Chromatin activity of IκBα mediates the exit from naïve pluripotency
- Program in Cancer Research. Hospital del Mar Research Institute, Spain
- Josep Carreras Leukemia Research Institute, Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain
- Institut Investigació Biomèdica de Bellvitge (IDIBELL), L'Hospitalet de Llobrega, Spain
- Centre for Genomic Regulationlation(CRG), The Barcelona Institute of Science and Technology, Spain
- Universitat de Vic - Universitat Central de Catalunya, Spain
- Catalan Institute of Oncology (ICO/IDIBELL), L'Hospitalet de Llobregat, Spain
- Spanish Network for Advanced Therapies (RICORS-TERAV). Carlos III Health Institute (ISCIII), Spain
- Department of Biomedicine. University of Barcelona, Spain
- Institucio Catalana de Recerca i Estudis Avançats(ICREA), Spain
- Universitat Pompeu Fabra, Spain
- Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Spain
Figures
Figure 1
IκBα is highly expressed in pluripotent cells.
(A) Schematic of pluripotent states in mouse embryonic stem cells (mESCs). mESCs were maintained regularly in naïve pluripotency by culturing them in serum/Leukemia Inhibitory Factor (LIF) medium, or they were polarized towards the ground state of naïve pluripotency by culturing them for two passages in LIF plus Gsk3β (CHIR99021 or Chiron) and MEK (PD0325091) inhibitors (2i/LIF). mESCs were further differentiated towards the primed pluripotent state (epiblast-like cells) by culturing mESCs in a medium containing 20 ng/mL Activin A and 10 ng/mL Fgf2 for 120 hr. (B–C) Relative mRNA levels (based on qPCR experiments) of the canonical IκB genes (Nfkbia, Nfkbib, Nfkbie) (B) or Nf-κB effector genes (Rela, c-Rel, Relb, Nfkb1, Nfkb2) (C) across the different states of pluripotency. Expression was normalized using the house-keeping gene Tbp relative to 2i/LIF. Data from three independent experiments. Dots indicate mean values and error bars refer to ± standard deviation (SD). (D) Western blot analysis of IκBα protein distribution in cytoplasm, nucleoplasm (Nucleus), or chromatin in cells at the ground-state of naïve pluripotency (2i/L), naïve pluripotency (S/L), or primed pluripotency (Epi). This experiment was independently repeated twice. (E) Transcriptome and proteome differences across naïve (2i/LIF) and primed (Epiblast-like cells or EpiSCs) pluripotent states. Enriched transcripts and proteins in naïve state are highlighted in red (Log2 fold-change < –1). Enriched transcripts and proteins in the primed state are marked in dark gray (Log2 fold-change > 1). Data from Atlasi et al., 2020. (F). Heatmap showing the expression levels of pluripotency (Dppa4, Pou5f1, Sox2, Prdm14, Klf4, Zfp42, Nanog), differentiation (Mesp1, Gata4, Gata6, Lhx1, Wnt3a, Fgf5, Nes, Axin2, Wnt3, Eomes, Foxa2, T, Mixl1, Lef1) NF- κB (Nfkbia, Nfkbib, Nfkbie, Nfkbiz, Rela, c-Rel, Relb), and Polycomb (Ezh1, Ezh2, Eed, Epop, Jarid2, Suz12, Mtf2, Aebp2) genes from the RNAseq samples at mESCs (Serum/LIF) and differentiating cells (embryoid bodies) at 48 hr and 96 hr of IκBα-WT cells. Normalized counts based on z-score are represented.
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Figure 1—source data 1
Annotated files for western blot analysis displayed in Figure 1D.
- https://cdn.elifesciences.org/articles/102784/elife-102784-fig1-data1-v1.zip
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Figure 1—source data 2
Raw files for western blot displayed in Figure 1D.
- https://cdn.elifesciences.org/articles/102784/elife-102784-fig1-data2-v1.zip
Figure 2 with 1 supplement
The absence of IκBα hinders the exit from the pluripotent state and prevents the activation of differentiation programs.
