doi: 10.1038/s41586-019-1041-6.
Epub 2019 Mar 20.
A NIK-SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB
Affiliations
- PMID: 30894749
- PMCID: PMC6812682
- DOI: 10.1038/s41586-019-1041-6
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A NIK-SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB
Nature.
2019 Apr.
Abstract
The non-canonical NF-κB signalling cascade is essential for lymphoid organogenesis, B cell maturation, osteoclast differentiation, and inflammation in mammals1,2; dysfunction of this system is associated with human diseases, including immunological disorders and cancer3-6. Although expression of NF-κB-inducing kinase (NIK, also known as MAP3K14) is the rate-limiting step in non-canonical NF-κB pathway activation2,7, the mechanisms by which transcriptional responses are regulated remain largely unknown. Here we show that the sine oculis homeobox (SIX) homologue family transcription factors SIX1 and SIX2 are integral components of the non-canonical NF-κB signalling cascade. The developmentally silenced SIX proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit the trans-activation function of the transcription factors RELA and RELB in a negative feedback circuit. In support of a physiologically pivotal role for SIX proteins in host immunity, a human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/P53-driven non-small-cell lung carcinomas from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents (small-molecule activators of the non-canonical NF-κB pathway). Our findings identify a NIK-SIX signalling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions.
Conflict of interest statement
COMPETING INTERESTS
The authors declare no competing interests.
Figures
a, b, Experiments were performed to exclude the possibility that the observed CD40L induced anti-bacterial function was specific to a particular cell type or protocol of cytokine induction. We reconstituted the CD40L signaling pathway in HEK293 cells. These cells do not express CD40, the endogenous receptor for CD40L (a, western blot). HEK293 cell are also unable to be stimulated by CD40L (a, graph). However, we found that overexpression of CD40 strongly induced NF-κB pathway activation (a, graph). Expression of CD40 restricted both Lm and Sf infection (b) to levels similar to those observed in CD40L treated U-2 OS cells (compare data to Fig. 1b). The NF-κB reporter activity assay in panel a was performed by co-transfecting empty vector (EV), CD40L or CD40 with 5×κB-LUC reporter gene into HEK293T cells. Luciferase activity was measured after 48 hours and normalized to EV (right). Data are mean±s.d. from 6 independent experiments. Experiment and quantification of panel b is presented as in Fig. 1b. Data are mean±s.d. from 6 independent experiments. c, d, Conformation of genetic knockout of the MAPK3K14 (here forward referred to as NIK) and MAPK3K7 (here forward referred to as TAK1) genes in STAT1−/− human fibroblasts. c, Schematic representation of In/Del base pairing and the sgRNA targets locus of Exon 1 in the NIK and TAK1 gene. NIK−/− contains −7bps, +G, and +CTCAC alleles (top). TAK1−/− contains +AT alleles, −2bps, and −409bps (bottom). (- means deletion, + means insertion). d, Western blot shows endogenous NIK and TAK1 expression in parental, NIK−/− and TAK1−/− cells. It is important to point out that NIK is constitutively degraded by cIAPs-TRAF2/3 E3-ligase complex in quiescent cells. To detect NIK expression, WT, NIK−/−, and TAK1−/− fibroblasts were treated with 2.5 μM BV6 (a SMAC-mimetics compound that antagonizes cIAPs and leads to NIK accumulation,) for 14 hours and then the endogenous proteins were probed with indicated antibodies by western blot. e, NIK is necessary for restricting Lm infection. Fibroblasts with the indicated genetic background were treated with vehicle control (DMSO) or 2.5 μM BV6 for 14 hours and then infected with LmGFP. The percent of bacterial infection was normalized to WT uninfected control. Black statistic markers denote the difference between WT and indicated cell lines and red markers denote the difference between DMSO and BV6 treatment. We noted that NIK−/− cells exhibited much greater levels of Lm infection than either WT or TAK−/− cells consistent with its role in preventing infection after cellular stimulation. However, BV6 treatment of cells, which suppressed Lm infection of WT cells, had no effect on NIK−/− cells further indicating that NIK activation is necessary for the anti-bacterial response. Data are mean±s.d. from 9 independent experiments. f, The kinase activity of NIK is required for its anti-bacterial