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. 2010 Aug 15;185(4):2324-30.
doi: 10.4049/jimmunol.0903052. Epub 2010 Jul 21.

XBP-1 couples endoplasmic reticulum stress to augmented IFN-beta induction via a cis-acting enhancer in macrophages

Ling Zeng  1 , Yi-Ping Liu, Haibo Sha, Hui Chen, Ling Qi, Judith A Smith
Affiliations

XBP-1 couples endoplasmic reticulum stress to augmented IFN-beta induction via a cis-acting enhancer in macrophages

Ling Zeng et al. J Immunol. .

Abstract

Perturbation of the endoplasmic reticulum (ER) results in a conserved stress response called the unfolded protein response (UPR). Macrophages undergoing a UPR respond to LPS with log-fold increased production of IFN-beta, a cytokine with diverse roles in innate and adaptive immunity. In this study, we found that thapsigargin-induced ER stress augmented recruitment of IFN regulatory factor-3, CREB binding protein/p300, and transcriptional machinery to the murine ifnb1 promoter during LPS stimulation. Although full synergistic IFN-beta production requires X-box binding protein 1 (XBP-1), this UPR-regulated transcription factor did not appreciably bind the ifnb1 promoter. However, XBP-1 bound a conserved site 6.1 kb downstream of ifnb1, along with IFN regulatory factor-3 and CREB binding protein only during concomitant UPR and LPS stimulation. XBP-1 physically associates with p300, suggesting a mechanism of multimolecular assembly at the +6.1 kb site. Luciferase reporter assays provide evidence this +6 kb region functions as an XBP-1-dependent enhancer of ifnb1 promoter activity. Thus, this study identifies a novel role for a UPR-dependent transcription factor in the regulation of an inflammatory cytokine. Our findings have broader mechanistic implications for the pathogenesis of diseases involving ER stress and type I IFN, including viral infection, ischemia-reperfusion injury, protein misfolding, and inflammatory diseases.

