EMBO/SourceData huggingface dataset:
Figure 2B-E) Expression of Hep-ID (n=13 genes), Hep-IDCONNECT (n=26 genes) and remaining TF-encoding genes from cluster G (Others; n=82 genes) was monitored in indicated transcriptomic data Box plots show log2 fold changes between adult versus newborn mouse livers (B), MPH of Hnf4ahep-/- (Hnf4a KO) versus wild-type mice (C), a meta-analysis of severe mouse liver injuries versus control livers and microdissected hepatocytes from alcohol-related human liver cirrhosis (alcoholic steatohepatitis) versus control livers (E). Box plots are composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Statistical significance was assessed using one-sided Wilcoxon rank sum test with Benjamini-Hochberg correction for multiple testing to determine if the mean log2 FC was statistically lower (B, D, E) or higher (C) than 0. *q<0.05.I) Binding of indicated Hep-ID and Hep-IDCONNECT TFs to the promoter of identity effector genes and a control group of non-TF-encoding genes of similar size (n=424) was monitored using mouse liver ChIP-seq data. A control group of non-TF-encoding genes (n=424) matched for their promoter mouse liver activity was also used The control groups used were selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of ChIP-seq signals is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. Pairwise one-sided Wilcoxon Rank Sum Tests with Benjamini-Hochberg correction was used to define whether the binding at identity effector genes versus control genes was significantly greater for each analyzed TF recruitment. *q<0.05.
Figure 3A) Binding of indicated Hep-ID and Hep-IDCONNECT TFs to the promoter of Hep-ID TF genes and a control group of non-Hep-ID TF-encoding genes matched for their promoter activity of similar size (n=13) was monitored using mouse liver ChIP-seq data. The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of ChIP-seq signals is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. One-sided Wilcoxon rank sum tests with Benjamini-Hochberg correction was used to define whether the binding on Hep-ID TF gene promoters was greater than on control genes for each individual TF ChIP-seq dataset. *q<0.05.B) Transcriptional modulation of Hep-ID TFs and a control group of non-Hep-ID TF-encoding genes matched for their promoter activity of similar size (n=13) in mouse liver/MPH of mice deleted for the indicated Hep-ID or Hep-IDCONNECT TF genes. The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of log2 fold changes is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line. Whiskers extent to the most extreme data point which is no more than 1.5 times the interquartile range from the box. One-sided Wilcoxon rank sum tests with Benjamini-Hochberg correction was used to define whether log2 fold changes for Hep-ID TF genes were lower than those of the control genes for each individual transcriptomic dataset. *q<0.05.D) Average gene expression levels of Mlxipl in the livers of WT and XBP1hep-/- mice across circadian time (n=2 mice per group). Error bars show standard deviations.
Figure 4B) Overall survival of patients with HCC expressing low (n=289) or high (n=73) levels of the Hep-IDCONNECT TF encoding genes. Differential overall survival analysis was assessed by Kaplan-Meier (KM) log rank adjusted for 100 permutationsE) Transcriptional modulation of Hep-ID TFs (n=13 genes) and a control group of non-Hep-ID TF-encoding genes matched for their promoter activity of similar size (n=13 genes) in Huh7 or HepG2 cells treated with GW3965, T3 or GC-1, respectively. The control group used was selected for providing data representative of those obtained with 1000 reiterations of this analysis The distribution of log2 fold changes is shown using box plots. Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction was used to define whether transcriptional regulation of Hep-ID TF genes was different from that of the control genes for each individual transcriptomic dataset. *q<0.05.mRNA expression of the indicated genes was monitored using RT-qPCR in HepG2 cells treated with T3 or GC-1 for 24h or 96h. Bar graphs show mean ±SD (n=3 biological replicates) of log2 fold changes in treated versus untreated HepG2 cells. Gray dots show the results obtained from the 3 independent biological replicates (each performed in technical triplicates). One-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0. *q<0.05.mRNA expression of the indicated genes was monitored using RT-qPCR in HepG2 cells treated with T3 or GC-1 for 24h or 96h. For Hnf4a, the log2 fold change in the ratio of P1 over P2 promoter-derived isoforms is also shown. Gray dots show the results obtained from the 3 independent biological replicates (each performed in technical triplicates). One-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log2 FC was statistically different from 0. *q<0.05.I) Western blots assays performed using antibodies against the indicated proteins on extracts from HepG2 cells treated or not with T3 for 24h. Rep#1-3 indicates the 3 independent biological replicates analyzed.L) Identity effector genes significantly downregulated in Nodules/Control (q<0.05) were split in 3 groups according to their log2 fold changes (i.e. low, intermediate and high repression; pink boxes) and then monitored for induction in Nodules+T3/Nodules (green boxes). Statistical differences between the High repression and the other groups regarding the Nodules/Control comparison, on the one hand, or the Nodules+T3/Nodules comparison, on the other hand, were defined using Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction for multiple testing correction. *q<0.05.