(A) Representative immunofluorescence of 216 hr embryoid bodies (EBs) stained with the pluripotency markers NANOG and OCT3/4. (B) Quantification of total number of alkaline phosphatase (AP) colonies after 216 hr EB differentiation and additional culture of 96 hr in Serum/LIF (S/L) medium. Dots represent values from two independent experiments from three different IκBα-WT or IκBα-KO clones. Unpaired two-sided t-test applied. Representative images from alkaline phosphatase (AP) colonies are included in the upper panel. (C) PC1 and PC2 from Principal Component Analysis (PCA) of normalized RNA-seq data from three different time points (mESCs, 48 hr EBs, and 96 hr EBs) of IκBα-WT and IκBα-KO cells (three independent clones from each genotype). (D) Heatmap showing the expression levels of naïve pluripotency genes (Gab1, Sox2, Esrrb, Zfp42, Dppa4, Klf2, Prdm14, Klf4, Tfcp2l1) from the RNAseq samples of IκBα-WT and IκBα-KO EBs at 96 hr. Normalized counts based on z-score are represented. (E) Z- score values for gene sets referring to Endoderm, Mesoderm, and Ectoderm formation from The Gene Ontology database (GO:0001706, GO:0001707, and GO:0001705 terms, respectively) and obtained from corresponding expression levels at mESCs, 48 hr, and 96 hr Ebs IκBα-WT or IκBα-KO cells (three independent clones of each genotype). Loess curves are also represented with 95% confidence intervals (shadowed area surrounding the curves). (F) Schematic of teratoma formation assay. 5×105 IκBα-WT or IκBα-KO murine Embryonic Stem Cells (mESCs) cultured in Serum/Leukemia Inhibitory Factor (LIF) were injected intramuscularly in the leg of immunocompromised NSG mice. Six weeks after the transplant, teratomas were formed, and mice were euthanized for further teratoma analysis. (G) Representative images of immunohistochemistry for OCT3/4 in IκBα- WT (left panel) or IκBα-KO (right panel) teratomas. Teratomas were derived from three independent clones of mESCs of both genotypes. (H) Percentage of OCT3/4+ cells in teratomas. Positive cells were counted from five different microscope fields from each IκBα-WT and IκBα- KO clone. Unpaired two-sided t-test was performed.
Bars indicate mean values and error bars refer to ± SD. Significance level of 0.05 is considered for all statistical tests.
Figure 2—figure supplement 1
Depletion of IκBα in murine Embryonic Stem Cells (mESCs) compromises the exit from pluripotency.
(A) Schematic of the approach followed to deplete IκBα protein in mESCs (upper panel) and western blot analysis of three independent IκBα-WT and IκBα-KO mESC clones (bottom panel). (B) Volcano plot from differential expression analysis between IκBα-KO vs IκBα-WT 96 hr EBs. Dashed vertical lines represent an absolute shrunken log2 Fold Change = 1 and dashed horizontal line represents an adjusted p-value = 0.05 (FDR). (C) Flow cytometry plots of SSEA-1 from IκBα-WT or IκBα-KO EBs 216 hr. (D) Schematic of Alkaline Phosphatase (AP) staining carried out in embryoid bodies (EBs). IκBα-WT or IκBα-KO 216 hr EBs were dissociated and seeded in Serum/Leukemia Inhibitory Factor (LIF) medium for another 96 hr. Attached cells were screened for AP+ total cell number. (E) Violin plot and individual values showing the quantification of relative size of 168 hr Ebs. Each dot represents an embryoid body. Unpaired two-sided t-test was applied. (F) Relative RNA levels of naïve pluripotency genes (Pou5f1, Gbx2, Klf2, Sox2, Rex1, Nanog) in IκBα-WT or IκBα-KO 216 hr EBs. RNA levels were normalized based on Tbp expression relative to IκBα-WT. Each dot represents an independent clone either from IκBα-WT or IκBα-KO genotypes. Unpaired two-sided t-test was applied. (G) Relative RNA levels of differentiation genes (Mixl1, Mesp1, T, KDR, Gata6, Zic1) in IκBα-WT or IκBα-KO 216 hr EBs. RNA levels were normalized on Tbp expression relative to IκBα-WT. Each dot represents an independent clone either from IκBα-WT or IκBα-KO genotypes.
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/102784/elife-102784-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Raw files for western blot analysis displayed in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/102784/elife-102784-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
IκBα-KO murine Embryonic Stem Cells (mESCs) retain the ground-state of naïve pluripotency under Serum/Leukemia Inhibitory Factor (LIF) culture.
