Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control

Arginine methylation widens the mechanism of control by E2F1 from a transcription factor to a regulator of alternative RNA splicing.


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Fig. S1. Generation of stable, inducible cell lines expressing E2F1 methylation site mutants. Fig. S2. Additional analysis of RNA-seq and rMATS datasets. Fig. S3. GO biological process enrichment analysis on spliced E2F1 target genes from the RNAseq data. Fig. S4. Additional analysis of E2F1 RIP-seq datasets. Fig. S5. Expression of E2F1 correlates with PRMT5 and MECOM V7 transcript expression in human cancer. Legends for tables S1 to S6

Other Supplementary Material for this manuscript includes the following:
(available at advances.sciencemag.org/cgi/content/full/5/6/eaaw4640/DC1)  An empty vector cell line was included as a control (pTRE). E2F1 expression and localisation was detected with an anti-HA antibody and nuclei were stained with DAPI.
B) U2OS stable cell lines were treated as above to induce expression of WT E2F1, R109K, or KK. An immunoprecipitation was performed using anti-HA antibody, and isolated chromatin was analysed by QPCR using primers targeting the indicated promoters. n = 3. C) U2OS stable cell lines were treated with doxycycline as described above, then subsequently exposed to etoposide for 48h as indicated. An immunoblot was performed to demonstrate E2F1 expression (i). Cells were also prepared for flow cytometry analysis, and the average percentage of cells in sub-G1 is displayed with standard error shown (ii). n = 3. D) 1000 cells of each U2OS stable cell line was plated and treated with doxycycline to induce E2F1 protein expression for 10 days. A colony formation assay was performed and the average number of colonies per well is displayed with standard error. n = 3. E) Proportion of E2F1 target genes (containing E2F1 binding motifs in their proximal promoters [-900 to +100]) from the RNA-seq analysis on each cell line that were up-or down-regulated over 2-fold (p adj value <0.01). The size of the dot reflects a percentage of the genes and the colour corresponds to the raw numbers, as indicated in the figure. See also table S1 and fig. S2A. F) Wild-type E2F1 (WT), R109K or KK protein expression was induced in the U2OS stable cell lines by addition of 1g/ml doxycycline for 24h. RNA was then isolated from cells and analysed by qRT-PCR using primers against target genes selected from the RNA-seq (i to iv). n = 3. G) U2OS cells were transfected with siRNA against E2F1 or non-targeting siRNA (NT) for 72h. RNA was then isolated and qRT-PCR was performed with primers targeting the indicated genes (i). An immunoblot is also included to show input protein levels (ii). n = 2. Enriched sets are represented as nodes and are coloured by significance (red indicating upregulation and blue indicating down-regulation). Nodes are connected based on the genes they share by grey lines, with edge thickness correlating with the number of shared genes.
Only differentially expressed, known E2F1 target genes with adjusted P-value < 0.01 were used in the GSA analysis. B) Magnitude of splicing changes observed in each cell line from the RNA-seq for different  ranges: strong (>50%), moderate (30-50%), weak (10-30%), expressed as a percentage of the total events for each cell line. This bar chart is an alternative representation of the data in Fig. 2C.

Fig. S3. GO biological process enrichment analysis on spliced E2F1 target genes from
the RNA-seq data. GO biological process enrichment analysis was performed on the unique genes in each cell line associated with statistically significant, non-overlapping alternative splicing events (From Fig. 2A). The heatmap was generated using automatic clustering of GO:BP terms, which were then manually assigned to broader functional groupings as depicted to the bottom of the heatmap (Function).

Fig. S4. Additional analysis of E2F1 RIP-seq datasets. A) U2OS cells (i) and HCT116
cells (ii) were transfected for 96h with 25nM non-targeting (siNT) or p100/TSN specific siRNA, prior to lysis in RIP buffer. Cell extracts were immunoprecipitated with E2F1 antibody and co-immunoprecipitating RNA was reverse transcribed prior to QPCR analysis with primers against U5 (i) and U6 (ii) snRNAs as indicated. Input protein levels were determined by immunoblot. B) U2OS cells were transfected with HA-tagged wild-type E2F1, E2F1 L132E, E2F1 R166H, or empty vector (pcDNA) for 48h as indicated. Expression and localisation of E2F1 was detected by indirect immunofluorescence using an anti-HA antibody, whilst nuclei were stained with DAPI. C and D) Schematic diagrams representing the exon structure for each of the indicated genes: P3H2 (C), and SPG21 (D). All annotated alternative transcripts expressed from each gene are also displayed, with transcription initiation sites highlighted by black arrows. Mining of the RIP-seq data for peaks which span exon boundaries identified a number of reads that permitted specific transcript variants to be identified. These exon spanning reads are indicated on the diagrams with red arrows. E) Sashimi plots of RNA-seq data for the MECOM and SENP7 gene are displayed to demonstrate that transcript variants observed in the RIP-seq (Figs. 4 and 5) could also be observed in the RNA-seq data (Fig. 2). The genomic coordinates for the highlighted regions of the gene, and a schematic of the splicing events occurring, are shown at the bottom of each graph. Exons are labelled as per the numbering system used in Fig. 4A (SENP7) and   Table S2. List of alternative splicing events in E2F1 target genes identified in the RNAseq rMATS analysis corresponding to the heatmap (Fig. 2A). Table S3. Differential expression of genes associated with RNA splicing, taken from the RNA-seq dataset (Fig. 1B). Table S4. List of RNAs identified in the anti-E2F1 RIP-seq analysis (Fig. 4). Table S5. List of overlapping E2F target genes between RIP-seq dataset (Fig. 4) and splicing analysis (Fig. 2A). Table S6. List of E2F1 RIP-seq reads that span exon junctions.