Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: (A) Scheme of SETD6 promoter and several human transcription factor binding sites that were predicted at the genomic region of the SETD6 promoter in the JASPAR database ( https://jaspar.genereg.net/ ) with a relative profile score threshold of at least 90%. (B) SETD6 promoter genomic region (blue highlight) with E2F1 [GSM1656410], ELK4 [GSM1424528], MYC [GSM1907203], H3K27ac [GSM1907213], H3K4me3 [GSM1907211] ChIP-seq data in prostate cancer cell lines. Data were extracted by the Cistrome Data Browser: ( http://cistrome.org/db/#/ ) and visualized using the IGV software. (C, D) DU145 cells were transfected with Flag-E2F1 WT (C) or with siRNA specifically targeting endogenous E2F1 (D) . 24 hours post-transfection, the SETD6 protein levels were assessed by WB (top) using the indicated antibodies, and the SETD6 mRNA expression levels were measured using RT- qPCR (bottom). mRNA expression levels were normalized to mRNA expression levels of GAPDH housekeeping gene. Error bars are s.e.m. Statistical analysis is based on 5 experimental repeats. **p≤0.002, ****p≤0.00001

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Binding Assay, ChIP-sequencing, Software, Transfection, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: The graphs were generated using the Human Protein Atlas resource ( https://www.proteinatlas.org/ ). For each cancer, color-coded bars indicate the percentage of patients (maximum 12 patients) with high and medium SETD6 protein expression level.

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Generated, Expressing

Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: (A) Dual-luciferase assay in DU145 cells transfected with siRNA targeting endogenous E2F and the full promotor region of SETD6 cloned to pGL3-plasmid (positions -650 to +150 from TSS). 24 hours post-transfection, the whole cell lysates were subjected to dual-luciferase assay (Promega). Luminescence was measured by Tecan Infinite M200 plate reader. Relative luminescence was calculated after normalization of the firefly luciferase signal over Renilla luciferase control. (B) Same as in A with over expression of Empty or Flag-E2F1 WT in control and SETD6 KO cells. Values are fold change over siControl. Error bars are s.d. statistical analysis was performed for 3 experimental repeats. *p≤0.03, ****p≤0.0001. (C) Chromatin immunoprecipitation (ChIP) assay in control and SETD6 KO DU145 cells (two independent SETD6 gRNAs). (D) Same as in C with overexpression of Flag-E2F1 WT in control or SETD6 KO cells. 24h after transfection the chromatin fraction of the cells were immunoprecipitated with endogenous E2F1 antibody (C) or Flag antibody (D). The bound DNA was purified and amplified by qPCR using specific primers to SETD6 gene promoter regions and a distal n.c. region. Graphs show the percentage input of the quantified DNA. Error bars are s.e.m. Statistical analysis was performed for 4 experimental repeats. ***p≤0.0002, ****p≤0.0001.

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Luciferase, Transfection, Clone Assay, Plasmid Preparation, Control, Over Expression, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Amplification

Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: DU145 control and SETD6 knock-out (SETD6 KO) were generated using the CRISPR/Cas9 system, following single-clone selection. Chromatograms of Sanger sequencing of control cells and two SETD6 KO cells generated from two independent gRNAs targeted for SETD6 exon 4 and exon 8.

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Control, Knock-Out, Generated, CRISPR, Selection, Sequencing

Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: His-SUMO-E2F1, MBP-RelA or BSA were bound to the surface of a 96-well plate, incubated with GST-SETD6, or GST protein as negative control, and binding was analyzed with anti-GST antibody. RelA served as positive control for interaction with SETD6, while BSA served as negative control. PBS served as background control. Error bars are s.d. based on 3 replicas.

