Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: Fig. 3 In vitro CRISPR/Cas9-mediated screen to identify E2F members important for HSPC expansion. A FACS analysis of the percentage of GFP+ (Cas9+) cells within the mCherry+ (sgRNA+) and mCherry- (sgRNA-) HSPCs on day two (top) and six (bottom) post transduction. B Heatmap of survival/proliferation scores in HSPCs treated with the indicated sgRNAs (n = 2, biological replicates). The color indicates decreased (orange) and increased (blue) survival/proliferation scores. C Western blot of TFDP1 and E2F4 three days post targeting with sgRNAs against Tfdp1 and E2f4. SgRosa26-1 was used as a negative control. Actin was used as a loading control. D Co-immunoprecipitation (Co-IP) using TFDP1 (top) and E2F4 as precipitating antibody (bottom) (n = 2, biological replicates).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: In Vitro, CRISPR, Transduction, Western Blot, Negative Control, Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: Fig. 5 TFDP1 and E2F4 regulate HSPC proliferation, but not apoptosis. A Scheme of assessment of HSPC proliferation and apoptosis. Cas9- HSPCs were labeled with Celltrace, cultured for one day and transduced with lentiviral particles expressing sgRNAs targeting Tfdp1, E2f4, or the Rosa26. B Representative FACS analysis of the percentages of Annexin V+DAPI- (early) and Annexin V+DAPI+ (late) apoptotic cells within mCherry- (sgRNA-, upper panel) or mCherry+ (sgRNA+, lower panel) HSPCs treated with the indicated sgRNAs. C Representative FACS analysis of active Caspase 3+ apoptotic HSPCs treated with the indicated sgRNAs three days post puromycin selection (top) and summary of the data (bottom) based on HSPCs from three mice (n = 3). D Representative FACS analysis of the proliferation rates of mCherry- (sgRNA-) and mCherry+ (sgRNA+) HSPCs infected with the indicated sgRNAs two and four days post cell-trace labeling. The number of cell divisions is indicated. E Percentage of cell division in mCherry+ (upper) and mCherry- (below) HSPC subpopulations treated with the indicated sgRNAs on day two and day four post cell-trace labeling (n = 3 independent experiments).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Labeling, Cell Culture, Transduction, Expressing, Selection, Infection

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis.

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: Fig. 7 Meta-analysis of the role of TFDP1 and E2F4 in gene activation in mouse HSPCs. A Venn diagrams depicting the overlap between the differentially expressed genes in Tfdp1-KO HSPCs (downregulated genes in blue and upregulated genes in red) and human TFDP1- (left; GSE80661; GSE105217; GSE127368), human E2F4- (middle; GSE31477; GSE170651), and mouse E2F4- (right; GSE48666) bound target genes. B Intersection between human TFDP1- and E2F4-bound genes downregulated in Tfdp1-KO HSPCs. C Density plots (upper panel) and heatmaps (lower panel) depicting the average tag densities around TSSs (−2/+2 kb) of up- and downregulated genes in Tfdp1 KO HSPCs. Data are derived from the ChIP-seq of RNA polymerase II S5P (RnapolII S5P; GSE34518), H3K4Me3 (GSE75426), and E2F4 (GSE48666) (together with a negative control) in mouse ES cells. Right panel: ATAC-seq signals (GSE100738) from mouse short-term (ST) HSCs in the same genomic regions. D Example of RnapolII S5P, H3K4Me3, E2F4 tracks in mouse ES cells and ATAC-seq in ST-HSCs at the mouse Cdk1 locus. E Example of E2F4 and TFDP1 ChIP-seq signals at the CDK1 locus in various human cell types. Mouse and human E2F4 and TFDP1 DNA binding sites derived from the Unibind database are shown and the core nucleotides involved in DNA binding are highlighted.

