anti e2f1  (Proteintech)

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: SETD6-mediated methylation of E2F1 at K117 regulates the binding site of E2F1 and genes expression. ( A ) The E2F1 transcription factor is methylated by SETD6 at K117. This research investigated whether methylation of E2F1 K117 by SETD6 has a role in regulating global gene expression programs. ( B ) Heatmap of RPKM-normalized E2F1 ChIP-seq signals at combined E2F1 peak regions (±0.8 kb) showing differential binding of E2F1 in DU145 prostate cancer cells stably expressing Flag-E2F1 in SETD6 WT or KO context. Separation into three clusters by k -means clustering revealed cluster 1 containing regions bound similarly by E2F1 in SETD6 WT and KO cells (596 regions), cluster 2 containing 10377 regions with stronger binding in the SETD6 WT cells, and cluster 3 containing 4799 regions with stronger binding in SETD6 KO cells. Data were merged from two experimental repeats, see . ( C ) Summary of the experimental strategy to identify candidate genes that are differentially bound and differentially expressed in a K117 methylation dependent manner. ( D ) RNA-seq experiments conducted in DU145 SETD6 WT and KO cells expressing Flag-E2F1 or E2F1 K117R. Differential gene expression was analyzed by Heatmapper ( http://heatmapper.ca/ ). The heatmaps display significant differential expressed genes (log 2 -fold change >1.5 and P -value <0.05) in SETD6-WT and KO cells either expressing E2F1 WT or E2F1 K117R. Green color represents up-regulated genes and red color represents down-regulated genes. Three replicates were analyzed for each condition. A scatter plot showing the results of a principal component analysis (PCA) of transformed count data from the RNA-seq replicates using DESeq2 is provided in . The source data to Fig. 1B and D are provided in Supplementary Data S1.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Methylation, Binding Assay, Expressing, Gene Expression, ChIP-sequencing, Stable Transfection, RNA Sequencing, Transformation Assay

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Integration of ChIP-Seq and RNA-Seq datasets identifies potential K117me dependent targets of E2F1 and corresponding cellular processes in prostate cancer cells. ( A ) Venn diagram showing the overlap genes of stronger ChIP-seq peaks in SETD6 WT and RNA-seq results. Fig. 1B cluster 2 related genes (8765) showing strong E2F1 binding in SETD6-WT (blue), 257 upregulated genes in SETD6 WT cells overexpressing Flag-E2F1 WT (green), and 222 upregulated genes in SETD6 WT cells overexpressing Flag-E2F1 K117R (yellow) have been identified. Finally, 40 genes show strong E2F1 binding and upregulation with E2F1 WT but not with E2F1 K117R. The Venn diagram is not drawn to scale. ( B ) Example browser views showing E2F1 binding to genes upregulation in E2F1 WT only in the context of SETD6 WT. ChIP-seq tracks of E2F1 in SETD6 WT (blue) and KO (red) cells are displayed. ( C ) GO biological pathway enrichment analysis on gene sets using the 40 genes identified in panel (A) conducted by ShinyGO 0.8. ( D ) GO biological pathway enrichment analysis on gene sets using the 40 genes identified in panel (A) conducted by Enrichr. The genes cluster in three GO groups related to roles in prostate gland morphogenesis and development (red), cell adhesion (green), and TGF-β-SMAD signaling (blue). For exemplary genes and gene functions see Table .

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: ChIP-sequencing, RNA Sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Integration of ChIP-Seq and RNA-Seq datasets identifies direct targets of E2F1 in SETD6 KO cells and corresponding cellular processes. ( A ) Venn diagram showing the overlap genes with stronger ChIP-seq binding in SETD6 KO and RNA-seq results. Fig. 1B cluster 3 related genes (4158) showing strong E2F1 binding in SETD6-KO (blue), 1081 upregulated genes in SETD6 KO cells overexpressing Flag-E2F1WT (green), and 280 upregulated genes in SETD6 KO cells overexpressing Flag-E2F1 K117R (yellow) have been identified. Finally, 210 genes show strong E2F1 binding and upregulation with E2F1 WT but not with E2F1 K117R. The Venn diagram is not drawn to scale. ( B ) Example browser views showing genes with strong E2F1 binding and upregulation in E2F1 WT only in the context of SETD6 KO. ChIP-seq tracks of E2F1 in SETD6 WT (blue) and KO (red) cells are displayed. ( C ) GO biological pathway enrichment analysis on gene sets using the 210 genes identified in panel (A) conducted by ShinyGO 0.8. ( D ) GO biological pathway enrichment analysis on gene sets using the 210 genes identified in panel A conducted by Enrichr. The genes cluster in three GO groups related to cell proliferation (red), ubiquitin transferase activity (green), and sphingolipid biosynthesis processes (blue). For exemplary genes and gene functions see Table .