function. Fluc, WT NIK or NIK-kinase dead mutant (NIKK429/430A referred to as NIKKD) lentivirus was transduced into fibroblasts or U-2 OS cell as indicated. Cells were then challenged with SfGFP. Quantification of bacterial infection is presented as described in Fig. 1b. Data are mean±s.d. from 4 independent experiments. g, NF-κB gene expression induced by NIK is kinase dependent. Empty vector (EV), NIK or NIKKD was co-transfected with 5×κB-LUC into HEK293T cells. NF-κB activity was measured after 48 hours and normalized to EV (right). Data are mean±s.d. from 4 independent experiments. h, Expression of NIK, but not TAK1, potently inhibits Lm and Sf infection. WT U-2 OS cells were transduced with combinational FlucRFP/FlucBFP, NIKRFP/FlucBFP, or TAK1RFP/TAB1BFP lentivirus. Cells were then challenged with LmGFP or SfGFP. Infection efficiency was quantified by flow cytometry. The infection efficiency was determined by gating GFP positive cells in both RFP and BFP positive cell populations. The relative percentage of pathogen infection was normalized to Fluc control. Data are mean±s.d. from 8 independent experiments. i, Control experiment showing NIK protein expression levels that correspond to experiments presented in Fig. 1c. Fluc and NIK transduced cells were lysed and probed with anti-NIK antibody. j, Overexpression of NIK does not cause cytotoxicity in fibroblasts. Previous studies suggest that ectopic expression of NIK causes cytotoxicity in A549 cells. To test if ectopic expression of NIK causes cytotoxicity in fibroblasts, we transduced WT fibroblasts with indicated lentivirus and measured the cell viability after 72 hours by measuring ATP. Data are mean±s.d. from 6 independent experiments. P values were measured using one-way ANOVA (GraphPad), ***P<0.001, ****P<0.0001, ns: no significant difference. The same statistics were used in the later figures unless otherwise stated. Western blot data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, Schematic of NIK-stimulated gene library design, cloning, and the multidimensional flow cytometry based high throughput screen. NIK-stimulated genes were determined by RNA-seq. The cDNAs of 237 NIK-stimulated genes were individually cloned into the lentiviral vector pTRIP upstream of the IRES-tagRFP (see methods). Fibroblasts were transduced with lentivirus in a one-gene to one-well format and were then infected with GFP expressing Lm, Sf, EAV, WNV, SINV, and PIV3 in independent experiments. The effect of a single gene expression on infection was quantified by flow cytometry. b, The relative expression levels of NIK-stimulated genes identified by RNA-seq. Fluc or NIK lentivirus was transduced into fibroblasts. Total RNA was isolated after 72 hours and gene expression level was determined by RNA sequencing. Graph shows gene expression levels that are significantly stimulated (red, 237 genes) or downregulated (green, 84 genes) by NIK expression compared to Fluc control. Fold change over 2 (Log2≥1) or less 0.5 (Log2≤−1) and FDR<0.05 (statistics test is presented in methods section). Bars were ranked numerically from low to high (see Table S1 for details). The expression levels of SIX1 and SIX2 are indicated. Data are representative of 2 independent experiments. c, Graph showing the efficiency of lentiviral expression of NIK-stimulated genes used in the high throughput bacterial and viral screen. NIK-stimulated genes were transduced into WT fibroblasts in a “one-gene per one-well” format. Transduction efficiency as measured % RFP positive cells was determined by flow cytometry and was ranked numerically from low to high (see source data for details, values are from the average of 2 technical replicates). 12 out of 237 genes were poorly transduced (less than 20% RFP+) and were excluded from subsequent analyses. d, Dot plots of Sf, Lm, EAV, WNV, SINV, and PIV3 infectivity in the presence of expressed NIK-stimulated genes (in c). Data were normalized to the average of each screen, indicating as the black dotted line. We chose to confirm hits in Fig. 1d based on two criteria: (1) the gene expression effect on inhibiting or enhancing pathogen infection by less than or greater than 50%, and (2) an adjusted Z-score less than −2 or greater than 2 (see Table S2 for details). NIK-stimulated genes that reproducibly and significantly inhibited (green) or enhanced infection (red) by these criteria are indicated. The genes shown in black font are hits that were not reproduced in the confirmatory experiments (Fig. 1d). Data are mean±s.d. from 2 (Sf and Lm) or 1 (EAV, WNV, SINV, and PIV3) independent experiments.
