Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: The pRb–E2F pathway regulates RNA splicing of E2F target genes. ( A ) Differential changes in splicing between WT and E2F1 Cr HCT116 cells, treated with DMSO or 1 μM T1-44 for 48 h are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event across all samples, as compared to the WT E2F1 HCT116 cells treated with DMSO (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. Black boxes indicate splice events that uniquely occur upon T1-44 treatment, only in the presence of WT E2F1. These data were generated from three independent biological samples. Venn diagram showing the overlap between statistically significant differential splicing events (FDR < 0.05) (AS) in each treatment, as compared to WT E2F1 HCT116 cells treated with DMSO. These data were generated from three independent biological samples. ( C ) A representative immunoblot displaying input protein levels of E2F1 and symmetric dimethylation (SDMe). Actin served as a loading control. ( D ) The bar chart displays the breakdown of statistically significant (FDR < 0.05) differential splicing events observed in each of the indicated treatments, as compared to WT E2F1 HCT116 cells treated with DMSO. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. and . ( E ) Annotation of genes which undergo splice events that uniquely occur upon T1-44 treatment in the presence of WT E2F1 (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle, stress responses, and DNA damage are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( F ) Differential changes in splicing between WT and Rb Cr MCF7 cells, treated with DMSO or 1 μM T1-44 for 48 h are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event across all samples, as compared to the WT Rb MCF7 cells treated with DMSO (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. Black boxes indicate splice events that uniquely occur upon T1-44 treatment, only in the presence of WT Rb. These data were generated from three independent biological samples. Venn diagram showing the overlap between statistically significant differential splicing events (FDR < 0.05) (AS) in each treatment, as compared to WT Rb MCF7 cells treated with DMSO. These data were generated from three independent biological samples. ( H ) A representative immunoblot displaying input protein levels of Rb and SDMe. GAPDH served as a loading control. ( I ) The bar chart displays the breakdown of statistically significant (FDR < 0.05) differential splicing events observed in each of the indicated treatments, as compared to WT Rb MCF7 cells treated with DMSO. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. and . ( J ) Annotation of genes which undergo splice events that uniquely occur upon T1-44 treatment in the presence of WT Rb (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle, stress responses, and DNA damage are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( K ) WT E2F1 and E2F1 Cr HCT116 cells treated for 48 h with 1 μM T1-44 or DMSO as indicated. An RT-PCR was performed to measure the inclusion of VCAN exon 7, MDM1 exon 4, METTL6 exon 3, or REV3L exon 3 in RNA transcripts from the cells. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. A diagram indicating the exon (boxed in grey) and intron (black lines) structure of each gene around the skipped exon (boxed in yellow) of interest is included. The splicing that gives rise to the exon included and excluded transcripts is also displayed, with specific primer pairs used in QPCR shown as blue arrows. A representative immunoblot is included to display input protein levels of E2F1 and SDMe. Actin was used as a loading control. (biological repeats: n = 4 for VCAN and METTL6, n = 3 for REV3L , and n = 8 for MDM1 ). See also . ( L ) A ChIP assay performed on WT E2F1 or E2F1 Cr HCT116 cells. Recruitment of E2F1 to the promoter regions of MDM1 and VCAN was tested. CDC6 acted as a positive control. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs. An immunoblot is included to display input protein levels of E2F1 and SDMe. Actin was used as a loading control (biological repeats: n = 3 for MDM1, VCAN , and CDC6 ).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Generated, Western Blot, Control, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: The pRb–E2F pathway regulates separate transcription and splicing gene networks. ( A ) Differential gene expression between WT and E2F1 Cr HCT116 cells, treated with DMSO or 1 μM T1-44 for 48 h, are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG across all samples, as compared to the WT E2F1 HCT116 cells treated with DMSO (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. Black boxes indicate DEGs that uniquely occur upon T1-44 treatment, only in the presence of WT E2F1. These data were generated from three independent biological samples. See also . ( B ) A Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) in each treatment, as compared to WT E2F1 HCT116 cells treated with DMSO. See also . ( C ) Annotation of genes which only undergo differential expression upon T1-44 treatment in the presence of WT E2F1 (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( D ) Differential gene expression between WT and Rb Cr MCF7 cells, treated with DMSO or 1 μM T1-44 for 48 h, are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG across all samples, as compared to the WT Rb MCF7 cells treated with DMSO (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. Black boxes indicate DEGs that uniquely occur upon T1-44 treatment, only in the presence of WT Rb. These data were generated from three independent biological samples. See also Supplementary Fig. S3E. ( E ) A Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) (DEGs) in each treatment, as compared to WT Rb MCF7 cells treated with DMSO. See also . ( F ) Annotation of genes which only undergo differential expression upon T1-44 treatment in the presence of WT Rb (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( G ) Venn diagrams displaying the overlap of total genes from the E2F1 Cr and Rb Cr RNA-seq datasets that score either as significantly differentially expressed (DEGs: padj < 0.05; log2FC > 0.58), significantly differentially spliced (AS: FDR < 0.05), or fall into both categories. These data were derived from the analyses in Figs and . ( H ) Venn diagrams showing the overlap between the total list of statistically significant DEGs (padj < 0.05, log2FC > 0.58), or the total list of statistically significant differentially spliced genes (AS: FDR < 0.05) in each of the indicated RNA-sequencing datasets, as compared to their corresponding wild-type cell lines.