Figure 5B-D) mRNA expression of the indicated genes was monitored in mouse livers using RT-qPCR. Mice treated with IL1B+T3 were subdivided into tertiles based on the mean expression of the Dio1 and Hectd3 genes and defined as low, intermediate or high T3 responsiveness groups (Low resp., Int. resp. and High resp., respectively). Fold change relative to the mean of the control group is shown using box plots composed of a box from the 25th to the 75th percentile with the median as a line (n=13 mice for the PBS group, 17 for the IL1B group and 10 for the other groups). Whiskers extend to the maximum and minimum values. Statistical differences between the High resp. and the other IL1B-treated groups were defined using Kruskal-Wallis with two-sided Wilcoxon pairwise comparison tests followed by Benjamini-Hochberg correction for multiple testing correction. *q<0.05.
Figure 1(C) Flow cytometry analysis of CD8 T cell infiltration in B16F10 tumors from WT or KO mice 14 days post-injection (DPI) (n = 7 per genotype). Statistical analysis was performed by 2-way ANOVA with Sidak correction (B, F), Mann-Whitney test The data are representative of 2-4 independent experimentssurvival curves (G) of Cre+ or TKO mice injected subcutaneously with B16F10 OVA cells (n = 7 per genotype). Survival time was defined as the time required for a tumor to reach a volume of 500 mm3.(J) Representative plots (left panels) and quantification (right panels) of IFNγ- and TNFα-producing CD8 TILs of B16F10 OVA tumors from Cre+ (n = 10) and TKO (n = 12) animals at day 14.
Figure 2(D) Representative flow cytometry plots (left panels) and quantification (right panel) of early activation markers CD25 and CD69 in CD8 cells activated ex vivo with CD3/CD28/IL-2 for 24 h (n = 7 mice per genotype). T cells were isolated as in A. (E) Representative flow cytometry plots (left panels) and quantification (right panel) of IFNγ+ CD8 cells activated ex vivo as in B (n = 9 mice per genotype). T cells were isolated as in A.(F) Oxygen consumption rate of Cre+ (n = 8 mice) or TKO (n = 7 mice) CD8 T cells activated ex vivo as in B after mitochondrial stress test analysis. Two representative WT and TKO animals are shown (left panel); the corresponding quantification for basal and maximum respiration is shown (right panels). Values are normalized to basal Cre+ measurements. T cells were isolated as in A.
Figure 3(C) qPCR of Cpeb4 mRNA in CD8 T cells resting or activated ex vivo with CD3/CD28/IL-2 at the indicated time-points; Tbp is used as endogenous control (n = 5 mice). CD8 T cells were isolated from the spleen and lymph nodes from WT mice.(D) Western blot (left panel) and quantification (right panel) of CPEB4 protein expression in CD8 resting or activated ex vivo with CD3/CD28/IL-2 at the indicated time-points; β-actin is used as loading control (n = 7 mice). CD8 T cells were isolated as in C.(F) Western blot (left panel) and quantification (right panel) of CPEB4 protein expression in OT-I T cells resting or activated ex vivo with OVA/IL-2 at the indicated time-points; β-actin is used as loading control (n = 4).
