(A) Schematic of the 7-gene human APOBEC3 locus with the A3B promoter magnified to depict five predicted E2F binding sites (A-E in blue) relative to the TSS at +1 (scales indicated). (B–E) Relative …
(A) Heatmap depicting high-to-low A3B expression levels in TCGA breast cancer specimens (n = 1,097) and correlations with two known RB/E2F target genes, MELK and FOXM1, and related APOBEC3 genes …
Complementary supporting data are in Figure 3—figure supplement 1. (A) Schematic of CRISPR/Cas9-mediated HDR of the predicted E2F binding site in A3B hemizygous MCF10A cells. Top: CRISPR/Cas9 …
(A) Four independent MCF10A clones bearing mutations within the predicted E2F binding site were obtained using two different gRNAs in parallel to three clones with a control lacZ gRNA. Immunoblot …
Immunoblot validations of representative binding proteins from proteomic experiments are in Figure 4—figure supplement 1. (A) Schematic of the SILAC DNA pull-down strategy used to identify proteins …
Immunoblots were done for the indicated proteins, which were pulled-down by wildtype A3B promoter sequence but not by the indicated mutant sequences.
(A) Schematics of the promoter regions interrogated by ChIP experiments. Wildtype E2F sites are depicted by blue boxes and the mutant E2F site in the A3B promoter by a white X-box. (B–C) E2F4, E2F6, …
Complementary analyses are presented in Figure 6—figure supplement 1. (A) Schematic depicting the bioinformatics workflow of TCGA breast tumor data sets based on the 20 genes most strongly …
(A–C) Reanalysis of the data sets in Figure 6, panels B-D. To facilitate pairwise comparisons, we first calculated a single relative mean expression value to represent the top 20 E2F-repressed/A3B-as…
Transcriptional repression of A3B through the combined activities of E2F4/DREAM and E2F6/PRC1.6 complexes. Other regulatory mechanisms including A3B transcriptional activation by PKC/ncNF-κB signal …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Homo sapiens, female) | MCF10A | ATCC | Cat#:CRL-10317 RRID:CVCL_0598 | |
Cell line (Homo sapiens, female) | MCF10A-4C10 | This study | Hemizygous for A3B | Request by contacting RSH |
Cell line (Homo sapiens, female) | MCF7 | ATCC | Cat#:HTB-22 RRID:CVCL_0031 | |
Cell line (Homo sapiens, female) | BT474 | ATCC | Cat#:CLR-7913 RRID:CVCL_0179 | |
Cell line (Homo sapiens, female) | Hs578T | ATCC | Cat#:HTB-126 RRID:CVCL_0332 | |
Cell line (Homo sapiens, female) | MDA-MB-453 | ATCC | Cat#:HTB-131 RRID:CVCL_0418 | |
Cell line (Homo sapiens, female) | HEK 293T | ATCC | Cat#:CRL-3216 RRID:CVCL_0063 | |
Antibody | Anti-RAD51 (rabbit monoclonal) | Abcam | Cat#:ab133534 RRID:AB_2722613 | WB (1:10,000) |
Antibody | Anti-E2F4 (mouse monoclonal) | Santa Cruz | Cat#:sc-398543 | WB (1:250) ChIP: 5 µg per 20 µg Dynabeads |
Antibody | Anti-E2F6 (rabbit polyclonal) | Abcam | Cat#:ab53061 RRID:AB_2097254 | WB (1:500) ChIP: 5 µg per 20 µg Dynabeads |
Antibody | Anti-HA (rabbit monoclonal) | Cell Signaling | Cat#:3724 RRID:AB_1549585 | WB (1:5000) |
Antibody | Anti-Rb (mouse monoclonal) | Santa Cruz | Cat#:sc-102 RRID:AB_628209 | WB (1:300) |
Antibody | Anti-E2F1 (mouse monoclonal) | Santa Cruz | Cat#:sc-251 RRID:AB_627476 | WB (1:1,000) |
Antibody | Anti-E2F3 (mouse monoclonal) | Santa Cruz | Cat#:sc-56665 RRID:AB_1122397 | WB (1:800) |