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Gene Expression, Expressing, Generated, Quantitative Proteomics, RNA Sequencing, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: Cell cycle regulated alternative splicing and transcriptional events dependent on E2F1. ( A ) On the left, a representative flow cytometry profile for wild-type (WT) E2F1 and E2F1 Cr HCT116 cells synchronized at the G1/S boundary with a double thymidine block (0 h timepoint), or released from the block to progress through S phase (3 h) into G2/M (6 h), and back into a subsequent G1 (9 and 12 h) are displayed. An RT-PCR was performed at each timepoint to measure the inclusion of MDM1 exon 4 in RNA transcripts. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. A representative immunoblot is included to display input protein levels of Rb, phosphorylated Rb and E2F1. GAPDH was used as a loading control ( n = 4 biological repeats). See also . ( B ) WT E2F1 and E2F1 Cr HCT116 cells were treated with 1 mM hydroxyurea (HU) for 24 h to synchronize cells in early S phase. Hydroxyurea was then washed out and cells were allowed to progress through S phase for the indicated number of hours. Alternatively, cells were treated with 20 μM etoposide (Etop) for 48 h where indicated, and an RT-PCR was performed to measure the inclusion of MDM1 exon 4 in RNA transcripts. Displayed is the mean inclusion/exclusion ratio, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 4 biological repeats). See also . ( C ) A representative immunoblot to display input protein levels of Rb, phosphorylated Rb, E2F1, HNRNPC, and SRSF2 for the experiments described in Fig. . GAPDH was used as a loading control. ( D ) Differential changes in splicing between WT E2F1 and E2F1 Cr HCT116 cells synchronized in G1/S (0 h) or G2/M (6 h) are displayed as a heatmap of delta PSI values (ΔΨ, PSI) for all significant differential splicing events (FDR < 0.05, ΔΨ > 0.1). Each column of the heatmap represents delta PSI values of one splice event in cells at 6 h, as compared to the same cells at 0 h (blue: reduced inclusion; red: increased inclusion). Data clustering used the Canberra distance method. These data were generated from four independent biological samples. A representative immunoblot is included to display the input protein levels for E2F1. GAPDH was used as a loading control. ( E ) Venn diagram showing the overlap between statistically significant differentially spliced genes (AS: FDR < 0.05) between the 6 h (G2/M) and 0 h (G1/S) timepoints, in WT E2F1 and E2F1 Cr HCT116 cells. These data were derived from the analysis in Fig. . ( F ) Annotation of genes which undergo splice events that uniquely occur in WT E2F1 cells between the 6 and 0 h timepoints (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. Biological terms connected with cell cycle and DNA damage/repair are highlighted in red. The number of genes enriched in each category is displayed to the right of each bar. ( G ) The bar charts display the breakdown of statistically significant (FDR < 0.05) differential splicing events observed between each of the indicated treatments. SE, skipped/cassette exon; RI, retained intron; MXE, mutually exclusive exons; A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice. These data were derived from the analysis in Fig. . ( H ) Differential changes in gene expression between WT E2F1 and E2F1 Cr HCT116 cells synchronized in G1/S (0 h) or G2/M (6 h) are displayed as a heatmap of log2 fold change (log2FC) values for all significant DEGs (padj < 0.05, log2FC > 0.58). Each column of the heatmap represents log2FC values of one DEG in cells at 6 h, as compared to the same cells at 0 h (blue: reduced expression; red: increased expression). Data clustering used the Canberra distance method. These data were generated from four independent biological samples. See also . ( I ) Venn diagram showing the overlap between statistically significant DEGs (padj < 0.05, log2FC > 0.58) between the 6 h (G2/M) and 0 h (G1/S) timepoints, in WT E2F1 and E2F1 Cr HCT116 cells. These data were derived from the analysis in Fig. . ( J ) Annotation of genes which undergo differential expression only in WT E2F1 cells between the 6 and 0 h timepoints (see Fig. ). GO biological process (GO:BP) and Reactome gene sets were used for pathway analysis in Metascape. The number of genes enriched in each category is displayed to the right of each bar. ( K ) Venn diagram displaying the overlap of total genes from the RNA-seq experiment in Fig. and H, that score either as significantly differentially expressed (DEGs: padj < 0.05, log2FC > 0.58), significantly differentially spliced (AS: FDR < 0.05), or fall into both categories.