a, Control experiment for Fig. 2a, c showing that Nik is expressed in WT BMDMs but not in BMDMs isolated from Map3k14−/− (here forward Nik−/−) mice. As mentioned in Extended Data Fig. 1d, Nik protein is constitutively degraded under quiescent condition. Thus, we employed MG132 proteasome inhibitor to stabilize endogenous Nik protein expression. To validate Nik protein expression, WT and Nik−/− primary BMDM cells were treated with mock or 30 μM MG132 for 12 hours and Nik protein was detected by western blot. b, c, Long term treatment of cells with TNF (b) or LPS (c) stabilized Six1 expression through activation of Nik in murine primary BMDM cells. WT and Nik−/− primary BMDMs were treated with 25 ng/ml TNF (b, graph showing quantification of Six1 protein expression in TNF treated cells mean±s.d. from 3 independent experiments as described in Fig. 2c) or 100 ng/ml LPS (c) for 24 hours. d, e, Human SIX1 and SIX2 protein reactivation by long term treatment of cells with both canonical (TNF) and non-canonical (LTα1β2) NF-κB agonists requires NIK, but not TAK1. WT, NIK−/− or TAK1−/− fibroblasts were treated with mock, 25 ng/ml TNF or 50 ng/ml LTα1β2 for 24 hours. LTα1β2 was employed as positive control of a non-canonical NF-κB agonist. TAK1−/− cells were included as control to show TNF and LTα1β2 could induce SIX1 and SIX2 accumulation in a TAK1 independent manner (e). f, g, Ectopic expression of NIK induces expression of recombinant SIX1 and SIX2 driven by the strong CMV promoter in HEK293 cells. Plasmids encoding CMV-driven GFP-SIX1 or GFP-SIX2 were co-transfected into HEK293T cells with empty vector (EV) or Flag-NIK. Western blot (f) and fluorescence microscopy (g) assays were performed to detect expression of GFP-SIX1 and GFP-SIX2 post 48 hours transfection. We estimate that SIX1 and SIX2 protein are expressed in 5–10% of untreated cells, whereas they are expressed in 60–70% of cells when co-transfected with NIK. Microscopy images were taken using a 10× objective (g). h, i, Experiments showing that activation of NIK by BV6 (h) or by inhibition of the proteasome with MG132 (i) stabilizes CMV-Flag-SIX2 expression in HEK293T cells. Flag-SIX2 was transfected into HEK293T cells for 24 hours, cells were then treated with mock or 5 μM BV6 for 24 hours or 30 μM MG132 for 12 hours. j, Inhibition of the 26S proteasome with MG132 induces endogenous Six1 protein expression in primary BMDMs. Cells were treated with 30 μM MG132 for the indicated time. k, Inhibition of the 26S proteasome promotes SIX1 and SIX2 expression in human fibroblasts and this expression occurs in NIK−/− fibroblasts. Experiments were performed as in j. l, Kinetics of cIAP1 degradation and NIK, SIX1 and SIX2 accumulation in human fibroblasts treated with BV6. WT fibroblasts were treated with 5 μM for indicated time. m, NIK potently suppresses SIX2 ubiquitination. HEK293T cells were co-transfected with HA-ubiquitin and Flag-SIX2 along with GFP-NIK as indicated and cells were incubated for 48 hours. SIX2 was immunoprecipitated with anti-Flag antibody. The ubiquitination status of the protein was determined by anti-HA western blot. n, Diagram showing the reactivation mechanism of SIX-proteins in response to non-canonical NF-κB activation. Details are explained in the main text. All data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, SIX2 enhances bacterial infection independent of interaction with Eya family transcriptional co-activators. Previous studies have shown that SIX-family transcription factors assemble gene co-activator complexes through interaction with Eyes Absent (Eya) family members. Structural studies indicate that SIX1 residues C16 and V17 are required for the interaction with EYA2. These residues are conserved in SIX2. Thus, using mutant SIX2C16RV17E protein, we found that SIX2 enhances Lm infection independent of EYA interactions. Fluc, WT SIX2, or SIX2C16RV17E lentivirus was transduced into WT fibroblast cells. Cells were then challenged with LmGFP. The percent of Lm infection was normalized to Fluc control. Data are mean±s.d. from 4 independent experiments. P is shown in the figure. b, Expression of SIX2 suppresses the anti-microbial function of NIK and CD40. WT fibroblasts were lentiviral transduced with a combination of cDNAs (FlucRFP/FlucBFP, NIKRFP/FlucBFP, NIKRFP/SIX2BFP, CD40RFP/FlucBFP, or CD40RFP/SIX2BFP). After 72 hours, cells were infected with LmGFP. The RFP-, BFP- and GFP-expressed cells were gated by flow cytometry. Quantification of infection was performed as described in Extended Data Fig. 1h. Data are mean±s.d. from 6 independent experiments. ***P<0.001, ****P<0.0001, ns: no significant difference. c, d, Characterization of SIX1−/−
SIX2−/− fibroblasts generated by CRISPR-Cas9. Schematic representation of In/Del base pairing and the sgRNA targets locus of Exon 1 in the SIX1 and SIX2 gene in fibroblasts (c). SIX1−/−
SIX2−/− contains a single T insertion in both alleles of the SIX1 and SIX2 genes. Western blot shows endogenous SIX1 and SIX2 expression in parental and SIX1−/−
