e2f4 cdna  (Addgene inc)

Journal: Nature communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression.

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: Fig. 3 | Deletion of Trim33 promotes E2f4 interactions with chromatin and with the Recql helicase. a Genome browser tracks of E2f4 ChIP-seq in p19/Nras Trim33- WT and Trim33-KO cells; n = 2. b Quantification of ChIP-seq tag coverage at E2f4 peaks in p19/Nras Trim33-WT and Trim33-KO cells. c Relationship between changes (log2FC) in E2f4 binding (normalized E2f4 ChIP-seq tags) and mRNA expression (log2FC) of the encoded gene for p19/Nras Trim33-KO vs Trim33-WT cells. Each dot is a bin of 5 genes sorted by the change in mRNA levels. Trendlines are derived from linear regression analysis. d LC-MS/MS analysis of E2f4 interactome in p19/Nras Trim33-WT and Trim33-KO cells. Known interaction partners are highlighted in blue. e PLA analysis of Recql-E2f4 association in p19/Nras Trim33-WT and Trim33- KO cells stably expressing HA-tagged E2f4 or vector Ctrl; from left, n = 49,49,50,50

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: ChIP-sequencing, Binding Assay, Expressing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a The top significantly represented transcription factors targeting Trim33-deregulated genes in p19/Nras cells; data were analyzed using Enrichr . b Cycloheximide chase assays in p19/Nras Trim33-WT and Trim33-KO cells; n = 3. Right panel shows the mean intensity (±SD) for three independent experiments with linear regression analysis. c Ubiquitin pulldown assays in HeLa cells transfected with vectors encoding E2f4, his-Ub, and wildtype (WT) or catalytically inactive (CA) Trim33; n = 3. d Immunoprecipitation assays with E2f4 antibodies in p19/Nras Trim33-WT cells treated with MG132 or DMSO; n = 3. e PLA assays in p19/Nras Trim33-WT with antibodies to Trim33 and E2f4. 50 cells per group were analyzed; n = 3. Scale bar = 2 μm. f Schematic of E2f4 domain structure and the variants used in the following experiments. g Immunoprecipitation analysis following transient transfection of the indicated E2f4 variants in HeLa cells; n = 2. h GST-pulldown assays with the indicated GST-E2f4 fusion proteins and U2OS whole cell lysates; n = 3. i DNA fiber assays in p19/Nras Trim33-WT and Trim33-KO cells transfected with siRNA against E2f4 or a non-targeting control. 100 fibers were analyzed per condition; n = 2. j DNA fiber assays in p19/Nras Trim33-WT and Trim33-KO cells, expressing indicated E2f4 variants, after release from a 4 h HU treatment. 140 fibers were counted per condition; n = 2. e , i , j Significance was determined by Kruskal–Wallis test with Dunn’s multiple comparisons. e , i , j Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Transfection, Immunoprecipitation, Control, Expressing

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Genome browser tracks of E2f4 ChIP-seq in p19/Nras Trim33-WT and Trim33-KO cells; n = 2. b Quantification of ChIP-seq tag coverage at E2f4 peaks in p19/Nras Trim33-WT and Trim33-KO cells. c Relationship between changes (log2FC) in E2f4 binding (normalized E2f4 ChIP-seq tags) and mRNA expression (log2FC) of the encoded gene for p19/Nras Trim33-KO vs Trim33-WT cells. Each dot is a bin of 5 genes sorted by the change in mRNA levels. Trendlines are derived from linear regression analysis. d LC-MS/MS analysis of E2f4 interactome in p19/Nras Trim33-WT and Trim33-KO cells. Known interaction partners are highlighted in blue. e PLA analysis of Recql-E2f4 association in p19/Nras Trim33-WT and Trim33-KO cells stably expressing HA-tagged E2f4 or vector Ctrl; from left, n = 49,49,50,50 cells; scale bar = 2 μm; n = 3 independent experiments. f Immunoprecipitation of endogenous E2f4 from benzonase-treated lysates of p19/Nras Trim33-WT and Trim33-KO cells using a mouse anti-E2f4 antibody. Quantification of three biological replicates shows mean intensity and SD. g GST-pulldown assay with the indicated GST-E2f4 fusion proteins and shTrim33 U2OS whole cell lysates; n = 3. h PLA analysis of Recql-E2f4 association in p19/Nras Trim33-KO cells stably expressing the indicated HA-tagged E2f4 variants; from left, n = 100,100,97,100 cells; scale bar = 2 μm; n = 2 experiments. e , h Significance was determined by Kruskal–Wallis test with Dunn’s multiple comparisons. e , h Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: ChIP-sequencing, Binding Assay, Expressing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Plasmid Preparation, Immunoprecipitation, GST Pulldown Assay