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Alternative Splicing, Flow Cytometry, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Generated, Derivative Assay, Gene Expression, Expressing, Quantitative Proteomics, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: Confirmation of cell cycle regulated alternative splicing and transcriptional events in cells. ( A ) RNA from WT E2F1 and E2F1 Cr cells either in G1/S (0 h) or G2/M (6 h) was used in an RT-PCR experiment to monitor the inclusion of exon 3 in TRPT1 , exons 2 and 3 in TRMT1 , exon 5 in DEPDC4 , and exon 17 in SORBS1 , each identified as being significantly differentially spliced in the analysis performed in Fig. . Displayed is the mean inclusion/exclusion ratio with SD. A diagram indicating the exon (boxed in grey) and intron (black lines) structure of each gene around the skipped exon (boxed in yellow) of interest is included. The splicing that gives rise to the exon included and excluded transcripts is also displayed, with specific primer pairs used in QPCR shown as blue arrows. Significance was calculated by ANOVA using Sidak’s multiple comparisons test. (biological repeats: n = 4 for TRMT1 ex2 + 3 and SORBS1 ex17, n = 5 for TRPT1 exon 3 and DEPDC4 exon 5). ( B ) RNA from WT E2F1 and E2F1 Cr cells either in G1/S (0 h) or G2/M (6 h) was used in an RT-PCR experiment to monitor the expression of genes ( THBS1, RCAN1 and IL18R1 ) identified as significantly differentially expressed in the RNA-sequencing experiment performed in Fig. . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 6 biological repeats). ( C ) A ChIP assay performed on wild-type E2F1 HCT116 cells. Recruitment of E2F1 to the promoter regions of the indicated differentially spliced genes ( TRPT1, TRMT1, DEPDC4 , and SORBS1 ) and DEGs ( THBS1, RCAN1 , and IL18R1 ) was tested. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs ( n = 3 biological repeats).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: RNA splicing factors associate with the E2F complex and are recruited to AS genes. ( A ) A representative immunoblot displaying the immunoprecipitation of E2F1 from WT HCT116 cells treated with 1 μM T1-44 or DMSO for 48 h where indicated. E2F1 Cr HCT116 cells were used in the E2F1 immunoprecipitation as a control. Rb is displayed on the immunoblots as a known interactor for E2F1, and SDMe is used as a control for T1-44 activity. GAPDH is presented as a loading control. The immunoprecipitated material was used in a downstream mass spectrometry analysis of the E2F1 interactome ( n = 3 bioligical repeats). ( B ) Volcano plots displaying log2 fold change (log2FC) and –log10 P -values for relative intensities of interacting proteins in E2F1 immunoprecipitates performed in T1-44 treated or untreated WT cells, as compared to immunoprecipitations performed in the control E2F1 Cr cell line. Red colour represents interacting proteins enriched in the WT E2F1 immunoprecipitates, whilst blue colour represents under-enriched proteins. Grey colour represents proteins that fell below the fold change or statistical cut-off applied ( P < 0.05, log2FC > 1). Marked on the figure are known E2F1 interacting proteins (Rb, E2F1, DP1, and DP2) and proteins implicated in RNA splicing and processing. These data were derived from the mass spectrometry experiment performed in Fig. ( n = 3 biological repeats). ( C ) Functional protein association networks for proteins identified as E2F1 interactors from mass spectrometry analysis performed on DMSO treated (i), or T1-44 treated cells (ii) were generated using STRING. Proteins that were enriched in E2F1 immunoprecipitations performed in WT cells, as compared to E2F1 Cr cells (fold change > 2), at a statistically significant level ( P < 0.05) were included. The edges indicate both functional and physical protein associations, with line thickness indicating the strength of data support. MCL clustering of proteins was performed. This figure was generated using the data from Fig. . ( D ) An immunoprecipitation experiment was performed in WT E2F1 HCT116 cells using the indicated antibodies against E2F1 (KH95 and G10 antibodies) or control IgG. Interacting SRSF2 and HNRNPC was detected using specific antibodies. Input protein levels are also displayed ( n = 2 bioligical repeats). See also . ( E ) An RIP assay was performed in (i) wild-type (WT) E2F1 HCT116 cells treated with 1 μM T1-44 or DMSO for 48 h as indicated, (ii) or in WT E2F1 and E2F1 Cr HCT116 cells. Anti-HNRNPC (HNC), -SRSF2 (SR2), -HNRNPH1 (HNH), or control IgG was used to immunoprecipitate the indicated splicing factors and bound RNA. Recruitment of splicing factors to the indicated exon regions (exon 3, 4, and 5) of MDM1 are shown. Displayed is the mean percentage enrichment of input, with SD. Significance was calculated by Student’s t -test between the indicated sample pairs. (iii) A diagram of the exon (boxed in grey) and intron (black lines) structure around the MDM1 skipped exon 4 (boxed in yellow) is displayed. Specific exon–intron flanking primer pairs used in the RIP analysis are indicated by blue arrows. (iv) A representative immunoblot is included to display input protein levels of E2F1, HNRNPC, SRSF2, HNRNPH1, and SDMe. α-Tubulin was used as a loading control ( n = 3 biological repeats).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Western Blot, Immunoprecipitation, Control, Activity Assay, Mass Spectrometry, Derivative Assay, Functional Assay, Generated

Journal: Nucleic Acids Research

Article Title: Separate transcription and splicing gene networks are linked and coordinated by the pRb–E2F pathway

doi: 10.1093/nar/gkag016

Figure Lengend Snippet: RNA splicing factors contribute to E2F-pathway dependent alternative splicing. ( A ) WT E2F1 HCT116 cells were transfected with control siRNA (siC) or siRNA against SRSF2 (siSR2) prior to treatment with 1 μM T1-44 or DMSO where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of SRSF2 . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). WT E2F1 and E2F1 Cr HCT116 cells were transfected with control siRNA (siC) or siRNA against SRSF2 (siSR2) where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of SRSF2 . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). ( C ) A representative immunoblot displaying input protein levels of E2F1 and SRSF2 for the experiments described in Fig. and . SDMe levels are also displayed and GAPDH served as a loading control. ( D ) WT E2F1 HCT116 cells were transfected with control siRNA (siC) or siRNA against HNRNPC (siHNC) prior to treatment with 1 μM T1-44 or DMSO where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of HNRNPC . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats: n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). WT E2F1 and E2F1 Cr HCT116 cells were transfected with control siRNA (siC) or siRNA against HNRNPC (siHNC) where indicated. (i) RNA extracted from these cells was used in an RT-PCR experiment to monitor the expression of HNRNPC . Displayed is the mean mRNA expression relative to the GAPDH internal calibrator, with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test ( n = 3). (ii) Alternatively, RNA was used to measure the inclusion of MDM1 exon 4 and HNRNPA2B1 exon 12 in transcripts. Displayed is the mean inclusion/exclusion ratio with SD. Significance was calculated by ANOVA using Sidak’s multiple comparisons test (biological repeats n = 4 for MDM1 and n = 3 for HNRNPA2B1 ). ( F ) A representative immunoblot displaying input protein levels of E2F1 and HNRNPC for the experiments described in Fig. and . SDMe levels are also displayed and GAPDH served as a loading control. ( G ) Model diagram indicating that the pRb–E2F complex, in concert with PRMT5 activity, regulates cell cycle dependent expression of E2F target genes both at the level of transcriptional control (i), and at the level of AS (ii). E2F target gene networks regulated by transcription or AS tend to be mutually exclusive. AS regulation is achieved in part through interactions between the pRb–E2F complex with RNA splicing factors, including SRSF2 and HNRNPC. E2F and PRMT5 activity regulate the recruitment of these splicing factors to mRNA around AS exons (ii).

Article Snippet: E2F1 chromatin immunoprecipitation (ChIPs) were performed as described previously [ ], using 3 μg of appropriate antibody [control rabbit IgG, anti-E2F1 (A300-766A), Bethyl Laboratories, Montgomery, USA] and pre-blocked protein A/G beads.