Antibody | Anti-E2F5 (mouse monoclonal) | Santa Cruz | Cat#:sc-374268 RRID:AB_10988935 | WB (1:800) |
Antibody | Anti-E2F6 (mouse monoclonal) | Santa Cruz | Cat#:sc-53273 RRID:AB_783163 | WB (1:300) |
Antibody | Anti-LIN9 (mouse monoclonal) | Santa Cruz | Cat#:sc-398234 | WB (1:300) |
Antibody | Anti-tubulin (mouse monoclonal) | Sigma-Aldrich | Cat#:T5168 RRID:AB_477579 | WB (1:20,000) |
Antibody | Anti-A3B (rabbit monoclonal) | NIH AIDS Reagent Program | Cat#:12397 RRID:AB_2721202 | WB (1:1,000) |
Antibody | Anti-L3MBTL2 (rabbit polyclonal) | Active Motif | Cat#:39569 RRID:AB_2615062 | ChIP: 5 µg per 20 µg Dynabeads |
Recombinant DNA reagent | pGL4.74 TK-RL renilla control (plasmid) | Promega | Cat#:E692A | Internal control for luciferase assays |
Recombinant DNA reagent | pGL3 basic (plasmid) | Promega | Cat#:E1751 | Base vector for luciferase assays |
Recombinant DNA reagent | pA3B-luciferase (plasmid) | This study | Wildtype A3B promoter + luciferase | Request by contacting RSH |
Recombinant DNA reagent | pLenti-lox-empty vector (plasmid) | Carpenter et al., 2019 | ||
Recombinant DNA reagent | pLenti-lox-BKPyV tTAg (plasmid) | Starrett et al., 2019 | ||
Recombinant DNA reagent | pLenti-lox-BKPyV tTAg LxCxE mutant (plasmid) | Starrett et al., 2019 | ||
Recombinant DNA reagent | pLenti4/TO-mCherry-T2A-MCS (plasmid) | This study | Base vector for E2F expression | Request by contacting RSH |
Recombinant DNA reagent | pLenti4/TO-mCherry-T2A-HA-E2F4 (plasmid) | This study | Lentiviral vector for expression of E2F4 | Request by contacting RSH |
Recombinant DNA reagent | pLenti4/TO-mCherry-T2A-HA-E2F5 (plasmid) | This study | Lentiviral vector for expression of E2F5 | Request by contacting RSH |
Recombinant DNA reagent | pLenti4/TO-mCherry-T2A-HA-E2F6 (plasmid) | This study | Lentiviral vector for expression of E2F6 | Request by contacting RSH |
Recombinant DNA reagent | pLentiCRISPR-LoxP-A3B-gRNA#1 (plasmid) | This study | Lentiviral vector for expression of gRNA targeting E2F site E | Request by contacting RSH |
Recombinant DNA reagent | pLentiCRISPR-LoxP-A3B-gRNA#3 (plasmid) | This study | Lentiviral vector for expression of gRNA targeting E2F site E | Request by contacting RSH |
Sequence-based reagent | Cas9-encoding modified RNA | TriLink Biotech | Cat#:L7206-100 | |
Commercial assay or kit | Dual Luciferase Reporter Assay | Promega | Cat#:E1960 | |
Commercial assay or kit | Neon Transfection System 100 µL Kit | ThermoFisher | Cat#:MPK10025 | |
Software, algorithm | MaxQuant version 1.5.2.8 | MaxQuant | RRID:SCR_014485 | |
Software, algorithm | Fiji | Fiji | RRID:SCR_002285 | |
Software, algorithm | GraphPad Prism 6 | GraphPad | RRID:SCR_002798 | |
Software, algorithm | Image Studio | LI-COR Biosciences | RRID:SCR_015795 | |
Other | Spark Multimode Microplate Reader | Tecan | ||
Other | Neon Transfection System | ThermoFisher | Cat#:MPK5000 | |
Other | LI-COR Odyssey FC | LI-COR | Cat#:2800 | |
Other | LightCycler 480 Instrument | Roche | Cat#:04640268001 | |
Other | EASY-nLC 1200 system | ThermoFisher | Cat#:LC140 | |
Other | Q Exactive HF mass spectrometer | ThermoFisher |
Genes associating positively with A3B expression in breast cancer.
Log-transformed SILAC values from proteomics experiments.
Gene expression values relative to TBP mRNA in TCGA breast tumors.
Sequences of oligonucleotides used in this study.