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Figures

Figure 1
Figure 1. Synergistic induction of IFN-β in macrophages by LPS and ER stress
A) Murine bone marrow macrophages were pre-treated with 1h thapsigargin (Tpg), and then stimulated with 6h LPS. Results were combined from 2 independent experiments. *P=0.001 vs. LPS. B) Murine marrow macrophages were pre-treated with ER stress inducers calcium ionophore (A23187), tunicamycin (Tu), dithiothreitol (DTT), 2-deoxyglucose (2-DG), Tpg, or no ER stress inducer (NT) prior to 3h of LPS. Relative mRNA expression was determined by quantitative PCR (qPCR). Bars represent fold induction of IFN-β mRNA by ER stress pre-treatment plus LPS compared to LPS without ER-stress pre-treatment (NT=1). Results were combined from 3 independent experiments. Except for homocysteine, p=<0.05 for other ER stress inducers vs. NT. (C, D) RAW264.7 macrophages were pretreated with Tpg for 1h and then LPS for up to 8h. Black boxes are LPS only and white boxes are Tpg+LPS. %XBP-1 splicing (C) represents ratio of spliced and spliced+unspliced PCR products. D) For fold induction of IFN-β mRNA, results were combined from 2 independent experiments by normalizing to LPS-induced IFN-β mRNA at 4h (=1). *P=0.003 vs. LPS. Representative experiments for B and D are shown in Supplemental Figure 1.
Figure 2
Figure 2. ER stress increases transcription factor and machinery occupancy of the ifnb1 promoter
RAW 264.7 macrophages were pre-treated with Tpg for 1h and then stimulated with LPS for the times indicated. Binding of IRF-3, CBP, TBP and Pol II to the ifnb1 promoter was detected by ChIP. Relative factor occupancy compared to input chromatin was determined by qPCR. Control IgG results were combined for all stimulation conditions (IgG, no symbol). Results were combined from 4 (IRF-3, TBP), 5 (CBP), and 2 (Pol II) independent experiments. *P<0.001 for LPS vs. Tpg+LPS.
Figure 3
Figure 3. XBP-1 knockdown decreases synergistic induction of IFN-β in macrophages
A) RAW 264.7 macrophages transfected with 300 nM control or XBP-1 siRNA (XBP-1i) were treated with 1h Tpg and then 3h LPS. XBP-1 (top) or actin (bottom) was detected by Western. Results are representative of 2 separate experiments. B) RAW cells transfected with 300 nM control RNAi or XBP-1i were stimulated as in (A) and relative IFN-β (top) and ERdj4 (bottom) mRNA was determined by qPCR. Results were combined from 2 (ERdj4) and 3 (IFN-β) independent experiments. *P=0.029, **p=0.021. C) RAW cells transfected with 200 nM control RNAi (black) or XBP-1i (gray) were stimulated as in (A) and relative expression of IFN-β, 18S rRNA, and IL-1β, and IL-6 mRNA was determined by qPCR. Results are representative of 3 independent experiments.
Figure 4
Figure 4. XBP-1 does not bind the ifnb1 promoter
RAW 264.7 macrophages were stimulated as described in Figure 2, and then ChIP was performed with anti-XBP-1. Relative occupancy of the ifnb1 (left) and ERdj4 (right) promoters was assessed by qPCR by comparison to input sample. For ERdj4 ChIP, occupancies of control IgG were combined for all stimulation conditions. Results were combined from 4 (ifnb1 promoter) and 2 (Erdj4 promoter) independent experiments.
Figure 5
Figure 5. Identification of an XBP-1-dependent enhancer site 6.1kb downstream of ifnb1
A) Genomic region containing ifnb1, XBP-1-binding site, and contiguous genes. Ifnb1 gene: 27796544–27797313 (Genbank)/88168698-88167929 (FASTA). B) Nucleotide sequence of the +6kb site, containing base pairs 27790246–27790479 (Genbank)/88161631–88161864 (FASTA). Conserved nucleotides between mouse and human are bolded. Predicted ifnb1 enhanceosome component binding sites (80–90% consensus identity) are denoted by gray box (IRF) dotted line (NF-κB) and underscore (AP-1). XBP-1 consensus binding sites are in unfilled boxes C) RAW cells were stimulated with 1h Tpg followed by LPS for the times indicated. Factor occupancy of the +6.1kb site was detected by ChIP. Results were combined from 3 (XBP-1) and 4 (CBP, IRF-3) independent experiments. *P<=0.01. D) RAW cells were transfected with luciferase reporters containing the ifnb1 promoter alone or promoter+6kb site with no deletions, deletions of either or both conserved XBP-1 core binding sites (Δ1, Δ2, or Δ1+Δ2). Cells were stimulated with 1h Tpg and/or 7h LPS. Results were normalized to Tpg+LPS stimulation (=100%). Results were combined from 2 (Δ1+Δ2), 3(Δ1, Δ2) and 6 (promoter vs. promoter+6kb enhancer) independent experiments. *P=0.00002, **p<0.04. A sample experiment is shown in Supplemental Figure 2.
Figure 6
Figure 6. XBP-1s associates with CBP/p300 and enhances ifnb1 promoter activity
A) Lysates from transfected HEK293 cells were immunoprecipitated with anti-Flag and Western blots probed with anti-HA HRP (top panel) or anti-Flag-HRP (lower panel). 1% of the input lysate was probed with HA-HRP (middle panel). Results are representative of 3 independent experiments. B) RAW 264.7 macrophages co-transfected with pCDNA3.1 vector, XBP-1s, or XBP-1u plus luciferase expression vectors containing either ifnb1 promoter only (Pro), +6kb site (Enh-Pro) or +Δ1 (Δ1 Enh-Pro, Figure 4D) were stimulated with 7h LPS. Results were combined from 4–5 independent experiments by normalization to maximum luciferase activity. P<0.002 for Pro vs. Enh-Pro constructs across all pCDNA3.1, XBP-1s and XBP-1u co-transfections. *P<0.00001. A representative set of experiments is shown in Supplemental Figure 3.
Figure 7
Figure 7. Model for XBP-1 enhanced factor recruitment to the ifnb1 promoter
In the presence of LPS stimulation alone, IRF-3 and CBP bind to the ifnb1 promoter (top). When macrophages undergoing ER stress (Tpg treatment) are stimulated with LPS, XBP-1, IRF-3, and CBP cooperatively bind the region 6.1kb downstream of the ifnb1 gene. Through chromatin looping, the enhancer bound IRF3 and CBP factors are delivered to the multi-molecular complex at the ifnb1 promoter, ultimately resulting in greater recruitment of transcriptional machinery.
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