Techniques: Alternative Splicing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Activity Assay

Journal:

Article Title: Distinct Cellular Factors Regulate the c- myb Promoter through Its E2F Element

doi:

Figure Lengend Snippet: (A) Schematic representation of several E2F-regulated cellular promoters. Arrows indicate transcription initiation sites. (B) Alignment of E2F elements from the DHFR, E2F1, cdc2, c-myc, and c-myb promoters.

Article Snippet: Antibodies against E2F1 (sc-193x), E2F2 (sc-632x), E2F3 (sc-879x), E2F4 (sc-512x), E2F5 (sc-1699x), Ets-1/Ets-2 (sc-112x), and SP-1 (sc-420x) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal:

Article Title: Distinct Cellular Factors Regulate the c- myb Promoter through Its E2F Element

doi:

Figure Lengend Snippet: EMSA of E2F elements from different promoters. Complex formation in nuclear extracts from the lymphoblastoid cell line X50-7 and the indicated radiolabeled E2F elements (Fig. ​(Fig.1)1) was analyzed by EMSA as described in Materials and Methods. Protein-DNA complexes are indicated as a to e. DHFR, c-myc, c-myb, and CDC2, oligonucleotides corresponding to the E2F sites within the DHFR, c-myc, c-myb, and cdc2 promoters, respectively; E2F1-A and E2F1-B, oligonucleotides corresponding to the two E2F sites found in the E2F1 promoter; n.s., a nonspecific complex.

Article Snippet: Antibodies against E2F1 (sc-193x), E2F2 (sc-632x), E2F3 (sc-879x), E2F4 (sc-512x), E2F5 (sc-1699x), Ets-1/Ets-2 (sc-112x), and SP-1 (sc-420x) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal:

Article Title: Distinct Cellular Factors Regulate the c- myb Promoter through Its E2F Element

doi:

Figure Lengend Snippet: Identification of a DNA-binding species which interact solely with the E2F elements within the c-myb promoter. Complexes formed with X50-7 nuclear extracts and radiolabeled E2F elements from the DHFR (A) and c-myb (B) promoters were analyzed by EMSA. Reaction mixtures were preincubated in the absence (None) or in the presence of a 100-fold excess of the indicated unlabeled competitor oligonucleotides. Positions of complexes a to e are indicated. DHFRwt, c-myc, c-myb, and CDC2, oligonucleotides corresponding to the E2F sites within the DHFR, c-myc, c-myb, and cdc2 promoters, respectively; E2F1-A and E2F1-B, oligonucleotides corresponding to the two E2F sites found in the E2F1 promoter; DHFRmut, mutant DHFR probe.

Article Snippet: Antibodies against E2F1 (sc-193x), E2F2 (sc-632x), E2F3 (sc-879x), E2F4 (sc-512x), E2F5 (sc-1699x), Ets-1/Ets-2 (sc-112x), and SP-1 (sc-420x) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Mutagenesis

Journal:

Article Title: Distinct Cellular Factors Regulate the c- myb Promoter through Its E2F Element

doi:

Figure Lengend Snippet: Activation of the c-myb promoter by E2Fmyb-sp and/or SP-1 through the E2F element during the G1 phase of the cell cycle. (A) The indicated myb-Luc reporter plasmids containing wild-type or mutant E2F elements were cotransfected with pCMV-βgal into NIH 3T3 cells. The cells were placed in low (0.5%) serum 4 h after the removal of the calcium phosphate precipitates. The cells remained in low serum for >48 h to induce quiescence, at which point serum was added (time zero). At the indicated time points, cells were removed for cell cycle analysis by flow cytometry and for determination of luciferase and β-galactosidase activities. (B) An E2F1 luciferase reporter plasmid containing wild-type E2F elements was analyzed in parallel as a control. Luciferase values from a representative experiment (normalized for β-galactosidase activity) (A and B) and percentages of cells in each phase of the cell cycle at the indicated time points (C) are shown.

Article Snippet: Antibodies against E2F1 (sc-193x), E2F2 (sc-632x), E2F3 (sc-879x), E2F4 (sc-512x), E2F5 (sc-1699x), Ets-1/Ets-2 (sc-112x), and SP-1 (sc-420x) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Mutagenesis, Cell Cycle Assay, Flow Cytometry, Luciferase, Plasmid Preparation, Activity Assay

Journal: Cell death and differentiation

Article Title: E2F1-regulated miR-30b suppresses Cyclophilin D and protects heart from ischemia/reperfusion injury and necrotic cell death.

doi: 10.1038/cdd.2014.165

Figure Lengend Snippet: Figure 6 E2F1 promotes necrosis through miR-30b and CypD. (a) E2F1 levels are increased in cardiomyocytes exposed to H2O2. Cardiomyocytes were exposed to H2O2. Cells were harvested at the indicated times for the analysis of E2F1 levels by immunoblot. (b) Knockdown of E2F1 reduces necrotic cell death induced by H2O2. Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc. Twenty-four hours after infection, cells were treated with H2O2. PI exclusion was analyzed. *Po0.05 versus H2O2 alone. (c) The levels of E2F1 are increased in myocardial I/R. Mice were induced to undergo cardiac I/R at the indicated times as described in Materials and Methods. E2F1 levels were analyzed by immunoblot. (d and e) E2F1 knockout mice attenuates myocyte necrosis and myocardial infarction upon I/R. WTand E2F1 knockout mice were subjected to I/R as described in Materials and Methods. Myocyte necrosis (d) and myocardial infarction (e) were analyzed. *Po0.05 versus WT+I/R. (f and g) CypD TP attenuates the inhibitory effect of E2F1 knockdown on CypD expression and necrotic responses induced by H2O2. Cardiomyocytes were infected with adenoviral E2F1-siRNA or E2F1-sc, transfected with the TP (CypD-TP miR-30b) or the control (CypD-TP control) and then exposed to H2O2. CypD expression (f) was analyzed by immunoblot. Necrosis was assessed by PI exclusion assay (g). *Po0.05. GAPDH, glyceraldehyde 3-phosphate dehydrogenase

Article Snippet: Mouse E2F1 cDNA was from Origene (Rockville, MD, USA).