(A) (Upper panel) Schematics of mESCs differentiation towards Epiblast Stem Cells (EpiSCs). mESCs cultured in Serum/LIF were then seeded in N2B27 medium supplemented with 10 ng/mL Fgf2 and 20 ng/mL Activin A for 120 hr (see Materials and methods section for further information). (Bottom panel) Relative RNA levels (based on qPCR experiments) of naïve pluripotency genes (Nanog, Sox2, Rex1, Klf4, Gbx2, Tbx3, Dppa3) in IκBα-WT or IκBα-KO EpiSCs at 120 hr. RNA levels were normalized based on Tbp expression relative to IκBα-WT. Data from three independent clones from either IκBα-WT or IκBα-KO genotypes. Unpaired two-sided t-test applied. Dots indicate mean values and error bars refer to ± SD. (B) GSEA results comparing IκBα-KO vs IκBα-WT mESCs cultured in Serum/LIF against ground (2i/LIF) and naïve (Serum/LIF) state pluripotency signatures retrieved from Ghimire et al., 2018. Adjusted p-value by Benjamini-Hochberg procedure and Normalized Enrichment Score (NES) indicated. (C) Relative RNA levels of the naïve pluripotency genes Rex1 (Zfp42), Klf2, and Tbx3 upon culture of IκBα-WT either in Serum/LIF or two passages in 2i/LIF. 2i/LIF IκBα-WT mESCs were compared with IκBα-KO mESCs cultured in Serum/LIF. Unpaired two-sided t-test was applied to calculate statistical significance. Each dot represents an independent clone from each of the two genotypes. Horizontal bars indicate mean values and error bars refer to ± SD. (D) Representative immunofluorescence images of DNA-methylation-related mark 5-methylcytosine (5 mC) in IκBα-WT (left) and IκBα-KO (right) mESCs cultured in Serum/LIF. (E) DNA methylation profile (using DNA methylation arrays; Zhou et al., 2022) in IκBα-WT vs IκBα-KO mESCs (upper) and 96 hr EBs (bottom panel) showing the distribution density of mean ß-values from all 261,220 CpGs under test per condition. Significance level of 0.05 is considered for all statistical tests.
Figure 3—figure supplement 1
Absence of IκBα favors the ground state of naïve pluripotency in Serum/Leukemia Inhibitory Factor (LIF) conditions.
(A) Brightfield images of IκBα-WT or IκBα-KO EpiSCs at 120 hr picture corresponds to an independent clone from either IκBα-WT or IκBα-KO genotypes. (B) Bright-field microscopy images of IκBα-WT and IκBα-KO murine Embryonic Stem Cells (mESCs) cultured in Serum/LIF conditions. Two independent clones from each genotype are shown. (C) DNA methylation levels (mean ß-values) across chromosomes in IκBα-WT vs IκBα-KO mESCs.
Figure 4 with 1 supplement
Absence of IκBα reinforces the pluripotency program at the epigenetic and transcriptional levels.
(A) ChIP-seq Enrichment Analysis (ChEA) of differentially expressed genes (DEGs; adjusted p-value <0.05) in IκBα-KO murine Embryonic Stem Cells (mESCs). P-value was calculated using Fisher exact test. (B) Number of differentially bound regions (adjusted p-value <0.05, FDR) for H3K4me3 and H3K27me3 histone marks in IκBα -/- vs IκBα +/+ mESCs cultured in Serum/Leukemia Inhibitory Factor (LIF). Enriched or reduced regions in IκBα -/- mESCs are distinguished (log2 Fold-change >0 or <0, respectively). Overrepresented WikiPathways, for (left) H3K4me3 and (right) H3K27me3 are indicated. Annotated genes to differential regions showing H3K4me3 enrichment or H3K27me3 reduction were considered. A one-sided hypergeometric test was conducted. (C) Venn Diagram of genes contained in regions with either increased H3K4me3 or reduced levels of H3K27me3. Statistical significance of the overlapping was calculated using the chi-squared method. Overrepresented pathways for genes containing increased H3K4me3 and reduced H3K27me3 levels are indicated (only pathways with an adjusted p-value <0.05 were considered). (D–E) Representative genomic regions of naïve pluripotency genes having either differential enriched H3K4me3-only (D) or reduced H3K27me3-only (E) levels in IκBα-KO vs IκBα-WT mESCs cultured in Serum/LIF. Shadowed regions highlight the differential levels of the histone marks. (F) Number of regions with differential enhancer activity in IκBα-KO vs IκBα- WT mESCs cultured in Serum/LIF. Increased/Reduced activity for (left) poised enhancers based on the H3K4me1 differential binding and absence of H3K27ac overlapping peaks in IκBα-KO mESCs and (right) active enhancers based on the differential binding of H3K27ac and presence of H3K4me1 overlapping peaks in IκBα-KO. Differential binding based on