Figure 4(C) Western blot analysis (upper panel) and quantification (lower panel) of eIF2α phosphorylation in WT (n=4) and TKO (n=4) CD8 T cells activated as in A; total eiF2 α and β-actin are used as loading control.(D) qPCR analysis of Atf4, Ddit3, Xbp1s and Hspa5 mRNA levels in Cpeb4+/+ (n = 5) and Cpeb4-/- (n = 6) CD8 cells activated as in a; Tbp is used as endogenous control (n = 5).(E) Western blot analysis (left panel) and quantification (right panels) of BIP and CHOP proteins in WT (n=7) and TKO (n=6) CD8 T cells activated as in A.(F) Ddti3 mRNA levels in CD8 T cells from B16F10 tumors and spleen of WT (n=8) and TKO (n=10) mice. The data are shown as ratio between tumors and spleen.(G) Flow cytometry analysis of apoptotic AnnexinV+ Cre+ (n = 4) or TKO (n = 6) CD8 cells activated as in A.
Figure 5(A) Western blot of CPEB4 in input and immunoprecipitated fractions using anti-CPEB4 antibody of Cpeb4+/+ and Cpeb4-/- CD8 T cells activated ex vivo with CD3/CD28/IL-2 for 48 h; β-actin is used as control (n = 3).(D) RIP-seq data from several 3′ UTRs depicting normalized RIP-seq coverage for inputs (light grey and blue) and IP (dark grey and blue) from Cpeb4+/+ and Cpeb4-/- CD8 T cells. Image obtained using the integrated genome viewer (IGV).(E) Western blot (left panel) and quantification (right panel) of DDIT4 protein levels in Cre+ and TKO CD8 T cells activated as in A; β-actin is used as loading control (n = 7). (F) Western blot (left panel) and quantification (right panel) of HERPUD2 protein levels in Cre+ and TKO CD8 T cells activated as in A; β-actin is used as loading control (n = 8).(G) Basal oxygen consumption rate (left panel) and maximal oxygen consumption rate (right panel) after mitochondrial stress test analysis. Cre+ and TKO CD8 T cells were activated as in A, but were treated with either Tauroursodeoxycholic Acid (TUDCA, T) 250 μM or vehicle for the last 24 h. Values are normalized to basal Cre+ measurements. n = 7 for all conditions in basal respiration; n = 6 for all conditions in maximal respiration.(I) Quantification of IFNγ+ CD8 cells activated and treated as in G (n = 9 for Cre+ and n = 12 for TKO in both conditions). *P = 0.0168, ** P = 0.0014 for Cre+ vs. TKO, P = 0.0029 for Cre+ vs. Cre+ TUDCA, **** P <0.0001.
Figure 1C. RBM3 E3a regulation is conserved in humans. HEK293 cells were incubated at the indicated temperatures for 12h (DMSO/CHX last 4h) and investigated for E3a inclusion as in B (mean ± s.d., n= 3, all individual data points are shown).
Figure 2A. CRISPR/CAS9 mediated removal of RBM3 E3a. One of two guide RNAs targeting the upstream intron (#1, #2) were co-transfected with a guide RNA targeting the downstream intron (#3). Below, genotyping PCR after clonal selection with px459 transfected cells serving as a negative control.RT-qPCR (B) analysis of RBM3 levels in edited cell lines. Clonal cell lines from A were incubated at the indicated temperatures for 24h. In B, isolated RNA was investigated by qPCR and rbm3 expression is shown relative to gapdh levels.(C) analysis of RBM3 levels in edited cell lines. Clonal cell lines from A were incubated at the indicated temperatures for 24h. In C, lysates from an independent experiment were investigated for RBM3 protein expression, hnRNP L served as a loading control, a representative gel (left) and quantification (right) are shown (mean ± s.d., n= 6 (n= 3 per clone, indicated in black/grey), all individual data points are shown).Manipulation of RBM3 E3a splicing directly controls RBM3 expression levels. In D, HEK293 cells were transfected for 48h at 37°C with a MO blocking the 5'ss of E3a. RBM3 expression is shown relative to GAPDH levels and normalized to a non-targeting MO (n= 4-6, line indicates median, whiskers min to max, all individual data points are shown).Manipulation of RBM3 E3a splicing directly controls RBM3 expression levels. In E, primary hippocampal neurons were transfected with the indicated MOs for 48 hours and investigated for RBM3 by Western blotting. GAPDH served as a loading control (n= 3; line indicates median, whiskers min to max, all individual data points are shown).