Techniques: Western Blot, Knockdown, Infection, Knock-Out, Expressing, Transfection, Control, Exclusion Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb B: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (%), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean ± SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey's HSD; different letters indicate p < 0.05. (D) Transfection efficiency (% GFP+) of a control plasmid (pMAX-GFP) quantified by flow cytometry. (E) JMP profiler for least-squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% Cls. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection, Control, Plasmid Preparation, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb C: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (6), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean = SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey’s HSD; different letters indicate p < 0.05. (D) JMP profiler for least-squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% Cls. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: High-throughput optimization of antibody production in CHO cells by tuning heavy- and light-chain promoter strength

doi: 10.3389/fbioe.2025.1747473

Figure Lengend Snippet: mAb E: effects of LC/HC promoter strength on titer, cell density, and viability post-transfection (72 hpt), and model profiling. (A) Viability (%), (B) viable cell density (VCD; cells/mL), and (C) titer (mg/L) for all LC/HC combinations. Promoter levels: 5, 40, and 100 RPU for both LC and HC. Bars show mean ± SD (n = 2). Statistical analysis was based on one-way ANOVA with Tukey’s HSD; different letters indicate p < 0.05. (D) JMP profiler for least- squares models of titer, VCD, and viability versus LC/HC promoter strength. Left: predicted means with 95% CIs. Right: composite desirability (0-1) balancing high titer with acceptable VCD and viability. Red dashed lines mark targets/constraints.

Article Snippet: CHO-S cells (R80007, Life Technologies, Waltham, MA, United States) CD CHO medium (10743029, Gibco, Thermo Fisher Scientific, Waltham, MA, United States) L-Glutamine (25030081, Thermo Fisher Scientific, Waltham, MA, United States) Freestyle MAX Transfection Reagent (16447100, Thermo Fisher Scientific, Waltham, MA, United States) OptiPRO serum free medium (12309019, Thermo Fisher Scientific, Waltham, MA, United States) Transfection positive control expressing GFP (pMax-E2F1, 16007, Addgene, Watertown, MA, United States) Solution 18 (910-3018, ChemoMetec A/S, Allerød, Denmark) NC-Slide A8 (941-0002, ChemoMetec A/S, Allerød, Denmark)

Techniques: Transfection

Journal: Molecular Cancer

Article Title: Regulation of DNA methyltransferase 1 transcription in BRCA1-mutated breast cancer: a novel crosstalk between E2F1 motif hypermethylation and loss of histone H3 lysine 9 acetylation

doi: 10.1186/1476-4598-13-26

Figure Lengend Snippet: Methylation patterns of the DNMT1 promoter in BRCA1-mutated and non-mutated breast cancer. Ai , location of CpG sites in the core promoter region of DNMT1. Genomic coordinates are shown, along with the primer-amplified fragments, GC percentage, location of individual CpG dinucleotides (dashes), the DNMT1 RefSeq gene (exon 1 shown as a blue box and intron shown as an arrowed line), and CpG island (green bar). The arrow indicates the direction of transcription. Aii and Aiii , comparative analysis of methylation patterns in the core promoter region of DNMT1 in non-mutated and BRCA1-mutated breast cancer, and their adjacent normal breast tissues (each group, n = 15). The circles correspond to the CpG sites denoted by black dashes in Figure Ai. Closed circles, methylation; open circles, unmethylated. Ten individual clones were sequenced for each sample. Arrow shows the methylation of a cytosine located in a CpG within the E2F1 motif (position +182, +1 is the transcription initiation site). B , summary of the methylation patterns of DNMT1 core promoter in BRCA1-mutated breast cancer and adjacent normal breast tissues. The y-axis shows the mean methylation sites. C and D , overall methylation percentage of the E2F1 motif and the DNMT1 core promoter region (-99 to +521) from BRCA1-mutated breast cancer and adjacent normal breast tissues. Bar graphs show mean ± SD.

Article Snippet: The shRNA lentiviral particles of GCN5 (sc-37946-V), PCAF (sc-36198-V) and E2F1 (sc-29297-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Methylation, Amplification, Clone Assay

Journal: Molecular Cancer

Article Title: Regulation of DNA methyltransferase 1 transcription in BRCA1-mutated breast cancer: a novel crosstalk between E2F1 motif hypermethylation and loss of histone H3 lysine 9 acetylation

doi: 10.1186/1476-4598-13-26

Figure Lengend Snippet: Repression of DNMT1 promoter activity by methylated E2F1 motif. Ai , comparative analysis of E2F1 motif methylation between BRCA1-mutated breast cancer and adjacent normal breast tissues. Aii , relationship between DNMT1 protein expression and promoter methylation in BRCA1-mutated breast cancer and adjacent normal breast tissues (U; unmethylated group, n = 46, M; methylated group, n = 39). Bi , the schematic shows that the nucleotide sequence of consensus E2F1 motif (Con.E2F1) was point mutated at position +182 (C to T) to generate the Mut.E2F1. Bii , 293 T cells, and 15 primary non-mutated and BRCA1-mutated breast cancer and their normal breast cells were transfected with Con.E2F1 and Mut.E2F1. 24 hours after transfection, whole-cell extracts were analyzed for luciferase activity. Each experiment was repeated four times for 293 T cells and primary breast cells of each patient. Bar graphs show mean ± SD. Ci , the schematic represents the selected nucleotide sequence containing the E2F1 motif, with or without a methyl group at the fifth position of the cytosine pyrimidine ring at position +182. Cii and Ciii , The CD spectra of the selected nucleotide sequence in the presence of 100 mM Na + or 100 mM K + are shown.