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Immunoprecipitation from p19/Nras Trim33-WT and Trim33-KO cells expressing HA-tagged Recql; n = 3. b Representative genome browser tracks of Cut&Run analysis of HA-Recql binding in p19/Nras Trim33-WT and Trim33-KO cells expressing HA-tagged Recql. Lower two tracks—E2f4 ChIP-seq. c Sequencing tag coverage for Recql Cut&Run signal from the experiment shown in b ) at all TSS and at E2f4 sites. d Immunoprecipitation analysis with Recql antibodies from formaldehyde-crosslinked p19/Nras Trim33-WT and Trim33-KO cells, transfected with E2f4-targeting or a control siRNA. e Cut&Run assays with Recql antibodies in p19/Nras Trim33-WT and Trim33-KO cells followed by qPCR analysis at the TSS sites of indicated genes. Mean values of percent input for three technical replicates and standard deviation are plotted. f Representative genome browser tracks of the Cut&Run assays with Recql antibodies in Trim33-KO p19/Nras cells expressing the indicated E2f4 variants. g Cut&Run sequencing tag coverage at all TSS for the experiment shown in f ). h DNA fiber assays in Trim33-KO cells, expressing siRNA against Recql or control siRNA, untreated or released from a 4 h HU treatment; n = 2. 100 fibers were counted per sample and the data were analyzed using Kruskal–Wallis test followed by Dunn’s multiple comparison. Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. i Immunoblotting analysis of Recql expression in Trim33-KO cells, used in h ). Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Immunoprecipitation, Expressing, Binding Assay, ChIP-sequencing, Sequencing, Transfection, Control, Standard Deviation, Comparison, Western Blot

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Immunoprecipitation of endogenous Recql from formaldehyde-crosslinked p19/Nras Trim33-WT cells, untreated or treated with HU for 10 or 120 min; n = 3. b PLA assays with antibodies to biotinylated EdU-labeled nascent DNA and Recql in Trim33-WT and Trim33-KO cells after release from a 4 h HU treatment; from left, n = 112,62,143,113 cells; scale bar = 5 µm; n = 3 experiments. Significance was determined using Kruskal–Wallis test with Dunn’s multiple comparisons. c Representative genome browser tracks of HA-Recql Cut&Run in p19/Nras Trim33-WT and Trim33-KO cells, untreated or treated with HU for 4 h. d Sequencing tag coverage for Recql Cut&Run experiment shown in c ). e Ubiquitin pulldown assay in HeLa cells, transfected with vectors for His-Ub, HA-tagged E2f4, and Trim33-WT or Trim33-CA; n = 2. f Immunoprecipitation of endogenous E2f4 from benzonase-treated lysates of control or HU-treated (2 h) p19/Nras Trim33-WT cells. Quantification of four independent experiments is shown as mean and S.D. of signal intensity g PLA assays with Trim33 and E2f4 antibodies in untreated and HU-treated (2 h) p19/Nras Trim33-WT cells. Significance was determined by a two-tailed t -test. From left, n = 98,117 cells; Scale bar = 5 µm. h Representative genome browser tracks of Cut&Run assay with E2f4 antibodies in untreated and HU-treated (4 h) p19/Nras Trim33-WT cells, expressing HA-Recql. The upper two tracks show HA-Recql binding in the same cells. i Relationship between E2f4 and Recql recruitment at 2000 transcription start sites with maximal E2f4 enrichment after HU treatment in Trim33-WT cells. The regions were sorted by E2f4 tags and binned at 50 genes/bin followed by linear regression analysis. j PLA assay with antibodies to PCNA and pS5-RNAPII or total RNAPII in untreated or HU-treated (4 h) p19/Nras Trim33-WT and Trim33-KO cells. From left, n = 50,50,50,46,39,46,50,50 cells. Significance was determined using Kruskal–Wallis test with Dunn’s multiple comparisons. b , g , j Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Immunoprecipitation, Labeling, Sequencing, Transfection, Control, Two Tailed Test, Expressing, Binding Assay

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Immunofluorescence analysis with 53bp1 antibodies in p19/Nras (Ctrl) and p19/Nras/Myc (Myc) cells expressing shCtrl or shTrim33. At least 24 cells were quantified. Scale bar = 2 μm. b Immunofluorescence analysis with pH2AX antibodies in p19/Nras/Myc cells expressing shTrim33 or shCtrl and siRNAs against E2f4, Recql or control. At least 133 cells were quantified per group. Scale bar = 5 μm. c Neutral comet assays in p19/Nras/Myc cells expressing shRNAs against Trim33 or a control sequence and siRNAs against E2f4, Recql, or control. Scale bar = 10 μm. At least 39 cells were quantified per group. d Schematic of the HDTV-based model of liver tumorigenesis. Transposon vectors encoding Myc, Nras, GFP, and shCtrl or shTrim33 are injected along with the SB transposase in the tail vein of C57BL/J mice. e Representative images of whole livers under daylight and ultraviolet light showing tumor nodules. f Kaplan–Meier survival plots for two cohorts of mice injected with shTrim33 or shCtrl (7 animals/group). Significance was determined using the log-rank test. g Representative images of IHC analysis with antibodies to Trim33 and pH2AX on FFPE sections of tumor-bearing livers. h Quantification of pH2AX-positive cells per view field in liver tumors expressing shTrim33 or shCtrl. Significance was determined by a two-tailed, unpaired t -test. a , b , c Significance was determined using Kruskal–Wallis test with Dunn’s multiple comparisons. a , b , c , h Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Immunofluorescence, Expressing, Control, Sequencing, Injection, Two Tailed Test