Figure 3D. ASOs targeting M2-9, M4-7 or the 5'ss (see Figure EV4A and Table EV2) prevent endogenous RBM3 E3a inclusion in human HeLa cells. ASO-transfected cells were kept for 24 hours at 40°C. Control samples at 37°C and 40°C are shown; CHX was added for the last 4 hours. Exon 3a inclusion was investigated by splicing sensitive RT-PCR, a representative gel and phosphorimager quantification are shown (mean ± s.d., n= 3). The hashtag marks the use of internal 5' and 3'ss that is promoted by all ASOs targeting the M4 region. ASOs targeting the 5'ss induced the usage of an internal 5'ss (marked by two asterisks).M2D induces RBM3 mRNA (E) expression in human HeLa cells. ASOs were transfected for 24 hours at 37°C (grey) or at 40°C (red). RBM3 induction was measured relative to GAPDH expression (mean ± s.d., n= 3, all individual data points are shown).M2D induces RBM3 protein (F) expression in human HeLa cells. ASOs were transfected for 24 hours at 37°C (grey) or at 40°C (red). RBM3 induction was measured relative to GAPDH expression (mean ± s.d., n= 3, all individual data points are shown).G. M2D induces RBM3 protein expression in vivo. Hippocampus samples from two mice per condition were analyzed by Western blotting (left) and RBM3 protein was quantified relative to actin and PBS (right, mean ± s.d., n= 2, all individual data points are shown).
Figure 4B. Western blot of hippocampal lysates from prion-infected mice treated with non-targeting control ASO (n= 5 mice) or M2D (n= 8 mice). M2D increases RBM3 expression by 2-fold compared to control ASO-treated mice, 9 weeks after ASO injection at 12 w.p.i. ** p= 0.0058, calculated using student's t-test.C. Representative images of haematoxylin and eosin stained brain slices from NBH (control), and prion-infected control ASO- and M2D-treated mice at 12 w.p.i. when control ASO-treated mice were culled for prion signs. M2D confers marked neuroprotection in the hippocampus, with conservation of CA1-3 pyramidal layer, protection from shrinkage of the whole hippocampus, as well as reduced spongiform change.D. NeuN counts of pyramidal neurons in NBH (n= 5 mice) versus control ASO-treated (n= 5 mice) and M2D-treated prion mice (n= 8 mice). M2D confers neuroprotection close to levels seen in NBH mice. control ASO-treated versus M2D-treated prion mice ** p= 0.0014. One-way ANOVA.F. Total PrP and proteinase K-resistant PrPSc levels in NBH-injected mice and in prion-diseased mice injected with control - or M2D ASOs. PrPSc levels are unaffected by M2D-mediated RBM3 induction.
Figure 1(Ci) The levels of WDR6 were determined and normalized against GAPDH in three groups. Normal, n=21; Noninvasive, n=15; Invasive, n=8. (Cii) The levels of WDR6 in 10 pairs of patients' tissues.(E) The DFS and OS assays of HCC patients (n=201) stratified by WDR6 expression.(F) Analysis of WDR6 using TCGA LIHC data. Fi and Fiii were plotted with GEPIA analyzing tool, and Fii were analyzed by UALCAN analyzing tool.