Article Snippet: The shRNA lentiviral particles of GCN5 (sc-37946-V), PCAF (sc-36198-V) and E2F1 (sc-29297-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Activity Assay, Methylation, Expressing, Sequencing, Transfection, Luciferase, Circular Dichroism

Journal: Molecular Cancer

Article Title: Regulation of DNA methyltransferase 1 transcription in BRCA1-mutated breast cancer: a novel crosstalk between E2F1 motif hypermethylation and loss of histone H3 lysine 9 acetylation

doi: 10.1186/1476-4598-13-26

Figure Lengend Snippet: Comparative analysis of histone modification and E2F1 enrichment around the E2F1 motif between BRCA1-mutated breast cancer and adjacent normal breast tissues. A , chromatin immunoprecipitation was performed using antibodies to H3K9Ac, H3K18Ac, H3K27Ac, H3K4me1, H3K4me2, H3K4me3, H3K36me3, H3K79me, H3K9me, H3K9me2, H3K9me3, H3K27me, H3K27me2, H3K27me3 and E2F1. PCR was performed for regions within the CpG islands and around the E2F1 motif. A negative control without antibodies is included for comparison. B , representative results of 15 primary BRCA1-mutated breast cancer and their normal breast tissues are shown. Bar graphs show mean ± SD.

Article Snippet: The shRNA lentiviral particles of GCN5 (sc-37946-V), PCAF (sc-36198-V) and E2F1 (sc-29297-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Modification, Chromatin Immunoprecipitation, Negative Control, Comparison

Journal: Molecular Cancer

Article Title: Regulation of DNA methyltransferase 1 transcription in BRCA1-mutated breast cancer: a novel crosstalk between E2F1 motif hypermethylation and loss of histone H3 lysine 9 acetylation

doi: 10.1186/1476-4598-13-26

Figure Lengend Snippet: H3K9ac and E2F1-mediated transcriptional regulation of DNMT1. Ai , RT-PCR showing GCN5, PCAF and E2F1 levels before and after knockdown by shRNAs, and normalized to β-actin expression. Aii , the results from three independent experiments are represented as mean ± SD. Bi , EdU labeling showing proliferation of GCN5, PCAF and E2F1-silenced and control cells. Blue, Hoechst 33342 labeling of cell nuclei; Red, EdU labeling of nuclei of proliferative cells. Bii , the EdU incorporation rate was expressed as the ratio of EdU positive cells to total Hoechst33342 positive cells. Ci and Cii , analysis of histone modification H3K9ac and transcription factor E2F1 enrichment around the E2F1 motif within the CpG islands after the deletion of GCN5, PCAF or E2F1. D , The interaction of E2F1 and GCN5 or PCAF were examined by the immunoprecipitation of cell extracts with an antibody to E2F1, and co-immunoprecipitation of E2F1, GCN5 and PCAF by western blot analysis. Results of Figure A-D were obtained in BRCA1-mutated breast cancer cells, and the same results were also obtained in 293 T cells and non-BRCA1 mutated breast cancer cells. Ei-Ev , the DNMT1 expression levels after deletion of H3K9ac and E2F1 around the E2F1 motif in 293 T cells, and 15 primary non-mutated and BRCA1-mutated breast cancer and their normal breast cells. Each experiment was repeated four times for 293 T cells and primary breast cells of each patient. Bar graphs show mean ± SD. * P < 0.05 vs. Control.

Article Snippet: The shRNA lentiviral particles of GCN5 (sc-37946-V), PCAF (sc-36198-V) and E2F1 (sc-29297-V) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Knockdown, Expressing, Labeling, Control, Modification, Immunoprecipitation, Western Blot

Journal: Nature communications

Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression.

doi: 10.1038/ncomms10028

Figure Lengend Snippet: Figure 3 | Accumulation of the H2a.z histone variant at E2f target genes in TKO HCC. (a) ChIP assay to detect the enrichment of common epigenetic marks at the promoter region of the panel of E2f target genes (see Fig. 2b for gene list) in TKO HCC cells, as determined by quantitative PCR (qPCR) analysis (n ¼ 3). Green: marks positively enriched; red: marks negatively enriched. (b) ChIP assay to detect the enrichment for the H3k9ac and the H3k4me3 marks (left) as well as the H2a.z histone variant (right) at the promoter region of the panel of E2f target genes in TKO HCC (black bar, long term Rb family loss) and in Rosa26 Cre-ERT2 TKO liver 4 days after Tamoxifen treatment (white bar, acute Rb family loss), as determined by qPCR analysis (n ¼ 3). The relative fold enrichment is compared for both conditions with the fold enrichment observed in control liver and arbitrarily set at 1 for the Acute Rb family loss condition. (c) ChIP-Seq was performed to identify the genomic sequences bound by E2f1 in TKO HCC. The graph determines the distance of E2f1 bound regions from the closest gene. (d) Venn diagram showing the overlap between E2f1 and H2a.z-bound genes in TKO HCC. P-value obtained by hypergeometric test. (e,f) E2f1 and E2f3 were pulled-down from TKO HCC lysates and the pull-down fractions were allowed to migrate on an acrylamide gel subsequently stained with Coomassie Blue for visualization (represented in f) and processed by mass spectrometry (n ¼ 3). The relative abundance of E2f1 (arbitrarily set at 100%), Dp1 (positive control) and Pontin/Reptin in the E2f1 pull-down fraction is indicated on the right of the display. All these proteins were isolated from the same band migrating at B50 kDa. (e) Summary of the principal findings of the MS performed with E2f1 and E2f3 in TKO HCC. The complete results are displayed in Supplementary Data 5. Error bars represent standard deviation *P o0.05; **Po0.01; ***Po0.001.

Article Snippet: To quantify the abundance of E2F1 and E2F3 proteins, we used the corresponding recombinant proteins (Origene) and loaded increasing amounts on a polyacrylamide gel, in parallel with protein extracts from TKO HCC.