SIX2−/− fibroblasts (d). Data are representative of 3 independent experiments. e, The anti-bacterial activity of NIK is enhanced in SIX1−/−
SIX2−/− fibroblasts. WT and SIX1−/−
SIX2−/− fibroblasts were transduced with Fluc or NIK lentivirus. After 72 hours, cells were then challenged with LmGFP. Black statistic markers denote the difference between WT (Fluc) and WT (NIK) or SIX1−/−
SIX2−/− (Fluc or NIK). Red marker denotes the difference between WT (NIK) and SIX1−/−
SIX2−/− (NIK). Relative infectivity was normalized to WT (Fluc) control. These data indicate that SIX-proteins oppose the function of NIK, potentially through suppression of non-canonical NF-κB gene expression (see f, below). Data are presented as mean±s.d. from 6 independent experiments. ****P<0.0001, ns: no significant difference. f, Model illustrating the relationship between NIK expression, SIX-proteins accumulation, and their roles in regulating anti-microbial immunity. g, Diagram describing RNA-seq experiments used to identify NIK-stimulated genes that are suppressed by SIX2. To identify the NIK-stimulated genes that are regulated by SIX2, the indicated lentiviruses (group I-IV) were transduced into WT fibroblasts. Total RNA was extracted for deep sequencing post 72 hours transduction (see Table S3). h, Diagram showing the group comparisons from data generated in g. Briefly, the NIK-stimulated genes that suppressed by SIX2 were determined from Group IV vs Group II comparison (Log2 ≤−1) and then adjusted to NIK-stimulated genes that are from Group II vs Group I comparison (we chose fold change greater than 4), FDR<0.01 (statistics test is presented in methods section). i, Representative raw data from RNA-seq experiments performed in g, h. RNA-seq data are presented as FPKM value (bars show mean from 2 independent experiments indicated as circle). j, Validation of RNA-seq data. Experiments were performed as described as in g. Gene transcription level was determined by qRT-PCR and relative gene expression was normalized to Fluc control. Data are mean±s.d. of 3 or 2 technical replicates and representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, b, Characterization of RelA−/− and RelB−/− in human fibroblasts. Schematic representation of In/Del base pairing and the sgRNA targeting locus of Exon 3 in RelA and Exon 1 in RelB gene. RelA−/− cells contain +C, −371 bps, and a 109 bps fragment that was replaced with 122 bps containing the GCGCTA with reverse complementary (orange fragment) (a up). RelB−/− cells contain the indicated deletions (a bottom). Western blot shows endogenous RelA and RelB expression in parental and RelA−/− or RelB−/− fibroblasts and response to stimulation by 24-hour application of TWEAK (b). c, Experiments evaluating the contributions of the canonical and non-canonical NF-κB subunits RelA and RelB, respectively, on expression of the indicated genes. Fluc or NIK was transduced into WT, RelA−/−, and RelB−/− fibroblasts to stimulate the non-canonical NF-κB signaling pathway and mRNA expression of the indicated genes were evaluated by qRT-PCR. We concluded that the IL-1β gene is specifically stimulated by RelA since we did not detect its expression in RelA−/− cells but did so in RelB−/− cells (which expression RelA). Similar logic was used to evaluate the 7 additional genes shown. Experiments were performed as described in Fig. 2g. Bars are mean of 2 technical replicates (shown as circle) and representative of 2 independent experiments. d, The human SIX-family consists of 6 unique isoforms. To determine which of these genes suppress NF-κB mediated gene expression, empty vector (EV), SIX1, SIX3, SIX4, SIX5, or SIX6 cDNAs were co-transfected with 5×κB-LUC into HEK293T cells. After 24 hours, cells were treated with mock or 25 ng/ml TNF for 24 hours. The luciferase activity was measured and normalized to EV untreated control. SIX1, SIX3, and SIX6 (SIX2 was not evaluated in this experiment) potently inhibited both basal and inducible activity of NF-κB. Data are mean±s.d. from 7 independent experiments. e, SIX2 inhibits LTα1β2- and TNF-induced NF-κB activation. EV or Flag-SIX2 was co-transfected with 5×κB-LUC into WT fibroblasts. After 24 hours, cells were treated with mock, 25 ng/ml TNF or 50 ng/ml LTα1β2 for 24 hours. Data were analyzed as described in d. Data are mean±s.d. from 6 independent experiments. f, The inhibitory potency of SIX2 is equivalent to A20 and IκBαSR. The Flag-tagged genes indicated were co-transfected with 5×κB-LUC into HEK293T cells. After 24 hours, cells were treated with mock or 25 ng/ml TNF for 24 hours. Data were analyzed as in d. Data are mean±s.d. from 9 independent experiments. Anti-Flag western blot was performed to determine expression levels, IκBα regulation was included as pathway activation control upon cellular stimulation with TNF. g, Experiments showing that SIX2 specifically inhibits the activity of both canonical and non-canonical NF-κB isoforms RelA and RelB, respectively. To evaluate the transcriptional activity of each NF-κB isoform independently, we transfected RelA cDNA into RelB−/− cells and RelB cDNA into RelA−/− cells along with SIX2, 5×κB-LUC. Transfection of both RelA and RelB potently induced NF-κB transcription of the indicated cells, and this transcription was suppressed by SIX2. Luminescence units were measured post 48 hours transfection. Data are mean±s.d. from 12 (left) and 9 (right) independent experiments. All western blot data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, In order to narrow down the possible mechanisms for SIX-proteins inhibition of inflammatory gene expression, we monitored the activation, processing and nuclear translocation of RelA and RelB upon cellular stimulation with TWEAK. WT and SIX1−/−
