PRIDE Assigned Tags:Biological Dataset
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TRANSCRIPTIONAL ACTIVATION BY E2F4 INDEPENDENT OF THE RB FAMILY
E2F transcription factors are central regulators of cell cycle progression and cell fate decisions in mammalian cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. E2F4 often represents the predominant E2F activity in cells. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. Importantly, this role for E2F4 is completely independent of the RB family. Accordingly, an unbiased analysis of the E2F4 interactome shows that E2F4 functionally interacts with chromatin regulators associated with gene activation in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that reveal novel insights into the biology of rapidly dividing cell types.
Sample Processing Protocol
Tagged mouse E2F4 was overexpressed in TKO mESCs by lentiviral infection. The lentiviral backbone vector (pWPXLd/LAP-C/puro/DEST) was created by inserting a DEST/TEV cleavage site-S tag- PreScission cleavage site-EGFP/puromycin resistance cassette into pWPXLd vector, a gift from Prof. Didier Trono (Addgene plasmid #12258). The Gateway entry vector for mouse E2F4 was generated by cloning E2F4 cDNA from the PiggyBac system into pDONR221. Then the N-terminally LAP (EGFP-TEV cleavage site-S tag-PreScission cleavage site)-tagged mouse E2F4 was generated by LR recombination between mE2F4 entry vector and pWPXLd/LAP-N/puro/DEST. Lentiviral infection was performed as in 76 . Following infection for 48 hours, GFP-positive cells were isolated by flow cytometry. Tagged human E2F4 was overexpressed in RPE cells using the Flp-In system 77 . Gateway entry vector for human E2F4 was obtained from Life Technologies (clone number IOH23241). Flp-In system compatible N-terminally LAP (EGFP-TEV cleavage site-S tag-PreScission cleavage site)-tagged human E2F4 was generated by LR recombination between the hE2F4 entry vector and pG-LAP6/puro. Flp-In system compatible RPE cells (RPE-FRT9) were cultured in DMEM/F-12 (Thermo Fisher 12400024) supplemented with 10% FBS (Gemini 100-106), 1XGlutaMax (Thermo Fisher 35050-079), 100 U/mL penicillin- streptomycin (Thermo Fisher 15140163). RPE-FRT9 cells were transfected with 150 ng of LAP-hE2F4 and 850 ng of pOG44, and selection was performed with 10 µg/mL puromycin. Tandem affinity purification was performed as previously described. The gel slices were then reduced and alkylated followed by destaining and in-gel digestion using 125 ng Trypsin/LysC (V5072, Promega) as previously described (Shevchenko et al., 2006) with the addition of Protease Max (V2071, Promega) to increase digestion efficiency. Tryptic peptides were extracted from the gel bands and dried in a speed vac. Prior to LC-MS, each sample was reconstituted in 0.1% formic acid, 2% acetonitrile, and water. NanoAcquity (Waters) LC instrument was set at a flow rate of either 300 nL/min or 450 nL/min where mobile phase A was 0.2% formic acid in water and mobile phase B was 0.2% formic acid in acetonitrile. The analytical column was in-house pulled and packed using C18 Reprosil Pur 2.4 uM (Dr. Maisch) where the I.D. was 100 uM and the column length was 20-25 cm. Peptide pools were directly injected onto the analytical column in which linear gradients (4-40% B) were of either 80 or 120 min eluting peptides into the mass spectrometer. Either the Orbitrap Elite or Orbitrap Fusion mass spectrometers were used, where a top 15 or “fastest” MS/MS data acquisition was used, respectively. MS/MS was acquired using CID with a collisional energy of 32-35.
Data Processing Protocol
RAW files were processed using Byonic (Protein Metrics) using 12 ppm mass accuracy limits for precursors and 0.4 Da mass accuracy limits for MS/MS spectra. MS/MS data was compared to an NCBI Genbank FASTA database containing all human proteomic isoforms with the exception of the tandem affinit y bait construct sequence and common contaminant proteins. Spectral counts were assumed to have undergone fully specific proteolysis and allowing up to two missed cleavages per peptide. All data was filtered and presented at a 1% false discovery rate (Elias and Gygi, 2007). Post processing using in-house MatLab and Excel scripts was used to further process the data for gene ontological analyses.
Hsu J, Arand J, Chaikovsky A, Mooney NA, Demeter J, Brison CM, Oliverio R, Vogel H, Rubin SM, Jackson PK, Sage J. E2F4 regulates transcriptional activation in mouse embryonic stem cells independently of the RB family. Nat Commun. 2019 10(1):2939 PubMed: 31270324
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