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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(alnz24) 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.

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Fig. 1: Progesterone confers resistance to nucleotide depletion in vivo.
Fig. 2: Pgr interacts with RNA helicase Ddx21.
Fig. 3: Loss of Ddx21 rescues neural crest defects under nucleotide depletion.
Fig. 4: Nucleotide stress alters binding of DDX21 to RNA.
Fig. 5: DDX21 mediates transcriptional changes during nucleotide stress.

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Data availability

Deep-sequencing (ChIP-seq, RNA-seq, PRO-seq, irCLIP) data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE128086. Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD014433. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data for Figs. 1, 2, 4 and 5, and Extended Data Figs. 1 and 3–8 are presented with the paper.

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Acknowledgements

We thank the following for their support and contribution: E. Fast, A. Choudhuri and J. M. Ordovas-Montanes for their critical reading of our manuscript; M. Rossman, E. Patton, J. Johansson, R. A. Young and J. Wysocka for discussions about the project; M. Brown, L. Krug, D. Grunwald, S. Spengler, M. Yuan, J. Asara, L. Rubin, A. Avanites and T. Schlaeger for help with reagents and experiments. This work was supported by NIH grant P50-GM107618 (M.K.), the Hope Funds for Cancer Research (B.J.A.), and the following grants to L.I.Z.: Cancer Biology R01 CA103846, NIH Melanoma PPG, P01CA63222, the Melanoma Research Alliance and a Starr Cancer Consortium grant.

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Authors and Affiliations

Authors

Contributions

C.S. designed the study. C.S., A.S., B.D., W.M., E.C.G., M.E.S. and M.F. performed experiments. A.L., M.S., B.J.A. and S.Y. provided formal data analysis. I.A. assisted with the zebrafish logistics. T.H., R.A.F., M.K. and E.C. performed experiments and analysed data. M.J., K.A., T.H. and Y.Z. provided insights on data interpretation. C.S., A.S. and L.I.Z. wrote the manuscript. L.I.Z. conceived and managed the study.

Corresponding author

Correspondence to Leonard I. Zon.

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Competing interests

B.J.A. is a shareholder of Syros Pharmaceuticals. L.I.Z. is founder and stockholder of Fate, Scholar Rock and Camp4 therapeutics and a scientific advisor for Stemgent. The other authors declare no competing interests.

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Extended data

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

(a) Chemical structure of leflunomide, the active metabolite of leflunomide known as teriflunomide or A77 1726 and an the independent DHODH inhibitor named here iDHODH1, or iDH1. (b) Lateral view of embryos at 24 h.p.f. treated with chemicals as indicated and subjected to in situ hybridization for crestin. Number of embryos displaying the presented 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.

Source data

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 of the workflow to sort neural crest cells, here defined as sox10:GFP positive cells. (b) qPCR on whole embryos, sorted GFP low neural crest cells, 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.

Source data

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 were injected with pgr:flag:T2A:gfp mRNA or mismatched pgr mRNA with a pgr morpholine (MO) to reveal MO specificity. Scale bars represent 200μm. (c) In situ hybridization for crestin at 24 h.p.f. in control and pgr mRNA injected embryos with or without leflunomide treatment. Number of embryos displaying the phenotype represented is indicated. Scale bars represent 200 μm.

Source data

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.

Source data

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 repeated independently for 4 times 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 25th and 75th 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.

Source data

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 24 hours. (b). Gene track of DDX21 binding at 14-kilobase 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.

Source data

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 are 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 non-DDX21 target genes in cells treated for 24 hours 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 25th and 75th percentiles. Whiskers are 10th and 90th percentile (n = 3 biologically independent experiments). Source data are available online.

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Santoriello, C., Sporrij, A., Yang, S. et al. RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells. Nat Cell Biol 22, 372–379 (2020). https://doi.org/10.1038/s41556-020-0493-0

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