Techniques: Variant Assay, Real-time Polymerase Chain Reaction, Control, ChIP-sequencing, Genomic Sequencing, Acrylamide Gel Assay, Staining, Mass Spectrometry, Positive Control, Isolation, Standard Deviation

Journal: Nature communications

Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression.

doi: 10.1038/ncomms10028

Figure Lengend Snippet: Figure 4 | E2f1 recruits the Pontin/Reptin complex in TKO HCC. (a) Immunoprecipitation (IP) for IgG, E2f1 and E2f3 in TKO HCC cells. Endogenous E2f1 and E2f3 were pulled-down and the presence of Reptin was detected in the pulled-down fractions by immunoblotting. (b) IP for HA and Reptin in TKO HCC cells. Endogenous Reptin was pulled-down and the presence of E2f1, E2f2, E2f3, Pontin and H2a.z was detected in the pulled-down fractions by immunoblotting. H2a.z could only be detected upon IP/western blot. (c) IP for IgG, E2f1 and E2f3 in TKO HCC cells. Endogenous E2f1 and E2f3 were pulled- down and the presence of Pontin was detected in the pulled-down fractions by immunoblotting. (d) IP for IgG and Pontin in TKO HCC cells. Endogenous Pontin was pulled-down and the presence of E2f1 and Reptin was detected in the pulled-down fractions by immunoblotting. (e–g) GST constructs encompassing domains of human E2f1 were generated (e) and their interaction with endogenous Reptin from TKO HCC cells was determined by immunoblotting (f). (g) The endogenous IP described in a was repeated and increasing amounts of GST-E2f1-1-121 were added to test its capacity to displace the E2f1/Reptin interaction. The presence of endogenous Reptin in the E2f1 pulled-down fraction was detected by immunoblotting. The E2f1 antibody used in this experiment (C-20) recognizes the C-terminal domain of E2f1. (h) Nuclear extracts from TKO HCC cells were deposited at the top of a gradient and allowed to migrate through the gradient by ultracentrifugation. Twenty fractions were collected from the gradient and aliquots were run on a polyacrylamide gel to detect the migration pattern of Pontin, Reptin, E2f1 and H2a.z by immunoblotting. (i,j) Detection by IP of the interaction between E2f1 and Pontin/Reptin in human cancer cell lines: HepG2, Hep3B and SNU4499 (hHCC), NBLS and Be2c (neuroblastoma), DAOY (medulloblastoma), U2OS (osteosarcoma), T47D (breast cancer), K562 (chronic myeloid leukemia-CML) and EW8 (Ewing sarcoma). Asterisk marks cell lines where Pontin and/or Reptin interacts with E2f1. (k) E2f1, Pontin and Reptin expression levels in the different cell lines used in i,j as detected by immunoblotting. Normalization was assessed by Ponceau staining to ensure equivalent loading. *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: To quantify the abundance of E2F1 and E2F3 proteins, we used the corresponding recombinant proteins (Origene) and loaded increasing amounts on a polyacrylamide gel, in parallel with protein extracts from TKO HCC.

Techniques: Immunoprecipitation, Western Blot, Construct, Generated, Migration, Expressing, Staining

Journal: Nature communications

Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression.

doi: 10.1038/ncomms10028

Figure Lengend Snippet: Figure 5 | Critical role for the chromatin remodelling functions of Reptin in TKO HCC. (a) Intensity of Reptin silencing in TKO HCC cells upon their transfection with either Scramble, Reptin1 or Reptin2 siRNAs, as determined by quantitative PCR (qPCR; n ¼ 3). (b) Expression of Reptin (upper panel), Pontin (middle panel) and Gapdh (lower panel) in TKO HCC cells transfected with either Scramble, Gapdh or Reptin1 or Reptin2 siRNAs, as detected by immunoblotting (n ¼ 3). (c) Average expression level of the panel of E2f target genes (see Fig. 2b for genes list) upon transfection of TKO HCC cells with Scramble (white), Reptin1 (grey) and Reptin2 siRNAs (black), as determined by reverse transcription–quantitative PCR analysis (n ¼ 3). (d,e) ChIP assay to detect the enrichment for E2f1, E2f3 (d) and H2a.z (e) at the promoter region of the panel of E2f target genes upon transfection of TKO HCC cells with either Scramble (white) or Reptin2 (black) siRNAs, as determined by qPCR analysis (n ¼ 3). (f) Decreased DNAseI hypersensitivity upon Reptin silencing in TKO HCC cells. Accessibility of promoter regions from cyclina2, mcm3, mcm6, glut4, gsk3b and pkm2 were assessed by qPCR, with non-promoter regions of gapdh as an internal control (n ¼ 3). (g) Growth curve of TKO HCC cells infected with the MigR1-IRES-GFP retrovirus either empty or expressing the wild- type (WT) and point mutant forms of human Pontin and Reptin. 50,000 cells were plated in six wells in triplicates and counted every day. This experiment has been performed three times independently and the panel displays the data from one representative experiment. (h) Representative image of cells expressing Reptin D299N point mutant. Cells expressing Reptin D299N (as identified by GFP þ expression, arrow) display a cellular crisis-like morphology and do not form colonies. They are outcompeted in the dish by rare GFP cells that display normal morphology and growth behaviour (colony on the right side of the picture). Normal morphology was also observed for all GFP þ cells expressing WT Pontin, WT Reptin and point mutant Pontin. Error bars represent standard deviation *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: To quantify the abundance of E2F1 and E2F3 proteins, we used the corresponding recombinant proteins (Origene) and loaded increasing amounts on a polyacrylamide gel, in parallel with protein extracts from TKO HCC.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Reverse Transcription, Control, Infection, Mutagenesis, Standard Deviation

Journal: Nature communications

Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression.

