Fig. 5: Inhibition of KDM5 induces TSS switching that remodels 5′UTRs and is associated with proteome changes.
From: Epigenetic alterations facilitate transcriptional and translational programs in hypoxia
a, Representative immunoblots of H3K4me3, and phosphorylated eIF2α (S51) and S6 (S240/244) from T47D cells treated with 10 μM C48 (24 h) or DMSO (0 h). H3, eIF2α, S6 and α-tubulin were used as loading controls (n = 4 independent experiments). b, Densitometry of H3K4me3 normalized to α-tubulin and H3 loading controls. Bars indicate mean ± s.d. P = 0.00018; two-sided t-test (n = 4 independent experiments). c, Kernel density estimation P value and FDR distributions for differential TSS usage between C48 and DMSO-treated T47D cells (n = 3,287). Dotted grey line indicates an FDR threshold of 0.15. d, Bar plot of protein-coding transcripts with significantly altered TSS usage between C48 and DMSO treatments (FDR < 0.15) in T47D cells. e, TSS-switching events under hypoxia with lower FDRs are more likely to be recapitulated by C48 treatment. The FDR range for TSS switching under hypoxia was divided into ventiles and the proportion of equivalent changes in 5′UTR isoforms between hypoxia versus normoxia and C48 versus DMSO comparisons was determined. Dashed line marks the proportion of changes expected to be the same by chance (21.8%, estimated by Monte Carlo simulation). The red line represents smoothed linear regression, with 95% CI shaded. f, Pie charts indicating the proportions of transcripts with the same changes in 5′UTR isoform expression under hypoxia versus C48 treatment (n = 682) with significantly (FDR < 0.15) increased (up), decreased (down) or unchanged overall expression between hypoxia and normoxia (left), and between C48 and DMSO (right). g, Bar plot of categories of 5′UTR sequences enriched in transcripts with significant TSS switching after C48 treatment, identified by change-point analysis (n = 3,287). h, Bar plot of categories of 5′UTR sequences enriched for transcripts with significant TSS switching between hypoxia and normoxia, identified by change-point analysis. The percentage recapitulated with C48 treatment is indicated. i, 5′UTR isoform expression for NM_001388028 (SFXN3 mRNA) in hypoxia and normoxia-treated (top), and C48 and DMSO-treated T47D cells (bottom). Change-point analysis (dotted orange lines) identified enriched and depleted 5′UTR segments in both comparisons (right). Both hypoxia and C48 treatment enrich shorter 5′UTR isoforms. j, The same as in i, but for NM_001251977 (RCAN3 mRNA). Both hypoxia and C48 treatment enrich longer 5′UTR isoforms. k, Scatter-plot of protein (from GPF-DIA proteomics analysis) versus total mRNA log2 fold changes in T47D cells (C48 versus DMSO, n = 4 independent experiments). Genes are coloured according to the mode of regulation assigned by anota2seq (FDR < 0.15). Protein up and down represent changes in protein level occurring independently of changes in mRNA level. Abundance up and down represent congruent changes in protein and mRNA levels. The number of regulated genes in each category is indicated in corresponding colours. l, Kernel density estimation P value and FDR distributions for anota2seq analysis (in k) of changes in protein, total mRNA and protein adjusted for mRNA between C48 and DMSO-treated T47D cells. m, The 400 most up- and downregulated genes were separated into 4 bins based on the quartiles of the fold changes in protein levels adjusted for mRNA levels (determined by anota2seq) in T47D cells treated with C48. Empirical distribution functions compare the TSS-switch scores (determined by change-point analysis) across the four bins. The set of background genes (that is not in bins) is also indicated (grey line). Differences in TSS scores between each bin compared to background were assessed using two-sided Wilcoxon rank-sum tests. P values and the magnitude of shifts at quartiles (q25–75) are indicated. Right-shifted curves indicate the sets of genes with more extensive TSS switching after C48 treatment compared to unregulated genes.
