR for (B) Egr2, (C) Pmp22, (D) IFNβ, and (E) panIFNα (all subtypes) using Ube2d2 as endogenous control. (B, C, D) Data are depicted relative to the WT 0 h control. (E) panIFNα mRNA could not be detected reliably in untreated cells (n.d.) and thus data normalised to the endogenous control only are depicted (not additionally calibrated to untreated cells). (B - E) Mean values ± SD of three independent experiments are shown. (F) WT and Oct-6^-/- FLMs were infected with MCMV (MOI = 1) for 90 min, washed with PBS and fresh medium was added. Supernatants were collected 0, 1, 3 and 6 days (d) after infection and virus titers were determined in a plaque forming assays using Stat1^-/- MEFs. Mean values ± SD of two independent experiments (each with FLMs from two embryos per genotype) are shown.

Figure 9

Expression levels of Stk40 and Lsm10 after poly(I:C) treatment are reduced in the absence of Oct-6. WT and Oct-6^-/- FLMs were treated with poly(I:C) for 8 h or incubated with medium alone (0 h). Expression levels of (A) Stk40 and (B) Lsm10 were determined by RT-qPCR using Ube2d2 as endogenous control. Data are depicted relative to untreated WT cells. Mean values ± SD of six experiments are shown.