Research ArticleCELL BIOLOGY

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

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Science Advances  26 Jun 2019:
Vol. 5, no. 6, eaaw4640
DOI: 10.1126/sciadv.aaw4640
  • Fig. 1 meR marks on E2F1 confer genome-wide effects.

    (A) Schematic representation of E2F1, highlighting the region of the protein targeted by PRMT1 and PRMT5. The arginine methylation-defective E2F1 derivatives [R109K and R111/113 K (KK)] used to generate U2OS stable cell lines for RNA-seq analysis are also indicated (i). An immunoblot displaying E2F1 protein expression in U2OS stable cells after 24 hours of doxycycline (1 μg/ml) treatment is also included (ii). See also fig. S1 (A to D). (B) Venn diagrams showing the crossover of genes up- or down-regulated over twofold (adjusted P value threshold < 0.01) in each cell line condition with respect to the pTRE empty vector cell line, filtered for genes containing an E2F1 motif in their proximal promoter region (−900 to +100). These data were generated from three independent biological samples.

  • Fig. 2 E2F1 affects alternative splicing of E2F target genes.

    (A) A heatmap displaying absolute values of ΔΨ (percent spliced in) for each cell line, corresponding to statistically significant alternative splicing event changes to E2F1 target genes (as determined by the presence of ChIP-seq peaks in their promoter and gene regions, retrieved from ENCODE data) with respect to the pTRE empty vector cell line, derived by analyzing the RNA-seq data with rMATS algorithm. Yellow color represents the lowest difference, and blue color represents the highest. Ivory blocks correspond to nonsignificant changes in splicing patterns (FDR > 0.01). See also table S2 and fig. S3. (B) Pie chart showing the percentage of genes identified in the rMATS splicing analysis, which are E2F1 target genes (as determined by the presence of ChIP-seq peaks in their promoter and gene regions, retrieved from ENCODE data) (i). The Venn diagram demonstrates the overlap of E2F1 target genes affected by alternative splicing events (FDR < 0.01) in each cell line (ii). These data were generated from three independent biological samples. (C) Bar chart displaying the statistically significant alternative splicing events to E2F target genes for each cell line, as compared to the pTRE vector control. The percentage of these alternative splicing changes corresponding to different types of splicing event is displayed in different colors. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice site. See also fig. S2B. (D) Venn diagrams showing overlap between E2F1 target genes identified in the differential expression analysis as being up- or down-regulated (regulated greater than twofold; Fig. 1B) and those identified as being differentially or alternatively spliced [(A) and table S2]. These data were generated from three independent biological samples. (E) Bar chart representing the average fold change in expression of differentially expressed E2F1 target genes (regulated greater than twofold), compared with the expression of those E2F1 target genes where alternative splicing occurred. Only 389 genes from the alternative splicing analysis met the significance threshold for differential expression (P < 0.01). The remaining 632 spliced genes had expression levels that were not significant from the pTRE empty vector cell line (P > 0.01) and were therefore assigned an arbitrary value of 1 for this analysis.

  • Fig. 3 E2F1 interacts with components of the splicing machinery.

    (A) U2OS cells were lysed in RIP lysis buffer, containing ribonuclease A (RNase A; 20 μg/ml) where indicated. Cell extracts were immunoprecipitated with E2F1 antibody, and coimmunoprecipitated RNA was reverse-transcribed before quantitative polymerase chain reaction (qPCR) analysis with primers against U6 (i) and U4 (ii) snRNAs as indicated. Input protein levels were determined by immunoblot (iii). n = 2. (B) U2OS cells were treated with 5 μM PRMT5 inhibitor (P5 inh), as indicated, before performing an anti-E2F1 RIP. Coimmunoprecipitated U6 (i) and U4 (ii) snRNAs were identified with specific primers by quantitative reverse transcription PCR (qRT-PCR). Input protein levels were determined by immunoblot (iii). n = 3. (C) An anti-E2F1 RIP was performed on U2OS cells, and coimmunoprecipitated U1 snRNA was detected by qRT-PCR. n = 2. (D) An anti-E2F1 RIP was performed on extracts prepared from U2OS or U2OS E2F1 CRISPR cell lines as indicated. Immunoprecipitated RNA was analyzed by qRT-PCR using primers specific to U1 (i), U6 (ii), or U5 (iii) snRNAs. Input protein levels are also displayed (iv). n = 2. (E) HCT116 cells were treated with 5 μM PRMT5 inhibitor, where indicated, before performing an anti-E2F1 RIP. Coimmunoprecipitated U1 (i) and U6 (ii) snRNA were detected by qRT-PCR. Input protein levels are also displayed (iii). n = 2. (F) As described above, although the experiment was performed in MCF7 cells. (G) U2OS cells were transfected with 1 μg of plasmid encoding WT E2F1, DNA binding domain mutant constructs (L132E and R166H) or empty vector (−) as indicated. Forty-eight hours later, cell extracts were used for ChIP analysis with the anti–hemagglutinin (HA) antibody. Immunoprecipitated chromatin was analyzed by qPCR using primers targeting the indicated promoters, where albumin served as the non-E2F target gene control (i to iii). Input protein levels are shown in (H). n = 2. See also fig. S4B. (H) U2OS cells were transfected as above. Forty-eight hours later, cell extracts were used for RIP analysis with anti-HA antibody. Immunoprecipitated RNA was analyzed by qRT-PCR using primers specific to U6 snRNA (i) or actin RNA (ii). Input protein levels were determined by immunoblot (iii). n = 3.