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: ChIP-sequencing, RNA Sequencing, Binding Assay, Ubiquitin Proteomics, Activity Assay

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Cellular processes in prostate cancer cells regulated by SETD6 activity. ( A ) Exemplary images of colony formation assays performed in DU145 SETD6 WT (Control) and KO cells. Images were captured 16 days after cell plating. ( B ) Apoptotic response in DU145 WT (Control) and three independent KO cell lines indicated as fraction of apoptotic cells. Black bars: DMSO treated cells used as negative control. Gray bars: 2.5 µM DOX-treated cells. ( C ) Apoptotic response in DU145 WT either empty (−) or with stable expression of WT Flag-E2F1 (WT) or its K117R mutant (K117R) indicated as fraction of apoptotic cells. Black bars: DMSO treated cells used as negative control. Gray bars: 2.5 µM DOX-treated cells. In panels (B) and (C), statistical analysis was performed for duplicates using two-way ANOVA. **** P < .0001. Error bars show the deviation of three technical replicates. For biological replicates see and . Cell migration was detected by wound healing tests in SETD6 WT cells and two SETD6 KO clones. Representative images are shown captured 0, 5, 10, 15, and 20 h after scratch producing with black dashed lines indicating wound borders. ( E ) Quantitative analysis of the data shown in panel (E). Blue dots: SETD6 WT (Control) cells. Green and red dots: two SETD6 CRISPR KO cells derived from two independent gRNAs clones. Exemplary primary cytometry data for panels (B) and (C) are shown in .

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Activity Assay, Control, Negative Control, Expressing, Mutagenesis, Migration, Clone Assay, CRISPR, Derivative Assay, Cytometry

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in vitro . ( A ) Crystal structure of the human BRD4 BD1 (sandy-brown) in complex with an acetylated K117ac/K120ac E2F1 peptide (blue) (PDB 6ULS) showing the key interaction of K117ac with BD1. ( B ) Coomassie BB stained 12% SDS gel of the purified GST tagged truncated BRD4 (2-477 aa) protein including BD1 and BD2 (BD1/2) as well as the purified BD1 domain (2-220 aa). The GST-tagged BRD4 proteins are marked with asterisks. ( C ) Binding of the GST-BRD4 BD1/2 to modified E2F1 peptides. 15 aa long E2F1 peptides with different combinations of unmodified, acetylated, and methylated K117 and K120 were synthesized on peptide SPOT arrays. The sequence of each peptide is listed in the table. Peptide arrays were incubated with 5 nM GST-BRD4 BD1/2 and binding was detected using a GST-specific antibody. The bar diagram shows the binding of E2F1-BRD4 to K117ac/K120ac and K117me/K120ac observed in three independent experiments. The bars represent the averages. The P- value was determined by two flanked t ‐test with equal variance. ( D ) Same as in panel (C), but GST-BRD4 BD1 was used. Additional data are provdied in .

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, In Vitro, Staining, SDS-Gel, Purification, Modification, Methylation, Synthesized, Sequencing, Incubation

