Fig 2: EGF stimulation induces locus-specific increases of H3K9me2S10ph.(A) Distributions of average H3K9me2S10ph z scores in chromatin regions that were significantly enriched (adjusted P < 0.005) for H3K9me2S10ph in a comparison between IMR90 cells that were untreated (PBS) or treated with EGF for 30 min. Boxplots show median, 25% and 75% quartiles. (B) Enrichment (fold change of observed over expected overlap for randomly permuted genomic regions of equal sizes) of EGF-dependent H3K9me2S10ph regions for each annotation category. Categories as defined in Fig. 3: “Enhancers of EGF-regulated genes” refers to the complete set of 320 genes that were significantly differentially expressed after either 30 or 60 min of EGF stimulation; “Enhancers of EGF60-Up genes” refers to the 219 genes uniquely up-regulated after 60 min of EGF stimulation; “Enhancers of MSK2-dependent genes” refers to the 59 genes defined as MSK2 dependent; “Enhancers of MSK2-independent genes” refers to the 228 genes defined as MSK2 independent. All enrichment analyses were subjected to 1000 simulations; all P values < 0.001. (C) UCSC Genome Browser view (hg38) showing EGF-dependent H3K9me2S10ph in a region around the EGF-regulated and MSK2-dependent gene SNX18 (blue highlight). Linked Enhancers track shows GeneHancer annotations of SNX18-associated enhancers (gray boxes). Purple highlights indicate significant EGF-dependent H3K9me2S10ph regions [as in (A) and (B)] that overlap annotated enhancers. (D) Schematic of proposed model: EGF stimulation leads to the activation of the nuclear kinase MSK2, which mediates H3K9me2S10 phosphorylation at enhancers of EGF-responsive genes, leading to transcriptional activation. Schematic created with BioRender.

Fig 3: Schematic representation for the role of free tRNALeu. Under amino acid starvation condition, free tRNALeu interacts with EBP1 and enhances the activation of ErbB2/ErbB2 pathway. Finally, the downstream signaling mediated by RSK1/MSK2 facilitates cancer cell proliferation.

Fig 4: RACO-1 stabilisation by MEK requires MSK1(a) Simplified scheme of MEK signalling pathway showing possible downstream effectors of Trim7 and RACO-1 regulation. Kinase inhibitors are shown in red. (b) Western blots showing the effect of the indicated kinase inhibitors on MEKR4F-induced FLAG–RACO-1 protein levels. Phosphorylated CREB (pCREB) is shown as a control for MSK inhibition. Tubulin is shown as loading control. Arrowhead indicates CREB band; asterisk indicates a non-specific band. (c) Western blots showing the effect of the MSK inhibitor Ro 31-8220 on FLAG–RACO-1 levels in the presence and absence of MG132. (d) Western blot showing the effect of the indicated inhibitors on endogenous RACO-1 levels in H727 cells. (e) siRNA-mediated depletion of MSK1 and MSK2 in H727 cells, showing the effect on endogenous RACO-1 levels. (f) Western blots showing the effect of MSK1 depletion on endogenous RACO-1 levels in H727 cells, in the presence and absence of MG132. (g) Western blot showing FLAG–RACO-1 levels in cells transfected with MSK1 or the catalytically impaired MSK1 mutant D565A. pCREB is shown as a control for MSK1 activity. Arrowhead indicates CREB/pCREB band; asterisk indicates a non-specific band. (h) Ubiquitin pulldown from cells expressing MSK1 or D565A mutant MSK1, showing ubiquitinated FLAG–RACO-1. (i) Western blots showing the effect of MSK1 depletion by siRNA on FLAG–RACO-1 levels in the presence or absence of TPA. (j) Western blots showing the effect of MSK1 depletion by siRNA on FLAG–RACO-1 levels in the presence or absence of Myc–Trim7 overexpression. Experiments were replicated at least three times.

Fig 5: A subset of EGF-induced transcriptional activation is MSK2 dependent.(A) Volcano plots showing the differential gene expression patterns of serum-starved IMR90 cells treated with EGF for 30 or 60 min. Up-regulated genes (log fold change > 0.5; adjusted P < 0.05; green), down-regulated genes (log fold change < 0; adjusted P < 0.05; brown). (B) Venn diagram depicting the overlap of genes that are significantly up-regulated as measured after EGF treatment of 30 or 60 min. (C) GSEA enrichment plot on genes up-regulated after 60 min of EGF treatment compared to PBS in serum-starved IMR90 cells. Normalized enrichment score (NES) and adjusted P value are shown for the NAGASHIMA_EGF_SIGNALING_UP gene set in the C2 category of Molecular Signatures Database (MSigDB). (D) Heatmaps display normalized expression for differentially expressed genes (DEGs; adjusted P < 0.05 and ranked by descending log fold change). Left panel shows the top 50 DEGs after 30 min of EGF treatment. Right panel shows the top 50 DEGs unique to 60 min of EGF treatment. Colors are scaled by row in each heatmap. (E) Heatmap of all significant DEGs (adjusted P < 0.05) between PBS control, EGF 60-min treatment, and EGF 60-min treatment in cells with MSK2 knockdown. DEGs were grouped into clusters via hierarchical clustering based on gene expression across the three different conditions. The top cluster of genes was additionally tested for significant differential expression (adjusted P < 0.05) after 60 min of EGF treatment with or without MSK2 knockdown. These MSK2-dependent genes (n = 59) are labeled in blue next to the top cluster. Colors are scaled by row in each heatmap. (F) Schematic showing that a subset of EGF-stimulated transcription is activated through MSK2.