  • Fig. 4 p100/TSN enables E2F1 to interact with alternatively spliced transcripts.

    (A) Schematic representation of exon structure for the SENP7 gene. Each alternatively spliced transcript expressed from this gene is displayed, with primer binding sites used to detect specific transcript variants in subsequent experiments indicated with black arrows. Note that forward primers were designed to span exon junctions. Mining of the RIP-seq dataset for exon spanning peaks identified reads around exons 4 and 7 (indicated by the red numbering), which occurs in SENP7 transcript V5 (highlighted in red text). (B) Anti-E2F1 RIP with U2OS cells treated with siRNA against E2F1, TSN, or nontargeting (NT) control, as indicated, for 72 hours. Cells were then immunoprecipitated with E2F1 antibody, and coimmunoprecipitated RNA was reverse-transcribed before qPCR analysis with primers against specific SENP7 transcript variants as indicated. n = 3. (C) HCT116 cells were treated with 5 μM PRMT5 inhibitor, where indicated, before performing an anti-E2F1 RIP. Coimmunoprecipitated SENP7 V5 transcripts were analyzed by qRT-PCR. Input protein levels are the same as those displayed in Fig. 3E. n = 2. DMSO, dimethyl sulfoxide. (D) U2OS cells were treated for 72 hours with 5 μM PRMT5 inhibitor. RNA was then isolated from cells and analyzed by qRT-PCR using primers targeting specific SENP7 transcript variants or total SENP7 RNA. Average (mean) fold change of each RNA species as compared to untreated U2OS cells was calculated and displayed with SE. Statistical analysis for each condition compared to untreated U2OS cells is also displayed over each bar (i). An immunoblot to demonstrate input protein levels is also included (ii). n = 3. (E) As described above, although the experiment was performed in HCT116 cells. n = 4. ns, not significant. (F) Examination of the promoter region of the SENP7 gene (–2 to +1 kb) identified an E2F1 DNA binding motif within +450 bp of the transcription start site, lying within the first intron (E2F1 motif marked in red) (i). An E2F1 ChIP was performed in the HCT116 E2F1 CRISPR and MCF7 TSN CRISPR cell lines. Immunoprecipitated chromatin was analyzed using primers spanning the identified E2F DNA binding motif in SENP7 or against the known E2F motif in the promoter sequence of CDC6 (ii). An immunoblot is included to demonstrate input protein levels (iii). n = 3

  • Fig. 5 E2F1 also interacts with alternatively spliced transcripts from the MECOM gene.

    (A) Schematic representation of exon structure for the MECOM gene. Each alternatively spliced transcript expressed from this gene is displayed, with primer binding sites used to detect specific transcript variants in subsequent experiments indicated with black arrows. Note that forward primers were designed to span exon junctions. Mining of the RIP-seq dataset for exon spanning peaks identified reads spanning exons 1 and 3 (indicated by the red numbering), which occurs in MECOM transcript V7 (highlighted in red text). (B) U2OS (i), MCF7 (ii), or HCT116 cells (iii) were treated with 5 μM PRMT5 inhibitor as indicated. An anti-E2F1 RIP was then performed, and coimmunoprecipitated MECOM transcript variant V7 was analyzed by qRT-PCR using specific primers. Input protein levels for the U2OS experiment are also included (iv), while the input protein levels for HCT116 and MCF7 cells are the same as those displayed in Fig. 3 (E and F). n = 2. (C) Examination of the promoter region of the MECOM gene identified an E2F1 DNA binding motif lying within the first intron of V7 or the second intron of V4 (E2F1 motif marked in red) (i). An E2F1 ChIP was performed in HCT116 or HCT116 E2F1 CRISPR cell lines. Immunoprecipitated chromatin was analyzed using primers spanning the identified E2F DNA binding motif in MECOM or against the known E2F motif in the promoter sequence of CDC6 (ii). Input protein levels are the same as those displayed in Fig. 4F. n = 3. (D) U2OS cells (i) or HCT116 cells (iii) were treated with 5 μM PRMT5 inhibitor, where indicated. RNA was then isolated from cells and analyzed by qRT-PCR using primers targeting specific MECOM transcript variants or total MECOM RNA. Average (mean) fold change of each RNA species as compared to untreated U2OS/HCT116 cells was calculated and displayed with SE. Statistical analysis for each condition compared to untreated cells is also displayed over each bar. Input protein levels for U2OS cells are also displayed (ii), while the input protein levels for HCT116 cells are the same as those displayed in Fig. 4E. n = 4.