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in cells. ( A ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) were transfected into DU145 SETD6 WT and KO cells. The GFP-tagged BRD4 was purified by GFP-trap and analyzed by western-blot with an anti-GFP antibody. Co-purification of Flag-E2F1 was determined by anti-Flag antibody. Equal loading of cell lysate isolated from transfected DU145 SETD6 WT or KO was verified by western-blot analysis against β-actin, GFP, and Flag. ( B ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) WT or K117R were transfected into in DU145 SETD6 KO cells. Some of the transfected cells were treated with JQ1-Bromodomain-Kac binding inhibitor (5 µM) or DMSO as control. GPF-trap and western-blot analysis was conducted as in panel A. (C–E) Interaction of BRD4 and E2F1 investigated by PLA. All experiments were conducted in DU145 cells. Exemplary microscopy images are shown. Scale bar: 10 µm. PLA signal quantification (PLA dots per nucleus, AU) for each sample is shown on the right. Statistical analysis was performed using Student’s t -test in GraphPad (**** P < .0001). ( C ) Interaction of endogenous BRD4 and Flag-E2F1 in the absence and the presence of the SAHA deacetylase inhibitor (20 µM) for 5 h (Flag-E2F1). Negative control (Neg) refers to reaction conducted without addition of Flag primary antibody. The interaction of BRD4 and E2F1 was detected and it was shown to be stimulated by increasing acetylation levels after SAHA treatment. Number of analyzed cells: 183, 132, 223. ( D ) Detection of the interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells (Control) and SETD6 KO cells (KO1 and KO2). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed cells: 67, 236, 104, 91. ( E ) Interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells and with overexpression of GFP (GFP empty) or GFP-SETD6 (GFP-SETD6). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed GFP positive cells: 19, 18, 28.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, Transfection, Purification, Western Blot, Copurification, Isolation, Control, Microscopy, Histone Deacetylase Assay, Negative Control, Over Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 co-occurence is observed in SETD6 KO but not SETD6 WT cells. ( A ) Heatmap of RPKM-normalized E2F1 ChIP-seq signals at E2F1 peaks (±0.8 kb) showing differential chromatin binding of E2F1 in SETD6 WT and KO cells stably expressing Flag-E2F1. The third heatmap shows BRD4 Chip-seq signals in a prostate cancer cell line (SRR1170714) using the same clustering. See also . ( B ) Example browser views showing ChIP-seq of BRD4 (SRR1170714, green) and E2F1 in SETD6 WT and KO cells. See also for additional examples. ( C ) Correlation analysis of E2F1 binding in SETD6 WT and KO cells with the literature BRD4 chromatin binding profile used in panel (A). E2F1 and BRD4 signals were determined in the E2F1 peak regions shown in panel (A) and their correlation was determined. ( D ) Bar-graph showing the slope of the correlation line of BRD4 and E2F1 binding signals in SETD6 WT or KO cells determined using three BRD4 ChIP-seq data sets (datasets SRR1170714, SRR5467129, and SRR5467130). The corresponding analyses are shown in panel (C) and . P -value determined by two-flanked t -test assuming equal variance.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Promoter and enhancer binding of BRD4 at genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. ( A ) Average of aggregated BRD4 signals at promoter and enhancer elements of genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. Note stronger binding in SETD6 KO cells. P -value determined by two-flanked t -test assuming equal variance. ( B ) Representative genome browser views showing co-occupancy of BRD4 and E2F1 at five genomic regions in SETD6 KO cells: C6orf226, TBCC, RPL21, RPL38, and MYC. ChIP-seq tracks were visualized using IGV (version 2.13.1) displaying BRD4 (SRR1170714, green), E2F1 in SETD6 WT (blue), and E2F1 in SETD6 KO (red) DU145 cells. ( C ) ChIP from SETD6 WT and KO DU145 cells performed using a BRD4-specific antibody to enrich BRD4-bound chromatin fragments. IgG was used as a negative control to assess the specificity of the immunoprecipitation. BRD4 occupancy was evaluated by qPCR at the same loci as shown in panel (B). Two independent biological replicates with three technical repeats were performed. Statistical significance was determined using a two-tailed t -test assuming equal variance. The negative controls RPL21 and RPL28 did not yield a detectable signal. Note the elevated BRD4 binding in SETD6 KO context. ( D ) RT-qPCR analysis of the relative expression of the five target genes shown in panel (B) in untreated SETD6 WT and KO DU145 cells (control) as well as after addition of DMSO and JQ1. Note the strong effect of JQ1 on gene expression in SETD6 KO cells.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, ChIP-sequencing, Negative Control, Immunoprecipitation, Two Tailed Test, Quantitative RT-PCR, Expressing, Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Summary of the results of this study. SETD6 monomethylates E2F1 at K117. This methylation disrupts the E2F1–BRD4 interaction leading to different target loci being bound by both factors. In the absence of K117 methylation, E2F1 is acetylated at K117 and K120 leading to BRD4 binding and a concerted engagement of both protein at genomic target sites. As a consequence, methylated and unmethylated E2F1 regulates distinct gene sets in prostate cancer cells.

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Methylation, Binding Assay

Journal: Science Advances

Article Title: Engineered anchoring aptamers induce relocalization and functional inactivation of transcription factors

doi: 10.1126/sciadv.adw6496

Figure Lengend Snippet: ( A ) Schematic illustration of relocalization and functional inactivation of E2F1 mediated by RTE. ( B ) Schematic diagram of the Ras ligand (Ra1, an aptamer targeting intracellular Ras), the E2F1 ligand (a stem-loop structure DNA sequence containing an E2F1-binding motif) and RCTE and RTE. ( C ) Western blot analysis of cytoplasmic and nuclear levels of E2F1 in HeLa cells transfected with library, RCTE, or RTE (all at 75 nM) for 24 hours. ( D ) Western blot analysis of cytoplasmic and nuclear E2F1 levels in HeLa cells transfected with RCTE (75 nM) or RTE (37.5 to 150 nM) for 24 hours. ( E ) HeLa cells were transfected with RCTE or RTE (all at 75 nM) for 24 hours. The subcellular localization of E2F1 was analyzed by immunofluorescence microscopy. Scale bars, 10 μm. ( F ) mRNA levels of CCNE and CDC6 in HeLa cells transfected with RCTE or RTE were measured by qPCR. ( G ) HeLa cells were transfected with RCTE or RTE (all at 75 nM) for 24 hours. Cell proliferation was measured by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay. Scale bars, 1000 μm. Error bars represent means ± SD from three independent experiments ( n = 3). P values are indicated by Student’s t test.

Article Snippet: The anti-Ras antibody (18295-1-AP), anti-E2F1 antibody (66515-1-Ig), anti-vinculin antibody (66305-2-Ig), anti-H3 antibody (68345-1-Ig), and anti-proliferating cell nuclear antigen (PCNA) antibody (60097-1-Ig) were obtained from Proteintech.

Techniques: Functional Assay, Sequencing, Binding Assay, Western Blot, Transfection, Immunofluorescence, Microscopy