Ancestral function of Inhibitors-of-kappaB regulates Caenorhabditis elegans development

Abstract

Mammalian IκB proteins (IκBs) exert their main function as negative regulators of NF-κB, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for IκBs has been shown to affect stemness and cell differentiation. However, the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB-1 are IκB homologs in Caenorhabditis elegans, which lacks NF-κB nuclear effectors. We found that nfki-1 and ikb-1 mutants display developmental defects that phenocopy mutations in Polycomb and UTX-1 histone demethylase, suggesting a role for C. elegans IκBs in chromatin regulation. Further supporting this possibility (1) we detected NFKI-1 in the nucleus of cells; (2) NFKI-1 and IKB-1 bind to histones and Polycomb proteins, (3) and associate with chromatin in vivo, and (4) mutations in nfki-1 and ikb-1 alter chromatin marks. Based on these results, we propose that ancestral IκB inhibitors modulate Polycomb activity at specific gene subsets with an impact on development.

Figure 1

Homology of NFKI-1 and IKB-1 with human IκB proteins and cellular and subcellular patterns of expression. (A, B) Image of the first-hit alignment of the C. elegans NFKI-1 (A) and IKB-1 (B) proteins obtained with the BlastP program. (C) Alignment of different IκB proteins, including NFKI-1, centered on the region that contain the consensus phosphorylation sites (residues 32 and 36 for IκBα, in red) for IKKβ kinase. Note the conservation of the upstream lysine residue that is either ubiquitinated or SUMOylated (in green) in IκBα. (D) Western blot analysis using the anti-p-S32-IκBα of C. elegans protein lysates of endogenous EGFP::nfki-1 strain (L4 stage) precipitated with α-GFP antibody. IgG precipitation is included as specificity control. Uncropped versions of the blots are included in Supplementary Figure S5. (E) Tissue-specific expression profiles of L2 animals^,. nfki-1 (green) is mainly expressed in the nervous system and ikb-1 (red) in the muscular system. Expression data is presented as transcripts per million reads (TPM). Plot was done using Graphpad Prism 8. (F) Microscopy image of an L1 animal carrying both EGFP::NFKI-1 and IKB-1::mCHERRY endogenous reporters. DAPI channel is merged to indicate gut autofluorescence. Scale bar: 50 µm. (G) Confocal microscopy images of transgenic animals showing the expression pattern of EGFP::NFKI-1 at the indicated developmental stages. mCHERRY::SFTB-1 is used as nuclear marker. Yellow arrowheads denote cells that show a nuclear and cytoplasmic EGFP::NFKI-1 signal. White arrowheads point cells that do not display a nuclear but only cytoplasmic localization at the same plane. Scale bars: 50 µm. Imaging was done with 63X magnification in Z-stacks with 0.25 µm of distance between planes. Images represent a single plane.

Figure 2

Phenotypes of nfki-1 and ikb-1 mutants. (A) Graphical representation of the nfki-1 and ikb-1 mutants used in the study. cer1 allele is a CRISPR-generated 368 bp deletion near nfki-1 exon 2. cer2 is a 434 bp deletion and 50 bp insertion at exon 2. Both modifications generate premature stop codons. cer9 allele is a 462 bp deletion that includes the ATG start codon of ikb-1. nr2027 is a 1911 bp deletion of ikb-1 that includes the ankyrin-repeat domains encoding sequence. (B) Survival curves of the indicated nematode genotypes growing under nutrient restriction. We included as experimental controls the lsm-1(tm3585) and lsm-3(tm5166) strains, null alleles mutants of two members of the Sm-like (LSm) family of proteins previously demonstrated to be sensitive to starvation stress. n = 100 animals per time point. N = 2. Error bars show standard deviation (SD) of 2 experiments. Statistically significant differences were calculated using two-tailed Spearman correlation test (CI 95%). (C) Differential Interference Contrast (DIC) images showing diverse aberrant morphologies observed in nfki-1mutants and an animal harboring a single copy of nfki-1 that rescues morphological defects. n > 3000. N = 2. Error bars show SD of 2 experiments. Statistically significant differences were calculated using two-sided Chi-square test (CI 95%). (D) Representative microscopy images of wild type and nfki-1(cer2) worms displaying gonad migration defects. The dashed white line depicts the typical U-shape of the normal gonad in the image of the wild type animal. Graph shows the percentage of animals with an aberrant gonad migration phenotype. n > 60. N = 3. Error bars show SD of 3 experiments. Statistically significant differences were calculated using two-sided Chi-square test (CI 95%) (E) Alterations in the number of DTCs were visualized using the LAG-2::GFP reporter line. Note the presence of two DTCs in the WT and a single DTC in the nfki-1 mutant (white asterisks). Scale bar: 100 µm. Graph display the percentage of animals with an abnormal DTC number. n > 1000. N = 3. Error bars show SD of 3 experiments. Statistically significant differences were calculated using two-sided Chi-square test (CI 95%). Plots in panels (B–E) were generated using Graphpad Prism 8.

Figure 3

C. elegans IκBs physically interact with histones and PRC2 proteins, but not mammalian NF-κB proteins in vitro and bind chromatin in vivo. (A) Pull-down assay using HA-tagged NFKI-1, IKB-1 or mammalian SUMO-IκBα expressed in HEK-293 T cells and the indicated GST-fusion histone constructs as bait (B–D) Pull-down assays from HA-tagged NFKI-1 or IKB-1 containing extracts using GST-fusion proteins containing the indicated fragments of MES-2 (B), MES-3 (C) or MES-6 (D). (E) Summary of the data shown in (B–D). (F) Extracts from HEK-293 T cells transfected with HA-tagged human SUMO-IκBα or C. elegans NFKI-1 and IKB-1 were used in co-immunoprecipitation experiments to measure association with the NF-κB proteins p65/RelA and p50. Western blot analysis of a representative experiment is shown. (G) Distribution of peaks from 3xFLAG::NFKI-1 and IKB-1::mCHERRY ChIP-seq across C. elegans chromosomes, indicating the localization of the peaks relative to the closest annotated gene. Data are presented relative to input. Plot done with ggplot2 package (version 3.2.1, R software version 3.6.1). (H) Table indicating the cell-type category distribution of genes identified in the ChIP analysis. Uncropped versions of gels and blots are included in Supplementary Figure S5.

Figure 4

RNA-seq and ChIP-seq indicate that gene expression and distribution of regulatory chromatin marks are affected in IκB-deficient mutants. (A) 2D plot illustrating the correlation between genes differentially expressed (adjusted p value < 0.01, log₂ fold-change ≥ 2) in nfki-1 and ikb-1 deficient animals at L4 larvae stage. R = 0.947, p value < 2.2e-16, Spearman test. (B) Doughnut charts showing the distribution of genes categorized according to whether their expression is ubiquitous (green), germline-enriched (dark blue), germline-specific (light blue), soma-specific (yellow) or unclassified (gray) for differentially expressed genes at L4 stage. Numbers in the center represent the number of genes in each dataset. Categories dataset was extracted from. Statistically significant differences between expected and observed distribution was calculated using Chi-square test for goodness of fit (p < 0.05). Doughnut charts were generated using Graphpad Prism 8. (C,D) Violin plots indicating the density and distribution of H3K27me3 (C) and H3K36me3 (D) peaks at germline and soma-specific genes differentially (color-filled/black lines) and not differentially (colored lines) expressed in the indicated genotypes (p adj < 0.05). x-axis represents the expression levels (log₂ fold-change) in mutants relative to the wild type. Plots in panels (A,C and D) were done with ggplot2 package (version 3.2.1, R software version 3.6.1).