SIX2−/− H1792 cells were treated with mock or 50 ng/ml TWEAK for 24 hours. Cells lysates from cytoplasmic or nuclear fractions were analyzed by western blot. Neither SIX1 nor SIX2 blocked RelA phosphorylation, p100/52 processing, or restricted NF-κB translocation to the nucleus. b, Domain analysis of SIX-family protein function. SIX-proteins are composed of a Six Domain (SD), Homeobox domain (HD), and Coiled Coil (CD) region (diagram). Full-length SIX1 and SIX2 have 80% identical amino acids over the entirety of the protein coding sequence. The SD and HD domains (residues 1–183, highlighted) are 96% identical. The indicated FLAG-tagged SIX2 fragments were transfected alone into U-2 OS cells and processed for microscopy (middle) or transfected with 5×κB-LUC into HEK293T cells and processed for κB reporter activity (bottom). The highly conserved SD-HD domain was the minimal fragment that inhibited κB reporter activity. This fragment strictly localized to the nucleus, together indicating that SIX-proteins inhibit nuclear activity of NF-κB. Graph data were analyzed as described in Extended Data Fig. 5d. Data are mean±s.d. from 9 independent experiments. c, SIX2 inhibits gene activation from the IL8-promoter. pIL8-LUC plasmid composed of the 1.5 kilobase (kb) promoter region of IL-8 cloned upstream of luciferase gene, was co-transfected with indicated plasmids into HEK293T cells. After 24 hours, cells were treated with mock or 25 ng/ml TNF for 24 hours. The luciferase activity was then measured and analyzed as described in Extended Data Fig. 5d. Data are mean±s.d. from 9 independent experiments. Together, these data suggested that SIX-proteins inhibit NF-κB activation at gene promoters. d, Chromatin immunoprecipitation (ChIP) experiment providing additional evidence that SIX-proteins bind to inflammatory gene promoters as shown in Fig. 3a. Chromatin was prepared from GFP-SIX1 stable cell lines (HCT116 cells) treated with mock or 25 ng/ml TNF for 2 hours. Anti-SIX1 antibodies (or anti-IgG control) were used to immunoprecipitate SIX1 from nuclear extracts. Co-eluted DNA was amplified by primer sets as shown in Fig. 3a. Relative promoter occupancy was normalized to each experimental IgG control. Bars are mean of 2 technical replicates (shown as circles) and data are representative of 3 independent experiments. e, Control experiments corresponding to Fig. 3a and Extended Data Fig. 6f showing GFP-SIX1 expression levels. WT and GFP-SIX1 stable fibroblasts were stimulated with 50 ng/ml TWEAK for 24 hours. GFP-SIX1 expression was measured by western bot. f, SIX1 expression does not affect recruitment of RelA to the IL-8 promoter. Chromatin was prepared from WT or GFP-SIX1 stable fibroblasts and then immunopreciptated with anti-RelA. Bound DNA was amplified and quantified by qPCR. Results were adjusted to “input DNA” that was saved prior to immunopreciptation. Relative enrichment was then normalized to each group’s untreated control. Data are presented as mean±s.d. from 3 independent experiments. g, Control experiments corresponding to Fig. 3c showing that SIX2 inhibits GAL4-RelA and GAL4-RelB induced 5×κB-LUC activity. To validate if GAL4-RelA and GAL4-RelB constructs were functional, GAL4 DNA-binding domain fused RelA or RelB construct was co-transfected with indicated plasmids into HEK293T cells. 48 hours post transfection, the luminescence units were measured. Data are mean±s.d. from 9 (left) and 12 (right) independent experiments. h, Model showing NIK-mediated reactivation of SIX-proteins function in a negative feedback loop to control inflammatory gene expression by targeting gene promoter and inhibiting NF-κB trans-activation function. In quiescent cells (top panel), NIK and SIX-proteins are constitutively ubiquitinated and degraded by the proteasome. Non-canonical NF-κB agonists (e.g. TWEAK, LTα1β2, or BV6) promote degradation of cIAPs, loss of NIK ubiquitination and subsequent NIK protein accumulation (middle panel). Stabilized NIK protein activates non-canonical NF-κB mediated inflammatory gene expression (middle panel). Under conditions of long-term cytokine exposure, NIK-mediated suppression of a currently unknown E3-ubiquitin ligase results in SIX-protein accumulation (bottom panel). Consequently, SIX-proteins suppress inflammatory gene expression by targeting gene promoters and directly inhibiting NF-κB trans-activation