Journal: Protein & Cell

Article Title: Unveiling E2F4, TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis

doi: 10.1007/s13238-021-00894-z

Figure Lengend Snippet: Transcriptional signatures and regulators of replicative senescence . (A) Schematic outlining the design of multi-omics analysis of cellular replicative senescence. Skin fibroblasts from 2-month-old mice were used, and samples were collected at P1, P3, P5 and P7 for RNA-seq, ATAC-seq and RRBS. (B) PCA of the transcriptome of skin fibroblast during RS. P1 (blue), P3 (dark red), P5 (dark green) and P7 (purple), ( n = 4). (C) Heatmap showing 6 major modules of differentially expressed genes during RS. Genes that differentially expressed in any group when compared with P1 are shown (adjusted P -value < 0.05 and fold change > 2). The number of genes within each cluster is given in parentheses, ( n = 4). (D) Transcription factor enrichment analysis based on the genes from each cluster. Only the top significantly enriched TFs are shown. AP-1 family TFs and E2F4 are highlighted. The analysis was performed with RcisTarget. (E) Selected biological process GO terms enriched in each gene cluster, with the top 5 most significantly enriched terms shown. The size of each point represents the gene count in the GO term, and the color of each point represents the significance of the enrichment. (F) GSEA showing cell cycle-related terms based on the transcriptome of P1 versus P7, ( n = 4). (G) GSEA showing inflammatory response-related terms based on the transcriptome of P5 versus P7, ( n = 4)

Article Snippet: For E2f4 overexpression vector construction, E2f4 cDNA (NM_148952.1) was cloned into lentiviral vector FU-tet-on-P2A-EGFP (Addgene) through homologous recombination using ClonExpress II One Step Cloning Kit (Vazyme, C112).

Techniques: Biomarker Discovery, RNA Sequencing

Journal: Protein & Cell

Article Title: Unveiling E2F4, TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis

doi: 10.1007/s13238-021-00894-z

Figure Lengend Snippet: E2F4 affects replicative senescence-associated phenotypes as a regulatory TF . (A) Structures of plasmids used for EGFP-labeled E2F4 expression under doxycycline induction and corresponding control. (B) RT-qPCR showing the change in the E2f4 expression level after overexpression with lentivirus, ( n = 3). (C) Immunoblot showing the change in E2F4 protein level after overexpression. (D) Change in SA-β-Gal activity after E2f4 overexpression. SA-β-Gal positive percentage was estimated based on 3 different views, and the points indicated the average value, ( n = 3). Scale bar = 200 μm. (E) Scatter plot showing the nuclear size distribution after E2f4 overexpression based on nuclear staining. Nuclear size was calculated using ImageJ, ( n = 3). (F) EdU staining showing the change in proliferating capacity after E2f4 overexpression. Red dots indicate EdU positive cells (white arrows), and the EdU-positive percentage was calculated with ImageJ. The positive percentage was estimated based on 5 different views, and the points indicated the average value, ( n = 3). Scale bar = 200 μm. (G) GSEA showing changes in cell cycle-related terms after E2f4 overexpression based on RNA-seq. (H) Heatmap showing changes in the expression levels of cell cycle-related genes, including positive regulators (up) and negative regulators (down). P3 samples were used as proliferating cell control, ( n = 2). (I) RT-qPCR detecting the change in E2f4 expression level after shRNA-mediated knockdown, ( n = 3). (J) Immunoblot showing the change in E2F4 protein level after knockdown. (K) EdU staining showing the change in proliferating capacity after E2f4 knockdown. Green dots represent EdU-positive cells (white arrows), ( n = 3). Scale bar = 200 μm. (L) Scatter plot showing the nuclear size distribution after E2f4 knockdown based on nuclear staining. Nuclear size was calculated using ImageJ, ( n = 3). (M) Bar plot showing the changes of RS-associated pathways after E2f4 knockdown through GSEA analysis. Blue bar indicates down-regulated pathways, and red bar indicates up-regulated pathways after E2f4 knockdown, ( n = 3). Data were analyzed by one-way ANOVA (I, K and L) and t -test (B, D, E and F). Error bars denote for the SD. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: For E2f4 overexpression vector construction, E2f4 cDNA (NM_148952.1) was cloned into lentiviral vector FU-tet-on-P2A-EGFP (Addgene) through homologous recombination using ClonExpress II One Step Cloning Kit (Vazyme, C112).

Techniques: Labeling, Expressing, Control, Quantitative RT-PCR, Over Expression, Western Blot, Activity Assay, Staining, RNA Sequencing, shRNA, Knockdown