Project PXD008796

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Biological Dataset
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Summary

Title

TRANSCRIPTIONAL ACTIVATION BY E2F4 INDEPENDENT OF THE RB FAMILY

Description

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.

Contact

Janos Demeter, Stanford University
Julien Sage, Department of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA ( lab head )

Submission Date

30/01/2018

Publication Date

26/04/2019

Publication

    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

Assay

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Showing 1 - 10 of 17 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 84878 160506_Blank_Pre_E2F4.raw_20160510_Byonic.mzid 429 1171 101 7404 0
2 84877 160506_NMooney_Jackson_5187_E2F4_01.raw_20160510_Byonic.mzid 561 5977 945 25469 0
3 84879 160506_NMooney_Jackson_5187_E2F4_02.raw_20160510_Byonic.mzid 574 9881 1104 27071 0
4 84874 160506_NMooney_Jackson_5187_E2F4_03.raw_20160510_Byonic.mzid 524 11032 1122 29722 0
5 84885 160506_NMooney_Jackson_5187_E2F4_04.raw_20160510_Byonic.mzid 572 10168 1117 30006 0
6 84873 160506_NMooney_Jackson_5187_E2F4_05.raw_20160510_Byonic.mzid 465 13460 1036 28594 0
7 84884 160506_NMooney_Jackson_5187_E2F4_06.raw_20160510_Byonic.mzid 547 10385 900 28230 0
8 84876 160506_NMooney_Jackson_5187_E2F4_07.raw_20160510_Byonic.mzid 687 7720 916 26641 0
9 84875 160506_NMooney_Jackson_5187_E2F4_08.raw_20160510_Byonic.mzid 447 4409 480 25200 0
10 84886 170629_JHsu_Sage_7200_E2F4_01.raw_20170707_Byonic.mzid 495 6958 1045 28449 0