  • Fig. 6 Biological consequence of SENP7 alternative splicing for E2F1 activity.

    (A) U2OS cells were treated with 5 μM PRMT5 inhibitor for 72 hours, where indicated, before ChIP analysis with anti-SUMO2/3–specific or control antibodies. Immunoprecipitated chromatin was analyzed using primers specific for the E2F site in the p73 promoter (i). An RT-PCR was also performed to monitor the levels of p73 transcripts in the cell (ii). An immunoblot for H4R3me2s is included to demonstrate the activity of the PRMT5 inhibitor (iii). n = 3. See also fig. S4 (F and G). (B) As described above, although cells were treated with the PRMT5 inhibitor for 24 or 48 hours as indicated. ChIP analysis was performed with anti-HP1α–specific or control antibodies (i). An immunoblot for H4R3me2s is included to demonstrate the activity of the PRMT5 inhibitor (ii). n = 2. (C) U2OS cells were transfected with SENP7 siRNA or nontargeting siRNA (siNT) for 96 hours as indicated. Cells were then prepared for ChIP analysis as described above (i). An immunoblot is included to demonstrate input protein levels (ii). n = 4. (D) ChIP analysis as described above, although U2OS cells were transfected with siRNA targeting E2F1, SENP7, or a combination of the two (siE2F1 + siSENP7). n = 3. (E) U2OS cells were transfected with siRNA targeting SENP7 or nontargeting siRNA for 96 hours, as indicated. Cells were subsequently transfected for 48 hours with an empty vector or a plasmid expressing Flag-tagged SENP7 V5. Cells were then prepared for ChIP analysis as described above (i). An immunoblot is included to demonstrate input protein levels (ii). n = 3. (F) U2OS cells were transfected with p73–luciferase (luc) or CDC6-luciferase reporter plasmids for 48 hours, along with empty vector (vec) or Flag-tagged SENP7 V5. Reporter activity was measured, and immunoblots were performed to monitor input protein levels. n = 2. (G) Model diagram where PRMT5-mediated methylation of chromatin-associated E2F1 mediates its interaction with p100/TSN, which permits the E2F1 complex to associate with a subset of RNAs, some being derived from E2F-target genes. By regulating the activity of the splicing machinery, it is proposed that the E2F1-p100/TSN complex can influence the alternative splicing of these RNAs. In the absence of E2F1 methylation (either under conditions of PRMT5 inhibitor treatment or in cells expressing E2F1-meR point mutants), a p100/TSN-dependent interaction with the splicing machinery is lost, and changes to alternative splicing of a subset of RNAs result.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaaw4640/DC1

    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 RNA-seq 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.

    Table S1. List of up- and down-regulated E2F1 target genes identified from the RNA-seq analysis for each cell line, corresponding to Fig. 1B.

    Table S2. List of alternative splicing events in E2F1 target genes identified in the RNA-seq 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.

  • Supplementary Materials

    The PDF file includes:

    • 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 RNA-seq 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

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of up- and down-regulated E2F1 target genes identified from the RNA-seq analysis for each cell line, corresponding to Fig. 1B.
    • Table S2 (Microsoft Excel format). List of alternative splicing events in E2F1 target genes identified in the RNA-seq rMATS analysis corresponding to the heatmap (Fig. 2A).
    • Table S3 (Microsoft Excel format). Differential expression of genes associated with RNA splicing, taken from the RNA-seq dataset (Fig. 1B).
    • Table S4 (Microsoft Excel format). List of RNAs identified in the anti-E2F1 RIP-seq analysis (Fig. 4).
    • Table S5 (Microsoft Excel format). List of overlapping E2F target genes between RIP-seq dataset (Fig. 4) and splicing analysis (Fig. 2A).
    • Table S6 (Microsoft Excel format). List of E2F1 RIP-seq reads that span exon junctions.

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