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. 2021 Apr 9:12:651254.
doi: 10.3389/fimmu.2021.651254. eCollection 2021.

High Dose IFN- β Activates GAF to Enhance Expression of ISGF3 Target Genes in MLE12 Epithelial Cells

Kensei Kishimoto  1   2 , Catera L Wilder  3 , Justin Buchanan  3 , Minh Nguyen  1   3 , Chidera Okeke  1   4 , Alexander Hoffmann  1   3 , Quen J Cheng  3   5
Affiliations

High Dose IFN- β Activates GAF to Enhance Expression of ISGF3 Target Genes in MLE12 Epithelial Cells

Kensei Kishimoto et al. Front Immunol. .

Abstract

Interferon β (IFN-β) signaling activates the transcription factor complex ISGF3 to induce gene expression programs critical for antiviral defense and host immune responses. It has also been observed that IFN-β activates a second transcription factor complex, γ-activated factor (GAF), but the significance of this coordinated activation is unclear. We report that in murine lung epithelial cells (MLE12) high doses of IFN-β indeed activate both ISGF3 and GAF, which bind to distinct genomic locations defined by their respective DNA sequence motifs. In contrast, low doses of IFN-β preferentially activate ISGF3 but not GAF. Surprisingly, in MLE12 cells GAF binding does not induce nearby gene expression even when strongly bound to the promoter. Yet expression of interferon stimulated genes is enhanced when GAF and ISGF3 are both active compared to ISGF3 alone. We propose that GAF may function as a dose-sensitive amplifier of ISG expression to enhance antiviral immunity and establish pro-inflammatory states.