adjusted p-value <0.05 (FDR). Increased or reduced regions in IκBα-KO are distinguished by log2 Fold-change>0 or<0, respectively, compared to IκBα-WT mESCs. Overrepresented WikiPathways for annotated genes to (left) poised enhancers with reduced activity and (right) active enhancers with increased activity in IκBα-KO vs IκBα-WT mESCs are indicated (adjusted p-value <0.05, FDR). A one-sided hypergeometric test was conducted. (G) Representative genomic regions of naïve pluripotency genes having an increase in active enhancer status (H3K27Ac enrichment) in IκBα-KO mESCs cultured in Serum/LIF. Shadowed regions highlight the differential levels of the histone mark. (H) Heatmap showing the expression levels of the naïve pluripotency genes from the RNAseq samples of IκBα-KO and IκBα-WT mESCs cultured in Serum/LIF. Normalized counts (z-score) from genes are represented.
Figure 4—figure supplement 1
Epigenetic rewiring characterization in IκBα-KO murine Embryonic Stem Cells (mESCs) cultured in Serum/Leukemia Inhibitory Factor (LIF).
(A–D) Volcano plots from differential binding analysis of H3K4me3 (A), H3K27me3 (B), H3K4me1 (C), and H3K27Ac (D) in IκBα-WT vs IκBα-KO mESCs cultured in Serum/LIF. Representative genes with enriched or reduced levels of H3K4me3 (A), H3K27me3 (B), H3K4me1 (C), and H3K27Ac (D) marks in IκBα-KO mESCs are highlighted. (E–F) Representative genomic regions of naïve pluripotency genes having higher H3K4me3 (E) or reduced H3K27me3 (F) levels in 2i/LIF compared to Serum/LIF medium. Displayed regions are the ones having differential H3K4me3 and H3K27me3 distribution in IκBα-WT vs IκBα-KO mESCs. Shadowed regions highlight the differential levels of the histone marks. Data from Galonska et al., 2015.
Figure 5 with 1 supplement
IκBα prevents the hyperactivation of naïve pluripotency program in an NF-κB- independent manner.
(A) Boxplots, violin plots, and individual values representing the normalized gene expression levels in three independent clones of IκBα-WT or IκBα-KO mESCs, embryoid bodies (EBs) 48 hr, and EBs 96 hr obtained from bulk RNAseq data. Considered genes are those annotated to NF-κB signaling pathway (KEGG PATHWAY mmu04064). Box Plots: center line, median; lower and upper hinges, first and third quartiles; whiskers, 1.5x interquartile range (IQR) extended to the largest/smallest value within 1.5x IQR; no outliers present. Unpaired two-sided Wilcoxon test applied. (B) Schematic of chromatin- specific/NF-κB-deficient (IκBαΔNF-κB) or NF-κB-specific/chromatin-deficient (IκBαΔChrom) IκBα mutants (Separation-Of-Function or SOF mutants). (C) Pull-down of GST-H2A and GST-P50 with cell lysates from different SOF IκBα mutant mESCs. (D) Western blot analysis of cytoplasmic protein extracts of doxycycline-inducible IκBα forms (i-IκBαΔWT, i-IκBαΔNF-κB, i-IκBαΔChrom) after 16 hr of doxycycline induction. (E) Western blot analysis of chromatin protein extracts of oxycycline-inducible IκBα forms (i-IκBαΔWT, i-IκBαΔNF-κB, IκBαΔChrom) after 16 hr of doxycycline induction. (F) Relative RNA levels of naïve pluripotency genes (Zfp42, Klf2, Sox2, Tbx3) in i- IκBαWT, i-IκBαΔNF-κB, and i-IκBαΔChrom mESCs after doxycycline induction. Unpaired two-sided t-test was performed. Dots indicate three independent experiments. Bars indicate mean values and error bars refer to ± SEM. (G) Representative immunofluorescence images of DNA-methylation-related mark 5-methylcytosine (5 mC) in i-IκBαΔWT (upper panel), i-IκBαΔNF-κB (medium panel), IκBαΔChrom (bottom panel) murine Embryonic Stem Cells (mESCs) cultured in Serum/Leukemia Inhibitory Factor (LI)F. (H) Flow cytometry analysis of the pluripotency surface marker SSEA-1 at 216 hr embryoid bodies (EBs) in i-IκBαWT, i-IκBαΔNF-κB and i-IκBαΔChrom cells. (I) Quantification of Alkaline Phosphatase (AP) staining in 216 hr EBs upon reconstitution with the different IκBα forms. Each dot represents an independent experiment. One-way ANOVA was applied followed by Tukey’s post-hoc test for all pairwise comparisons. Bars indicate mean values and error bars refer to ± SEM. Representative images of Alkaline Phosphatase (AP) staining in 216 hr EBs are included in the upper panel. (J) Representative immunofluorescence for NANOG and OCT3/4 at 216 hr EBs in i-IκBαWT (upper panel), i-IκBαΔNF-κB (medium panel), and i-IκBαΔChrom (bottom panel) cells. Significance level of 0.05 is considered for all statistical tests.
