RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells

Abstract

The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo¹. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(aln^z24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.

Extended Data Fig. 1. A chemical suppressor screen for Leflunomide

(a) Chemical structure of the Leflunomide, the active metabolite of leflunomide known as A77 1726 and an the independent DHODH inhibitor named here iDHODH1, or iDH1. (b) Lateral view of embryos at 24hpf treated with chemicals as indicated and subjected to in situ hybridization for crestin. Number of embryos displaying the indicated phenotype is indicated in the lower right corner. Scale bars represent 200μm. (c) Metabolite profiling in A375 melanoma cells. Upper panel: A375 cells exposed to A77 1726, Esomeprazol or both compounds for 24 hours. Lower panel: cells exposed to A77 1726, Aphidicolin or both chemicals. (n = 3 independent experiments, Mean ± SD, Two-Way ANOVA with Bonferroni Comparison, ** = p < 0.01, *** = p < 0.0001). Source data are available online.

Extended Data Fig. 2. Effects of Leflunomide and Progesterone on neural crest during embryonic development.

(a) In situ hybridization for the neural crest cell markers sox10, foxd3 and pax3 in zebrafish embryos at the 15-somite stage upon treatment with indicated chemicals. (b) Lateral view of embryos subjected to in situ hybridization for crestin at 5, 8, 12 and 15 somites. Number of embryos displaying the presented phenotype is indicated in lower right corner. Scale bars represent 200μm.

Extended Data Fig. 3. Progesterone restores transcriptional changes caused by DHODH inhibition.

(a) Schematic representation workflow to sort neural crest cells, here defined as sox10:GFP positive cells. (b) qPCR on whole embryos, sorted GFP low and sorted GFP high neural crest cells for neural crest (mitfa, foxd3, sox10, crestin, snail2) and non-neural crest (neurogenin and myf5) genes (n = 3 technical replicates, pooled from 1 experiment, Mean ± SD). (c, d) Hierarchical clustering heatmap of genes down-regulated or up-regulated in sox10:GFP high cells sorted from leflunomide-treated zebrafish (c) or ctr9 morphants (d). Differentially expressed genes criteria: log2 fold change ≥ 1.5 or ≤ 1.5. (n = 3 biologically independent experiment). Source data are available online.

Extended Data Fig. 4. Progesterone receptor expression and perturbation effects.

(a) RT-PCR for pgr using two primer sets to reveal receptor expression during early development (1 experiment). (b) GFP positive embryos injected with pgr:flag:T2A:gfp mRNA or mismatched pgr mRNA with a pgr morpholine (MO) reveals MO specificity. Scale bars represent 200μm. (c) In situ hybridization for crestin at 24hpf in control and pgr mRNA injected embryos with or without leflunomide treatment. Number of embryos displaying the phenotype represented is indicated. Scale bars represent 200μm.

Extended Data Fig. 5. DDX21 interacts with PGR and loss of pgr function rescues crestin expression in vivo.

(a, b) DDX21 associates with PGR in A375 melanoma cells containing a doxycycline inducible PGR expression and in T47D breast cancer cells (2 independent biological experiments per cell line). (c) RT-PCR for ddx21 to reveal receptor expression during early development (3 independent experiments). Source data are available online.

Extended Data Fig. 6. DDX21 relocalizes from the nucleolus to the nucleoplasm upon nucleotide depletion.

(a, d) DDX21 and TCOF1 immunofluorescence staining in A375 melanoma cells. Experiment was 4 times repeated independently with similar results. Scale bar represents 100μm. (b, e) Quantification of nucleoli to nucleoplasm ratio (n = 5 sections per condition, Two-sided Wilcoxon-Mann-Whitney test). Box plots represent median value and 25^th and 75^th percentiles. Whiskers are minima and maxima. Red asterisks indicate outliers. (c) Western blot analysis for DDX21 in A375 cells treated for 24 hours with DMSO, A77 1726 or A77 1726 plus nucleotides. Actin was used as loading control. Immunoblot are representative of at least 2 independent experiments. Source data are available online.

Extended Data Fig. 7. Genome wide annotation for DDX21 bound regions in A375 cells.

(a) Pie chart indicating the location of DDX21 ChIP-Seq peaks relative to gene locations in A375 melanoma cells treated with DMSO for 24hrs. (b). Gene track of DDX21 binding at 14kb rDNA region in A375 melanoma cells treated for 24 hours with DMSO, A77 1726 or and A77 1726 plus nucleotides. Source data are available online.

Extended Data Fig. 8. Gene Ontology analysis and PRO-Seq analysis in A375 cells.