Keywords: GAF; ISGF3; JAK-STAT signaling pathway; gene regulation; interferon; signal transduction.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
IFN-β Induced STAT1 binds to GAS and ISRE motifs. (A) EMSA of GAS and ISRE binding in MLE12 cells treated with IFN-β (100 U/ml).NFY shown as loading control. Data are representative of >5 independent experiments. (B) STAT1 ChIP-seq heatmap of inducible STAT1 peaks (FDR< 0.01 for peak calling & > 2-fold induction in at least two time points) after IFN-β (10U/ml) stimulation. Peaks were clustered using k-means clustering. (C) Top three hits from de novo motif analysis on STAT1 peaks in Cluster 1 vs. 2 as defined in (B). (D) IFN-γ (100 ng/ml) induced STAT1 ChiP-seq signal comparing locations in Cluster 1 vs. 2. (E) Stacked bar graph of number of peaks per Cluster that contain ISRE, GAS, or BOTH motifs by searching the HOMER database (see Figure S1 ). (F) Re-clustered STAT1 ChIP-seq heatmap based on motif categories. (G) Genome browser tracks of representative promoter-bound STAT1 ChIP-seq peaks from BOTH (Isg15), GAS (Ripk1), and ISRE (Usp18) categories.
Figure 2
Figure 2
Low-dose IFN-β preferentially activates ISGF3 over GAF. (A) Heatmap of STAT1 ChIP-seq with two doses of IFN-β (1vs 10 U/ml) for 1h, showing GAS and ISRE clusters identified in Figure 1F . (B) Boxplot of STAT1 ChIP-seq signals for GAS cluster peaks in (B). n.s., not significant, *** = p < 2.2 e-16 by one-way Wilcox ranked sum test. (C) Genome browser tracks of representative promoter-bound STAT1 ChIP-seq peaks from "Both" (Isg15I), GAS (Ripk1), and ISRE (Usp18) clusters across two doses of IFN-β.
Figure 3
Figure 3
GAF does not induce expression of nearby genes. (A) Schematic for linking peaks to genes. Promoter defined as -1000 to +100 bp from TSS. (B) Heat map of log2 fold­ change of genes linked to STAT1 peaks, clustered by STAT1 peak motifs. Genes are ordered by distance to STAT1 peak. (C) Dot plot of genes in each category showing number of genes above 2-fold induction threshold. (D) Genome browser tracks of representative STAT1 peaks with GAS motifs at promoters of three genes. (E) Gene expression response to IFN-β (10 U/ml) for same genes as in (D).
Figure 4
Figure 4
In macrophages, GAF does induce expression of nearby genes. (A) Heat map of ChIP-seq data in macrophages (21), categorized by presence of GAS or ISRE motif. (B) Schematic for linking peaks to genes in macrophages. Promoter defined as -1000 to +100 bp from TSS. (C) Heat map of log2 fold-change of macrophage genes linked to macrophage STAT1 peaks. Three replicates of 2-hour stimulation with IFN. Genes are ordered by distance to STAT1 peak. (D) Dot plot of genes in each category showing number of macrophage genes above 2-fold induction threshold. (E) Bar graph of the percentage of genes near motif-categorized peaks that are induced upon IFN-β stimulation, comparing MLE12 cells vs. BMDMs.
Figure 5
Figure 5
Combined activation of GAF and ISGF3 enhances expression of ISGs In respiratory epithelial cells.(A) Heat map of 179 MLE12 ISGs as defined by induction with 10 U/ml IFN-β using thresholds of FDR<0.5 and fold­ change>2. (B) EMSA of ISRE and GAS binding in response to IFN-β (1 vs.100 U/mll), IFN-γ (1 vs.100 ng/ml), or a mixture of the low doses. Representative gel from five replicates. (C) Table of relative ISGF3 and GAF activation strengths in response to different doses of IFN-β and IFN-γ, based on EMSA data. (D) Principal component analysis of ISGs in response to IFN-β low dose (1 U/ml), IFN-γ low dose (1 ng/ml), IFN-β + IFN-γ low doses ("Mixed"), or IFN-β high dose (10 U/ml). Four-hour stimulation, two replicates. (E) Heat map of ISGs showing response to IFN-β low-dose, IFN-γ low dose, and Mixed. First three levels of hierarchical clustering are highlighted. Row annotations indicate Enhancement score, defined as RPKMmixed I (RPKMbeta + RPKMgamma).
Figure 6
Figure 6
Properties of strongly enhanced ISGs. (A) Density plot showing distribution of Enhancement Scores (ES) across 179 ISGs. Genes in the Top quartile (ES > 1.156) and Bottom quartile (ES < 0.774) are shaded (n=45 genes each). (B) Heat map of top and bottom quartile genes in a time course of stimulation of IFN-β low, IFN-γ high, Mixed, and IFN-β high (10 U/ml). (C) Box plots of absolute gene expression in Log2RPKM for top and bottom enhancement genes by stimulus. (D) Box plot of GC content at TSS (-300bp to +300bp) of top vs. bottom enhancement genes. (E) Percentage of top vs. bottom enhancement genes that have a STAT1ChiP-seq peak in promoter (-1000bp to +100bp). (F) Box plots of inducible expression of top vs. bottom enhancement genes in response to high doses of IFN-β (10 and 100 U/ml). (G) Line plots of representative genes of Top (Cxcl10, lfit1) vs. Bottom enhancement score (Tor1aip1).
Figure 7
Figure 7
Gene-specific mechanisms of GAF enhancement. (A) Schematic of GAF and ISGF3 colocalized at promoters to enhance ISG expression. (B) Violin plots of Enhancement scores for genes with STAT1 promoter peaks containing BOTH motifs (GAS+ISRE) vs. ISRE motif alone. (C) STAT1 ChIP-seq tracks at Mx2 promoter which has a BOTH peak in the proximal promoter as well as an ISRE peak at the TSS. (D) Schematic of distal GAF binding looping to promoters of ISGs to enhance ISG expression. (E) Density plot of distance from all STAT1 peaks to nearest TSS, separated by peak motif category. (F) Genome browser tracks of STAT1 ChIP-seq data and Hi-C data (46) at Cxcl10 locus demonstrating contact of two BOTH peaks with the TSS.
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