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Activation Assay, Derivative Assay, ChIP-sequencing, Negative Control, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Hierarchical Requirement of SWI/SNF in Retinoblastoma Tumor Suppressor-mediated Repression of Plk1

doi: 10.1074/jbc.m400395200

Figure Lengend Snippet: FIG. 3. SWI/SNF is dispensable for E2F chromatin/promoter associa- tion. A, SW13 (lanes 1 and 2) and TSUPr-1 (lanes 3 and 4) cells were in- fected with either GFP (lanes 1 and 3) or p16ink4a (lanes 2 and 4) encoding adeno- viruses. Total protein was resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting. B, SW13 cells were transfected with cyto- megalovirus -gal, the reporter construct 3XE2F-Luc, and either vector or E2F2 as indicated. The relative luciferase activity was normalized to -gal activity for trans- fection efficiency, and vector control was set to 1. C, U2OS cells were transiently transfected with free GFP alone or GFP- E2F2. Total protein was resolved by SDS- PAGE, and GFP-E2F2 protein was de- tected with antibodies specific for GFP and E2F2. D, SW13 cells on 25-mm cov- erslips were cotransfected with GFP, GFP-Histone H2B, or GFP-E2F2 and ei- ther empty vector or wild type BRG1. The coverslips were transferred to live cell im- aging chambers, and nuclear fluorescence recovery after photobleaching analysis was performed 18 h post-transfection. E, SW13 (lane 1) and TSUPr-1 (lane 2) cells were infected with p16ink4a encoding ad- enoviruses. ChIP assays were performed with antibodies for E2F4 and Dbf4 (non- specific). Input and immunoprecipitated DNA was amplified by PCR with primers specific for the Plk1 promoter.

Article Snippet: The following antibodies were used: RB-purified mouse anti-human (554136, BD Biosciences), p107 (sc-318, Santa Cruz), p130 (sc-317, Santa Cruz), Plk1 (sc 17783, Santa Cruz; 06–813, Upstate Biotechnology, Inc.), BRG1 (sc-17796, Santa Cruz), p16ink4a (sc-759, Santa Cruz), E2F1 (gift from A. Yee), E2F2 (sc-9967, Santa Cruz), E2F4 (sc-1082, Santa Cruz), Lamin B (sc-6217, Santa Cruz), and Vimentin (gift from Dr. Wallace Ip).

Techniques: SDS Page, Western Blot, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Control, Fluorescence, Infection, Immunoprecipitation, Amplification

Journal: Journal of Biological Chemistry

Article Title: Hierarchical Requirement of SWI/SNF in Retinoblastoma Tumor Suppressor-mediated Repression of Plk1

doi: 10.1074/jbc.m400395200

Figure Lengend Snippet: FIG. 6. Schematic diagram of transcriptional regulation of Plk1. Here we show that activation of the Plk1 promoter is regulated by E2F and pocket proteins. In SWI/SNF-deficient cells, E2F4 and pocket proteins are recruited to the Plk1 promoter. Despite the presence of the pocket proteins, histones remain acetylated and the promoter retains activity. By contrast, in the presence of SWI/SNF, the E2F4 and pocket protein recruitment results in histone deacetylation at the Plk1 pro- moter and subsequent promoter repression. In summary, our data support a model of hierarchy between SWI/SNF and histone deacety- lation of the Plk1 promoter.

Article Snippet: The following antibodies were used: RB-purified mouse anti-human (554136, BD Biosciences), p107 (sc-318, Santa Cruz), p130 (sc-317, Santa Cruz), Plk1 (sc 17783, Santa Cruz; 06–813, Upstate Biotechnology, Inc.), BRG1 (sc-17796, Santa Cruz), p16ink4a (sc-759, Santa Cruz), E2F1 (gift from A. Yee), E2F2 (sc-9967, Santa Cruz), E2F4 (sc-1082, Santa Cruz), Lamin B (sc-6217, Santa Cruz), and Vimentin (gift from Dr. Wallace Ip).

Techniques: Activation Assay, Activity Assay