Gene Ontology (GO) term enrichment analysis of genes down-regulated (a) and down-regulated DDX21 target genes (b) in A375 melanoma cells 48 hours post treatment with A77 1726 (n = 3 independent biological experiments, hypergeometric test and Benjamini-Hochberg correction). The number of genes associated to each GO term is shown at the end of each bar within the graph. (c) PRO-seq in A375 cells. Nascent transcription at the transcription start site (TSS) and at the gene body of DDX21 target and not-DDX21 target genes in cells treated for 24hrs with DMSO, A77 1726 or A77 1726 plus nucleotides (n = 3 biologically independent experiments). (d) Box plot of PRO-Seq signal shows no difference of nascent transcription in DDX21-bound and non-bound genes, as no changes are observed in promoter to gene body ratio between DDX21 targets and non-targets. Box plots represent median value and 25^th and 75^th percentiles. Whiskers are 10^th and 90^th percentile (n = 3 biologically independent experiments). Source data are available online.

Figure 1.. Progesterone confers resistance to nucleotide depletion in vivo.

(a) Schematic overview of chemical suppressor screen performed in zebrafish embryos with the DHODH inhibitors leflunomide, A77 1726, and iDHODH1 (iDH1). Crestin expression was assessed at 24hpf. (b) In situ hybridization for crestin at 24hpf in embryos treated with the indicated DHODH inhibitors plus or minus progesterone. Number of embryos displaying the represented phenotype is noted. Scale bar represents 200μm. (c) Pyrimidine biosynthesis pathway highlighting the enzymatic conversion inhibited by the leflunomide, A77 1726 and iDH1. Metabolite profiling of A375 melanoma cells exposed to indicated chemicals for 24 hours. (n = 3 biologically independent experiments, Mean ± SD, Two-Way ANOVA with Bonferroni Comparison, * = p < 0.001, ** = p < 0.0001, ns = p > 0.05). (d) In situ hybridization for crestin at 24hpf in Ctr9 morpholine (MO) injected or paf mutant zebrafish embryos exposed to DMSO or progesterone. Number of embryos displaying the presented phenotype is indicated. Scale bars represent 200μm. Source data are available online.

Figure 2.. The progesterone receptor interacts with RNA-helicase DDX21.

(a, b) In situ hybridization at 24hpf for crestin in control, pgr morpholine (MO) and pgr mRNA injected embryos exposed to leflunomide in the presence and absence of progesterone. Number of embryos displaying the presented phenotype is indicated. Scale bars represent 200μm. (c) qPCR for pgr on zebrafish embryos at 24hpf using two different primer sets. (pgr primer set 1; n = 4, pgr primer set 2; n = 3 biologically independent experiments, Mean ± SD, Unpaired T-test with Welch Correction, * p = 0.017, ** p = 0.002, *** p = 0.021). (d) Co-Immunoprecipitation followed by mass-spectrometry of pgr:flag:T2A:gfp injected embryos identified complex association partners of Pgr in vivo. (e) DDX21 associates with PGR in A375 melanoma cells (2 biologically independent experiments). Source data are available online.

Figure 3.. Loss of Ddx21 rescues neural crest defects under nucleotide depletion.

(a, b) In situ hybridization at 24hpf for crestin in control, ddx21 morpholine (MO) and ddx21 mRNA injected embryos exposed to DHODH inhibitors. Number of embryos displaying the presented phenotype is indicated. Scale bars represent 200μm. (c) Lateral view of pigmentation of zebrafish embryos at 48hpf. Number of embryos displaying the phenotype represented is indicated in lower right corner. Scale bars represent 250μm.

Figure 4.. Nucleotide stress alters binding of DDX21 to RNA.

(a) Annotation of DDX21-associated RNAs identified by irCLIP in A375 melanoma cells after DHODH inhibition with or without nucleotide supplementation. (b) Venn diagram comparing overlap in mRNA binding targets of DDX21 under different conditions. (c) Gene tracks showing enhancer irCLIP signal of DDX21 at the CAD and CDKN1A locus during nucleotide depletion. (n = 3 biologically independent experiments). Source data are available online.

Figure 5.. Ddx21 mediates transcriptional changes during nucleotide stress.

(a) ChIP-Seq for DDX21 and CDK9 in A375 melanoma cells after 24 hours of treatment. Upper panel: Metagene analysis of DDX21-bound regions. Normalized peak intensities ± 2Kb from the center of DDX21 ChIP-seq are shown. Lower panel: Metagene representation of normalized CDK9 occupancy at transcription start site (TSS), along gene bodies and at transcription end site (TES) are shown. (b) Percentage of differentially expressed genes (DEGs) in A375 melanoma cells after 24 hours and 48 hours in the presence of A77 1726. DEG criteria: FPMK ≥1 after treatment, Log2 fold change ≥1 or ≤ −1. (n = 3 biologically independent experiments). (c) Hierarchical clustering heatmap of FPKM values from genes up-regulated (2028) and down-regulated (2961) at 24 hours and 48 hours post DHODH inhibition plus or minus nucleotide supplementation. (d) Venn diagram of DDX21 target genes with reduced DDX21 binding upon A77 1726 treatment as defined by ChIP-Seq (orange); genes up-regulated by RNA-Seq (blue) and genes down-regulated by RNA-Seq (red) at 48 hours post treatment with A77 1726 in A375 melanoma cells (n = 3 biologically independent experiments, Hypergeometric Test, ns = not significant; under enrichment p-value = 2.7e-58). Source data are available online.