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Incubation, Negative Control, Binding Assay, Positive Control, Control

Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: (A) Methylation of a peptide SPOT array for determination of the target lysine of SETD6 on E2F1. 15 aa long peptides containing all lysine residues of E2F1 and variants in which individual lysine residues were replaced by alanine were synthesized on a peptide SPOT array, and incubated with recombinant SETD6 in the presence of radioactively labelled AdoMet. The RelA peptide (in spot A1) served as positive control, while the mutated RelA K310A in spot B1 served as the negative control. Peptide sequences of the individual spots are presented on the right. (B) Same experiment as described in panel A was performed with a peptide array containing unmodified (WT), or modified (Kme1, Kme2, Kme3 and a K to A mutated) E2F1 K117 peptides (aa 111-125, same as in spot A3). (C) Coomassie stain of purified His-SETD6, GST-E2F1 WT and GST-E2F1 K117R mutant to show equal loading of substrate proteins and enzyme used for in-vitro methylation experiments. (D) In-vitro methylation assay of GST-E2F1 (WT and the mutant K117R) by recombinant His-SETD6 in the presence of radioactively labelled AdoMet. The transferred radioactively labelled methyl groups were detected by autoradiography after different exposure times.

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Methylation, Synthesized, Incubation, Recombinant, Positive Control, Negative Control, Peptide Microarray, Modification, Staining, Purification, Mutagenesis, In Vitro, Autoradiography

Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: (A) DU145 cells were transfected with Flag-E2F1 WT or the respective K117R-mutant. After 24 hours, whole cell lysates were immunoprecipitated with Pan-Kme1 antibody, followed by WB analysis with indicated antibodies. (B) For validation of the specificity of the custom made E2F1-K117me1 antibody, a peptide SPOT array with 15 aa long unmodified (WT), or modified (Kme1, Kme2, Kme3 and a K to A mutated) E2F1 peptides (aa 111-125) was incubated with the antibody and binding analyzed by a secondary antibody and ECL. (C) In-vitro methylation assay GST- E2F1 (WT and the mutant K117R) by recombinant His-SETD6 using unlabelled AdoMet as cofactor. After methylation WB analysis was performed using the E2F1-K117me1 antibody. (D) DU145 cells were transfected with the indicated plasmids. 72 hours post-transfection, the methylation levels of EGFP-fused E2F1 were assessed by WB using the E2F1-K117me1 antibody. WB against GFP was included as input control. (E) DU145 control and SETD6 KO cells were transfected with the indicated plasmids. After 24 hours the whole protein lysate was analyzed by WB with the E2F1-K117me1 antibody. WB against H3 was included as input loading control.

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Modification, Incubation, Binding Assay, In Vitro, Methylation, Recombinant, Control

Journal: bioRxiv

Article Title: E2F1 methylation by SETD6 regulates SETD6 expression via positive feedback mechanism

doi: 10.1101/2023.06.27.546651

Figure Lengend Snippet: (A) Dual-luciferase assay 24h after transfection with empty plasmid, Flag-E2F1 WT or Flag-E2F1 K117R mutant and the full-length SETD6 promoter luciferase construct. Values are fold change over empty control. E2F1 protein levels were assessed by WB (left). (B) DU145 cells were transfected with empty plasmid, Flag-E2F1 WT or Flag-E2F1 K117R mutant. SETD6 mRNA expression levels were measured using RT-qPCR. mRNA expression levels were normalized to mRNA expression levels of GAPDH housekeeping gene. (C) Chromatin immunoprecipitation (ChIP) assay 24h after transfection with empty plasmid, Flag-E2F1 WT or Flag-E2F1 K117R mutant. The chromatin fractions of the cells were immunoprecipitated with anti-E2F1 K117me1. Error bars are s.e.m. Statistical analysis was performed for 5 experimental repeats. **p≤0.002, ***p≤0.0002, ****p≤0.00001. (D) Graphical model of our findings. E2F1 methylation by SETD6 regulates the mRNA expression of SETD6 in a positive feedback mechanism.

Article Snippet: 1.6 µM of GST-tagged E2F1 WT or mutant was incubated with 0.2 μM of His-SUMO-tagged SETD6 WT or mutant in methylation buffer (20 mM Tris/HCl pH 9 and 5 mM DTT), supplemented with 0.76 µM labelled [methyl- 3 H] -AdoMet (Perkin Elmer) for 3 hours at 25°C.

Techniques: Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Construct, Control, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation, Methylation