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Figure 5—source data 1
Annotated files for western blot analysis displayed in Figure 5C, D, E.
- https://cdn.elifesciences.org/articles/102784/elife-102784-fig5-data1-v1.zip
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Figure 5—source data 2
Raw files for western blot displayed in Figure 5C, D, E.
- https://cdn.elifesciences.org/articles/102784/elife-102784-fig5-data2-v1.zip
Figure 5—figure supplement 1
Reversion of IκBα-KO-related hyper pluripotent status is NF-κB independent.
(A) Relative expression level of Nfkbia gene after overnight doxycycline induction in i-IκBαΔWT, i-IκBαΔNF-κB, and IκBαΔChrom murine Embryonic Stem Cells (mESCs). (B) Schematic representation of reconstitution of IκBα-KO mESCs with either of the two doxycycline-inducible Separation-Of-Function (SOF) mutants (i- IκBαΔNF-κB or IκBαΔChrom). mESCs were then kept in Serum/Leukemia Inhibitory Factor (LIF) medium supplemented with 1 μg/mL Doxycycline for four consecutive passages. mESCs were characterized at the pluripotent level and then their potential to be further differentiated was addressed. (C) Percentage of differentiated cells (determined as SSEA-1) at 216 hr EBs 216 hr derived from i-IκBαΔWT, i- IκBαΔNF-κB, and IκBαΔChrom mESCs. (D) Quantification of EB differentiation status (percentage of pluripotent cells) at 216 hr after induction of SOF IκBα mutants. Data from three independent experiments. (E) Representative immunofluorescence for NANOG and OCT3/4 at 216 hr EBs that have not been treated with doxycycline in i-IκBαWT (upper panel), i-IκBαΔNF-κB (medium), and i- IκBαΔChrom (bottom) cells.
Author response image 1
Heatmap including all the genes from Figure 1F of the manuscript and clustering is simultaneously conducted over the four categories.
Author response image 2
Heatmap of NF-κB genes expression at the different time points of differentiation (mESCs, 48h EBs, 96h EBs).
Highlighted region marks the genes that are differentially expressed between both genotypes at 96h EBs.
Author response image 3
Violin plot of genes from the NF-κB pathway which are differentially expressed at 96h EBs.
Additional files
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MDAR checklist
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Supplementary file 1
Oligonucleotides for PCR primers-sgRNAs.
- https://cdn.elifesciences.org/articles/102784/elife-102784-supp1-v1.xlsx
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Supplementary file 2
ChiPseq results for Histone marks.
- https://cdn.elifesciences.org/articles/102784/elife-102784-supp2-v1.xlsx
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Supplementary file 3
Table with RNAseq analysis.
- https://cdn.elifesciences.org/articles/102784/elife-102784-supp3-v1.xlsx
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Supplementary file 4
Table with methylation array results.
- https://cdn.elifesciences.org/articles/102784/elife-102784-supp4-v1.xlsx
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Chromatin activity of IκBα mediates the exit from naïve pluripotency
eLife 14:RP102784.
https://doi.org/10.7554/eLife.102784.3
