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. 6 Transcriptional regulation of TFDP1 in HSPCs. A Experimental scheme of the RNA-seq experiment in Tfdp1-KO HSPCs. As a negative control, sgRosa26 was used. B Volcano plot depicting changes in gene expression in Tfdp1-KO HSPCs; y-axis represents the log10 transformation of the adjusted p value and x-axis the log2 transformation of the fold change. Red and blue dots represent up and downregulated genes, respectively. REACTOME pathway (C) and transcription factor enrichment analysis (D) of the downregulated genes in Tfdp1-KO HSPCs. The bubble plots depict the top 10 most significantly enriched gene sets. The bubble size corresponds to the number of genes and the color intensity reflects the adj. p value for each geneset. The total number of genes found within each dataset and the number of genes present in the downregulated genes are shown on the right.

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: RNA Sequencing, Negative Control, Gene Expression, Transformation 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. 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: The Journal of Cell Biology

Article Title: Retinoblastoma tumor suppressor protein–dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit

doi: 10.1083/jcb.200705051

Figure Lengend Snippet: pRb-dependent trimethylation of H3K27 at cell cycle gene promoters. (A) Histogram indicating H3K27Me2/3 levels in C2C12 myotubes transfected with a nonspecific control or pRb-silencing siRNA. The ChIP signal intensity is reported as a fraction of the signal obtained in cells transfected with the nonspecific siRNA. (B and C) Histograms depicting H3K27Me2/3 (B) and H3Ac (C) levels in C2C12 myotubes transfected with siRNAs targeting p130 or pRb or with a nonspecific control siRNA as indicated in A. Error bars represent SD of three independent experiments. (D) ChIP assay performed in C2C12 myoblasts and myotubes using antibodies directed against Suz12, Ezh2, and Bmi-1. Two regions of the Hoxd10 gene were surveyed to evaluate PRC binding. The results of two independent experiments are shown.

Article Snippet: The antibodies used were rabbit anti-E2F4 (Santa Cruz Biotechnology, Inc.) and mouse anti-p130 (Becton Dickinson).

Techniques: Transfection, Control, Binding Assay