function in a negative feedback loop (bottom panel). All western blot and microscopy data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, Schematic representation of the doxycycline-induced HA-SIX1 bitransgenic mouse model system. CAG-rtTA3 mice were intercrossed with Tet-on driven HA-SIX1 mice to obtain rtTA3+/−-SIX1+ mice. In principle, doxycycline bound rtTA3 targets the Tet-on operator and drives broad HA-SIX1 expression across multiple tissues. Primer sets used to genotype CAG-rtTA3 on chromosome 8 and Tet-on HA-SIX1 are shown. Electrophoresis gels shows genotyping of a representative rtTA3+/−-SIX1+ bitransgenic mouse. b, Anti-SIX1 western blot of whole cell lysates from BMDMs isolated from rtTA3+/−-SIX1+ mice. HA-SIX1 is not expressed in the absence of doxycycline under quiescent condition (left lane). TNF was administered to these cells as a control showing that endogenous murine Six1 is stimulated in these cells (right lane). c, Whole cell lysates from doxycycline treated BMDMs isolated from rtTA3+/− or rtTA3+/−-SIX1+ mice. BMDMs were stimulated with TNF, as indicated, and probed with anti-SIX1 antibody by western blot. Dox induced HA-SIX1 expression (lane 1), and this induction is potentiated by TNF (lane 2). We noted that HA-SIX1 ran as a doublet, which potentially represents unmodified and a mono-ubiquitinated form of the SIX1 protein. Neither endogenous Six1 nor HA-SIX1 was detected in BMDMs isolated from Dox treated rtTA3+/− mice (lane 3). In control experiments, TNF induced endogenous murine Six1 in BMDMs isolated from Dox treated rtTA3+/− mice (lane 4). d, HA-SIX1 is expressed in liver and spleen. rtTA3+/−-SIX1+ and rtTA3+/− mice were given 2 mg/ml doxycycline through drinking water for 10 days. Cell lysates from liver and spleen were used to probe HA-SIX1 expression by anti-SIX1 western blot. All western blot data are representative of 3 independent experiments. e, Peritoneal macrophages were isolated from rtTA3+/−-SIX1+ and rtTA3+/− littermate control mice. The adherent macrophages were incubated with 2 μg/ml doxycycline for 24 hours and then treated with mock or 100 ng/ml LPS for 4 hours. Total RNA was isolated for RT-qPCR. Relative gene expression was normalized to rtTA3+/− untreated control. Bars show mean from 2 technical replicates (shown as circles). Data are representative of 3 independent experiments. f, Diagram showing the experimental procedures corresponding to Fig. 4a–c. Further experimental details are provided in the methods section. For gel source data, see Supplementary Figure 1.
To further validate the observation that SIX1−/−
SIX2−/− sensitized NSCLCs cell lines to combined BV6 and TNF induced cell death, we chose SV40 immortalized STAT1−/− fibroblasts for additional studies. a, b, Fibroblasts of the indicated genotype were treated with either BV6 (a) or TNF (b) alone. Cell viability was determined by measuring ATP after 24 hours. Cell survival rate was normalized to each genotype untreated control. Neither BV6 nor TNF (10ng/ml) alone induced fibroblast cell death. c, Knockout of NIK or SIX1/SIX2 sensitized fibroblasts to combined BV6/TNF induced cell death. Experiments were performed and data were analyzed as in a (graph). Representative images showing that the cell death phenotype induced by BV6/TNF in fibroblasts of the indicated genotype (right panels). d, Time course of combined BV6 (2.5 μM) and TNF (25 ng/ml) treatment of the indicted fibroblast genotypes. NIK−/− and SIX1−/−
SIX2−/− cells exhibited increased cleavage of poly ADP-ribose polymerase (PARP) and the executioner Caspase-3 in BV6/TNF treated fibroblasts. We also noted that BV6/TNF induced NIK-dependent expression of both SIX1 and SIX2 proteins suggesting this cascade may be responsible for resistance to this treatment. e, f, We introduced a silent mutations in gRNA recognition sequence of the SIX2 cDNA that cannot be targeted by CRISPR-Cas9 (SIX2R, e, diagram). Expression of SIX2R in SIX1−/−
SIX2−/− fibroblasts rescued the cell death phenotype (e) and suppressed both PARP and Caspase-3 cleavage (f) induced by BV6/TNF. WT and SIX1−/−
SIX2−/− fibroblasts were transduced with Fluc or SIX2R lentivirus. After 72 hours, cells were treated with mock or 0.2 μM BV6 plus 10 ng/ml TNF for 24 hours and cell viability was determined by measuring ATP. Cell survival rate was normalized to each untreated control (e, bottom). For western blot, cells were treated with 2.5 μM BV6 plus 25 ng/ml TNF for 6 hours (f). All quantified data are mean±s.d. from 9 independent experiments. **P<0.01, ****P<0.0001. Western blot data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a-d, Knock out NIK or SIX1/SIX2 in U-2 OS cells using the CRISPR-Cas9 system. Western blot shows endogenous NIK, SIX1, or SIX2 expression in parental, NIK−/− and SIX1−/−
SIX2−/− U-2 OS cells (a, c). We employed MG132 to stabilize endogenous NIK protein in WT and NIK−/− U-2 OS cells. Schematic representation of In/Del base pairing and the sgRNA targets locus of Exon 1 in the NIK in U-2 OS cells (b). NIK−/− contains +G, −5bps, and −18 bps (disrupted the alternative splicing site) alleles. Schematic representation of In/Del base pairing and the sgRNA targets locus of Exon 1 in the SIX1 and SIX2 gene in U-2 OS cells (d). SIX1−/−