doi: 10.1038/ncomms10028

Figure Lengend Snippet: Figure 6 | Progressive recruitment of Pontin/Reptin by E2f1 in TKO HCC. (a) E2f1, E2f2, E2f3, E2f4 and Gapdh (normalization) expression levels in control liver and TKO HCC, as detected by immunoblotting. (b) IP for HA, E2f1 and E2f3 in primary liver extracts from cTKO (d0), Rosa26-CreERT2 TKO 4 days after Tamoxifen treatment (d4) and TKO HCC. Reptin (upper panels) and Pontin (middle panels) were detected in the pull-down fractions at each time point by immunoblotting. Expression levels of E2f1, E2f3, Reptin and Pontin were detected at the different time points in the corresponding input fractions by immunoblotting (lower panels). (c) H2a.z, Pontin, Reptin and Actin protein expression in control liver and TKO HCC, as detected by immunoblotting. Ponceau staining is used for normalization purpose. (d) mRNA expression for Reptin and Pontin in control liver (n ¼ 5) and TKO HCC (n ¼ 9), as detected by qPCR. (e) mRNA expression for H2a.z in control liver (n ¼ 5) and TKO HCC (n ¼ 9), as detected by qPCR. (f) Binding of E2f1 to the promoter region of H2a.z (following the identification of an E2f-binding site in the promoter of H2a.z), as shown by ChIP assay in TKO HCC cells (n ¼ 3). (g) Repression of H2a.z expression in two clones of TKO HCC cells (2.1 and 1.1) upon infection with retroviruses either control (MigR1) or expressing the Rb-7LP protein (n ¼ 3). (h) Left: Frequency of E2F1 expression in tissue microarray (TMA) encompassing human HCC cases from stages I–IV. Right: Representative E2F1 staining of two independent HCC cases classified as stage IV HCC. Error bars represent standard deviation *P o0.05; **P o0.01; ***Po0.001. WB, western blot.

Article Snippet: To quantify the abundance of E2F1 and E2F3 proteins, we used the corresponding recombinant proteins (Origene) and loaded increasing amounts on a polyacrylamide gel, in parallel with protein extracts from TKO HCC.

Techniques: Expressing, Control, Western Blot, Staining, Binding Assay, Clone Assay, Infection, Microarray, Standard Deviation

Journal: Nature communications

Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression.

doi: 10.1038/ncomms10028

Figure Lengend Snippet: Figure 7 | Distinct classes of E2f target genes based on E2f affinity for their promoter regions. (a,b) ChIP assays for E2f1 (a) and E2f3 (b) to detect their enrichment at the promoter regions of representative cell cycle or metabolic target genes (see Fig. 2b for gene lists) at different time points upon Rb family loss, as determined by quantitative PCR (n ¼ 3). (c,d) Quantification of E2f1 and E2f3 proteins expressed in TKO HCC. Protein extracts from TKO HCC tumours were run on a polyacrylamide gel in parallel with increasing amount of recombinant E2f1 (c, upper panel) and E2f3 (c, lower panel) proteins. Expression of E2f1 and E2f3 was quantified using the standard curve established with recombinant proteins. The average expression is displayed in d for better comparison. The box represents the data included within the 25th to 75th percentile. The horizontal bar represents the average. (e) Alignment of ChIP validated E2f-binding sites from cell cycle and metabolic target genes. Canonical E2f-binding site is found within the red rectangle and thymine residues are in bold. (f) Identification of consensus binding sites for early and late sets of E2f target genes. The consensus site for the early genes was obtained by collapsing the sequences of the E2f-binding sites identified in the set of 296 genes (see Fig. 2c for details). The consensus site for the late genes was obtained by collapsing the sequences of the E2f-binding sites located in the promoter region of the metabolic target genes and the Notch signalling genes11. The red box identifies the high-affinity binding site, which includes the T-stretch. The blue box identifies the minimal sequence, 50-SSCGC-30, required for low-affinity binding. (g) Gel shift assay to determine E2f1- and E2f3-binding affinity to probes containing either the early consensus sequence, the Pfkl sequence and a T-stretch mutated Pfkl sequence. Complexes were supershifted with increasing amount of E2f1 or E2f3 antibodies. Ss: supershift. Free probe is displayed at the bottom of the panel. (h) Gel shift assay with increasing amount of recombinant GST–E2f1 incubated with constant amount of the Pfkl and Pfkl T-stretch probes. (i,j) Intersection of genes transactivated in TKO HCC with gene sets identified in the E2f1 ChIP-Seq experiment performed in TKO HCC (i) and in MCF7 cells stably expressing HA-E2f1 by the Farnham group (j). P-value obtained by hypergeometric test. Error bars represent standard deviation *P o0.05; **P o0.01; ***Po0.001.

Article Snippet: To quantify the abundance of E2F1 and E2F3 proteins, we used the corresponding recombinant proteins (Origene) and loaded increasing amounts on a polyacrylamide gel, in parallel with protein extracts from TKO HCC.

Techniques: Real-time Polymerase Chain Reaction, Recombinant, Expressing, Comparison, Binding Assay, Sequencing, Gel Shift, Incubation, ChIP-sequencing, Stable Transfection, Standard Deviation

Journal: Nature communications

Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression.

doi: 10.1038/ncomms10028

Figure Lengend Snippet: Figure 8 | Progressive amplification of E2f response during cancer progression. Model for the evolution of E2f transcriptional output in TKO HCC. Upon Rb family loss, TKO liver cells enter cell cycle activity. Early lesions composed of proliferative Sca1 þ progenitor-like cells can be detected after 2–5 weeks and gradually turn into late lesions macroscopically visible by B4 months of age (TKO HCC). In quiescent cells, Rb family represses the transcriptional activity of E2f and the dimerization partner Dp1. Upon acute Rb family loss, E2f2 and E2f3 activate the transcription of cell cycle genes and E2f1. During TKO HCC progression, E2f1 gradually binds to the promoter region of E2f target genes and recruits Pontin and Reptin to incorporate H2a.z and amplify E2f transcriptional response. This mechanism leads to increased transactivation of cell cycle target genes (early genes displaying high-affinity E2f-binding sites in their promoter regions) and activation of non-cell-cycle target genes (late genes displaying low-affinity E2f-binding sites in their promoter regions), including genes that regulate the Warburg effect, Notch signalling and most likely other oncogenic features.

Article Snippet: To quantify the abundance of E2F1 and E2F3 proteins, we used the corresponding recombinant proteins (Origene) and loaded increasing amounts on a polyacrylamide gel, in parallel with protein extracts from TKO HCC.

Techniques: Activity Assay, Binding Assay, Activation Assay