Figure 2(A) Western blots confirmed WDR6 knockdown in HCC cells transduced with shWDR6 or shcontrol lentiviral vector.(B) The effects of WDR6 silencing on the invasive and proliferative ability of HCC cells, respectively. Left panel: HCC-LM3: P=0.912 shControl vs shWDR6-#1, P=0.889 shControl vs shWDR6-#2; HepG2: P=0.826 shControl vs shWDR6-#1, P=0.796 shControl vs shWDR6-#2; right panel: HCC-LM3: P=0.856 shControl vs shWDR6-#1, P=0.814 shControl vs shWDR6-#2; HepG2: P=0.726 shControl vs shWDR6-#1, P=0.745 shControl vs shWDR6-#2.
Figure 3(C) The survival plot of Albumin-Cre/+; Wdr6f/f mice with DEN-induced HCC.(F) Flow cytometric analysis of MDSC and T cells in tumors from BALB/c mice with H22-WDR6 or empty vector cells orthotopically implantation (n=6/group).(G) α-Gr1 antibody injection markedly decreased the tumor volume and lung metastatic nodules' number in BALB/c mice with H22-WDR6 cells orthotopically implantation when compared to IgG control (n=5/group).
Figure 4(Ai) ATAC-sequencing showed the effects of WDR6 knockdown on the chromatin accessibility of IL10, IL1β, IL6, and TNFα loci. (Aii) quantitative real-time polymerase chain reaction (qRT-PCR) analyzing cytokines' mRNA levels in Hepa1-6 and H22 cells treated with or without shWDR6 or WDR6, respectively.(B) TNFα ELISA assays were performed in the conditioned medium from Hepa1-6 and H22 cells with WDR6 knockdown or overexpression, respectively.(D) The effects of TNFα knockdown in H22-WDR6 cells on MDSC recruitment (Dii), tumor size (Diii), and the ratio of IFN-γ+ CD8+ T cells (Div) in tumors from syngeneic mice (n=5/group).(E) TNFα supplementation decreased MDSC infiltration induced by WDR6 knockdown in tumor tissues from syngeneic mice (n=6/group).
Figure 5(A) The effects of NF‐κB inhibitor IMD0354 or IκBα‐SR on TNFα expression in MHCC-97L cells with or without WDR6 overexpression.(B) The effects of WDR6 knockdown on the protein levels of p65, RelB, c-Rel, p52, and p50 in HCC cells.(Di-ii) The effects of autophagic deficiency using 3-MA on the levels of p65 protein and TNFα secretion in HCC cells with WDR6 knockdown.(Ei) WDR6 mRNA levels in Hep3B and MHCC-97L cells treated with or without TNFα in culture media. (Eii) Schematic diagram showing three candidate NF‐κB-binding sites in WDR6 promoter. (Eiii) The impact of IMD0354 on the luciferase reporter of WDR6 proximal promoter in HCC cells. (Eiv-v) ChIP assays using HCC cells or HCC tissues from patients (P#36 and P#54). Da
Figure 6(C) Co-IP analysis indicated the effect of WDR6 mutant (R123A) or (R203A) on the binding of CUL4A to endogenous UVRAG in MHCC-97L cells.(Di) UVRAG ubiquitination assay in MHCC-97L cells expressing WT-WDR6 or its mutant WDR6-R123A. (Dii) UVRAG ubiquitination in HCC-LM3 cells treated as shown.(Ei) MS assay identified K176 of UVRAG as major ubiquitination site. (Eii) K176R mutant abolished UVRAG ubiquitination promoted by WDR6.
Figure 7(A) WDR6 or its mutant (Ai) or WDR6 shRNAs (Aii) or WDR6 with CUL4A siRNA (Aiii) affected the levels of endogenous UVRAG in HCC cell lines.(B) CHX assays of UVRAG stability in HCC-LM3 cells transfected with WDR6 shRNA.(C) The effects of WT-WDR6 or WDR6-R123A mutant on UVRAG levels in MHCC-97L cells in the presence of MG132.(Di-iii) UVRAG knockdown in Hepa1-6 cells reverted WDR6-deficient-induced chromatin accessibility of TNFα locus and TNFα secretion in culture medium. The cells in (Di) were orthotopically implanted into C57BL/6J mice.(F) Heat-map showing the expression of altered genes related to functional signatures in MDSCs after WDR6 alone or with UVRAG knockdown.