SIX2−/− contains +T of SIX1 and SIX2. e, SIX1−/−
SIX2−/− and NIK−/− U-2 OS cells are sensitive to BV6/TNF-induced apoptosis. WT, NIK−/−, or SIX1−/−
SIX2−/− U-2 OS cells were treated with 25 ng/ml TNF alone or in the presence of indicated concentrations of BV6 for 48 hours. The cell viability was measured by ATP. Cell survival rate was normalized to the absence of BV6 control. f, g, Expression of cDNAs SIX2R (see Extended Data Fig. 8e) or SIX2 protected SIX1−/−
SIX2−/− or NIK−/− cells, respectively, from BV6/TNF induced apoptosis. WT, NIK−/−, or SIX1−/−
SIX2−/− U-2 OS cells were transduced with Flu, SIX2 or SIX2R lentivirus as indicated. Cells were then treated with 2.5 μM BV6 plus 25 ng/ml TNF for 24 hours. h, j, We confirmed that cell death was mediated by the extrinsic apoptotic pathway, as both the pan-Caspase inhibitor (z-VAD) and specific Caspase-8 inhibitor (z-IETD) blocked BV6/TNF-induced cell death SIX1−/−
SIX2−/− fibroblasts (h) and U-2 OS cells (j). WT and SIX1−/−
SIX2−/− fibroblasts were treated with 1 μM BV6 plus 10 ng/ml TNF alone or in the presence of 20 μM z-VAD or z-IETD for 24 hours. For U-2 OS experiments, cells were treated with 2.5 μM BV6 plus 25 ng/ml TNF alone or in the presence of 20 μM z-VAD for 48 hours. The cell viability was measured by ATP. Cell survival rate was normalized to each untreated control. i, Western blot data showing PARP/Caspase-3 cleavage and the effect of Caspase inhibitors on BV6/TNF induced cell death of WT and SIX1−/−
SIX2−/− fibroblasts. Cells were treated with mock, 2.5 μM BV6 plus 25 ng/ml TNF alone or in the presence of 30 μM z-VAD or 40 μM z-IETD for 6 hours. All quantified data are mean±s.d. from 9 (e, f, h, and j) and 6 (g) independent experiments. *P<0.05, ****P<0.0001. Western blot data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, To validate cluster 1 genes from RNA-seq data shown in Fig. 4f, WT and SIX1−/−
SIX2−/− H1792 cells were treated with mock or 5 μM BV6 plus 25 ng/ml TNF for 24 hours. Total RNA was then isolated for qRT-PCR. Gene expression was normalized to WT untreated control. Data are mean±s.d. of 3 technical replicates and are representative of 3 independent experiments. b, Ingenuity pathway analysis (IPA) of Cluster 1 genes was performed as described in the methods. Pathway enrichment bar plots are shown. Data are from 2 independent experiments. The significance values for the canonical pathways are calculated by Fisher’s exact test right tailed. c, Experiment showing that inflammatory gene transcription is highly induced in SIX1−/−
SIX2−/− H1792 cells (compared to WT controls) upon specific stimulation of the non-canonical NF-κB signaling pathway. WT and SIX1−/−
SIX2−/− H1792 cells were treated with mock or 50 ng/ml TWEAK for 3 hours. Total RNA was extracted for qRT-PCR. Gene expression level was normalized to WT untreated control. Data are mean±s.d. of 3 technical replicates and representative of 2 independent experiments.
a, b, Diagram and data showing lentiviral delivery of CD40L/RFP into U-2 OS cells and its effect on GFP-expressing Lm infection. Representative flow cytometry scatter plots showing (1) luciferase (Fluc) RFP transduced uninfected cells, (2) untransduced LmGFP infected cells, (3) FlucRFP transduced cells infected with LmGFP, and (4) CD40LRFP transduced cells infected with LmGFP (a). Quantification of Lm and Sf infection of CD40L transduced cells as indicated (b). Bacterial infectivity was normalized to Fluc control (shown as 100%). Data are mean±s.d. from 6 independent experiments, ****P<0.0001, P values were derived from biological replicates using one-way ANOVA (GraphPad). The same statistics were applied to later studies unless otherwise stated. c, Fibroblasts transduced with Fluc or NIK lentivirus and then infected with GFP expressing Lm and Sf as indicated. Quantification of infection is presented as in b. Data are mean±s.d. from 6 independent experiments, ****P<0.0001. Representative flow cytometry scatter plots showing infection efficiency as described in a. d, Repeated trials of NIK-stimulated genes that inhibit (green) or enhance (red) infection by bacterial pathogens (Lm and Sf), +ssRNA viral pathogens (EAV: equine arteritis virus, WNV: West Nile virus, and SINV: Sindbis virus), and +ssRNA viral pathogen (PIV3: parainfluenza virus type 3). The relative percentage of pathogen infection was normalized to Fluc control (black dotted line). Data are presented as box and whisker plots, box is percentiles, black line is the population median, whiskers indicating the highest and lowest values (6 independent experiments). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: no significant difference. e, Representative flow cytometry scatter plots showing Lm infection efficiency in Fluc and SIX2 expressed fibroblasts as described in a. Data are representative of 6 independent experiments.