Figure 8(A) HEK293T cells with indicated constructs were treated with 5 μM Pep2-WDxR or Pep2-con or Pep2-WDxA mutant for 16 hours, respectively. Then co-IP assay was used to assess the impacts of these peptides on the WDR6-UVRAG binding.(C) BALB/c mice introduced with H22-WDR6 overexpression cells were administered with Pep2-WDxR or Pep2-con with or without anti-PD-L1 antibody. The images and sizes of HCC (Ci), hematoxylin-eosin (H&E) staining and metastasis nodules of lung were presented (Cii). The survival analysis of the mouse after treatment was performed (n = 9/group) (Ciii). In the BALB/c mice from (C), Flow cytometry was performed to analyze PMN-MDSC, M-MDSC, and IFN-γ+ CD8+ T cells in tumors (Civ).(D) The survival test of DEN- and CCl4-induced HCC using WDR6-knockout and WT mice. At twenty-six weeks, mice were administered with or without anti-PD-L1 antibody (n = 6/group).
Figure 2A Growth of indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3). B Growth of indicated Y. pestis strains in zinc-replete medium (cPMH2 + 10μM ZnCl2) (n=3). Data information: One-way ANOVA with Dunnett test, *p≤0.05, **p≤0.001, ****p≤0.0001. For A, B growth of mutants was compared to znuBC. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.C Growth of indicated Y. pestis strains in iron-limited (cPMH2 + 0μM FeCl2) or iron-replete (cPMH2 + 10μM FeCl2) media (n=3).D Growth of znuBC y0702-y0703, znuBC y0704, or y0702-y0703 complemented mutant (pBCSK+y0702-70703) in zinc-limited medium (cPMH2) (n=3). Data information: One-way ANOVA with Dunnett test, *p≤0.05, **p≤0.001, ****p≤0.0001. D, growth of mutants was compared to znuBC. Data represents the mean ±SD of three biological replicates. For some points, error bars are too small to visualize on the graph.
Figure 3B Growth of znuBC irp2 pGENlux in co-culture with indicated Y. pestis strains in zinc-limited medium (cPMH2) (n=3). Data information: For B, Data represents the mean ±SD relative luminescent units (RLU) from three biological replicates. For some points, error bars are too small to visualize on the graph. One-way ANOVA with Dunnett test compared to znuBC irp2 + Y. pestis; ****p≤0.0001.C Absorbance of PAR at 500nm incubated with indicated Y. pestis supernatants (n=3). D OD600 of bacterial cultures at 8 h for LC-MS (n=3). Data information: For One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.E LC-MS analysis of Ybt in culture supernatants (note: both peaks are Ybt). F LC-MS quantification (n=3). Data information For One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.G OD600 of bacterial cultures at 8 h harvested for GFP quantification (n=3). Data information: For , One-way ANOVA with Tukey test, **p≤0.01, ***p≤0.001, and ****p≤0.0001; ns=not significant. Mean ±SD of three biological replicates.
Figure 4A IC50 of calprotectin for indicated Y. pestis strains (n=3). Data information: For A, mean ±SD of three biological replicates. One-way ANOVA with Tukey's comparison: ns= not significant, **p<0.01, ***p=0.001.B C57BL6/J mice (n=10) subcutaneously challenged with 102 CFU of indicated Y. pestis strains. C C57BL6/J mice (n=10) intranasally challenged with 104 CFU of indicated Y. pestis strains. Data information: For A, mean ±SD of three biological replicates. One-way ANOVA with Tukey's comparison: ns= not significant, **p<0.01, ***p=0.001. For B and C, differences in survival were calculated by log rank test compared to Y. pestis. For results are combined data from 2 independent experiments (n=5 mice per experiment).