a, qRT-PCR of total RNA isolated from WT or Nik−/− primary BMDMs infected with Lm (MOI=0.1) or treated with 25 ng/ml TNF for 24 hours. The relative gene expression was normalized to untreated control. Bars are the mean from 2 independent experiments and circles are the average of 2 technical replicates from each experiment. b, c, Levels of Six1 protein in WT or Nik−/− primary BMDMs infected with Lm (MOI=~0.1) for indicated time points (b) or for 24 hours (c). Whole cell lysate or nuclear extracts were probed with indicated antibodies by western blot. Quantification of Six1 protein levels (mean±s.d. from 3 independent experiments) in the indicated samples were normalized to WT cells (1.0). d, e, SIX1 expression levels in WT and NIK−/− fibroblasts treated with 50 ng/ml TWEAK (d) or 5 μM BV6 (e) for 24 hours and processed for western blot analysis as in b. f, qRT-PCR of total RNA isolated from WT and SIX1−/−
SIX2−/− fibroblasts treated with 50 ng/ml TWEAK for 24 hours. The relative gene expression was normalized to WT untreated control and shown as mean±s.d. of 3 technical replicates from one experiment. Data are representative of 3 independent experiments. g, qRT-PCR of total RNA isolated from WT and SIX1−/−
SIX2−/− fibroblasts transduced with Fluc or NIK lentivirus for 72 hours. The relative gene expression was normalized to WT Fluc transduced control and shown as mean±s.d. of 3 technical replicates from one experiment. Data are representative of 3 independent experiments. All western blot data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, ChIP qPCR analysis of SIX1 occupancy of the indicated genes from fibroblasts treated with mock or 50 ng/ml TWEAK for 2 hours. Location of each primer set compared to the gene start sites (ATG) and κB sites are shown (diagram). IgG control samples were normalized to 1.0 and relative fold enrichment of SIX1 is shown as mean±s.d. of 3 technical replicates from one experiment. Data are representative of 3 independent experiments. b, Western blot showing input and Co-Immunoprecipitation (Co-IP) of Flag-SIX2 and association with HA-RelA (upper blot) or HA-RelB (lower blot) expressed in HEK293T cells. c, Graph showing luminescence units from 5×GAL4-Luciferase reporter gene driven by RelA (upper) and RelB (lower) fused to GAL4 DNA-binding domain. Reporter constructs were co-transfected with or without SIX2 and measured after 48 hours as indicated. Data are mean±s.d. from 9 independent experiments. d, Yeast two hybrid analysis of SIX2 binding to RelA TAD domain. Diagram shows RelA domains (RHD: Rel homology domain, TAD: Transactivation domain). Yeast transformed with the indicated plasmids were serial diluted and spotted on SD/UWL− (SD+His) or SD/WHULK− (SD-His) used to detect His-reporter gene activation by protein-protein interactions (bottom). e, The relative fold enrichment of RNA Pol II on the indicated genes in mock or 50 ng/ml TWEAK (2 hours) treatment of WT or SIX1−/−
SIX2−/− fibroblasts as in a. Mock treated WT fibroblasts were normalized to 1.0 by adjusting to “input DNA” that was saved prior to immunopreciptation. Relative fold enrichment of RNA Pol II is shown as mean±s.d. of 3 technical replicates from one experiment. Data are representative of 3 independent experiments. All western blot and yeast two hybrid data are representative of 3 independent experiments. For gel source data, see Supplementary Figure 1.
a, b, rtTA3+/−
SIX1+ mice or littermate controls (rtTA3+/−) were challenged with LPS as described in Extended Data Fig. 7f. LPS-induced survival curve (a) and clinical score outcome (b) are representative of 3 independent experiments. The number of animals (n) are shown. Data are mean±s.e.m and P value was measured by two tailed unpaired Student’s t-test (b). c, serum Il-1β and Cxcl2 production in the indicated genotypes treated with LPS as described in Extended Data Fig. 7f. The number of animals (n) are shown. P value was measured by two tailed unpaired Student’s t-test. d, e, H1155 (left), H1792 (middle), or H2087 (right) cells were treated with 10 ng/ml TNF alone or in the presence of BV6 for 24 hours. Cell survival rate was normalized to the absence of BV6 control. Data are presented as box and whisker plots (9 independent experiments). Box is percentiles, line is the population median, and whiskers indicate the highest and lowest values. ****P<0.0001 (d). Western blot showing endogenous SIX1 and SIX2 expression in the indicated cells (e). Data are representative of 3 independent experiments. f, RNA-seq data from WT and SIX1−/−
SIX2−/− H1792 cells treated with mock or 5 μM BV6 plus 25 ng/ml TNF for 24 hours. 1024 genes were significantly induced by BV6/TNF treatment in WT cells (statistical test is presented in methods). 120 out of 1024 genes were significantly upregulated (left), 804 genes were unchanged (middle), and 100 genes were downregulated (right) in SIX1−/−
SIX2−/− cells. The representative genes were listed. Data are from 2 independent experiments and presented as box and whisker plots. Box is percentiles and line is the population median from the indicated number of genes. For gel source data, see Supplementary Figure 1.
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