title: p53-Regulated Networks of Protein, mRNA, miRNA, and lncRNA Expression Revealed by Integrated Pulsed Stable Isotope Labeling With Amino Acids in Cell Culture (pSILAC) and Next Generation Sequencing (NGS) Analyses.
abstract: We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3'-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the mechanisms of p53-mediated tumor suppression.
authors.affiliation: From the ‡Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Thalkirchner Straβe 36, 80337 Munich, Germany;
authors.forename: Sabine
authors.initials: S
authors.lastname: Hünten
authors.affiliation: From the ‡Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Thalkirchner Straβe 36, 80337 Munich, Germany;
authors.forename: Markus
authors.initials: M
authors.lastname: Kaller
authors.affiliation: ‖Department of Biochemistry and Functional Proteomics, Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany;
authors.forename: Friedel
authors.initials: F
authors.lastname: Drepper
authors.affiliation: ‖Department of Biochemistry and Functional Proteomics, Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany;
authors.forename: Silke
authors.initials: S
authors.lastname: Oeljeklaus
authors.affiliation: ‡‡Institute for Informatics, Ludwig-Maximilians-University Munich, 80337 Munich, Germany;
authors.forename: Thomas
authors.initials: T
authors.lastname: Bonfert
authors.affiliation: ‡‡Institute for Informatics, Ludwig-Maximilians-University Munich, 80337 Munich, Germany;
authors.forename: Florian
authors.initials: F
authors.lastname: Erhard
authors.affiliation: §§Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Dueck
authors.affiliation: §§Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Eichner
authors.affiliation: ‡‡Institute for Informatics, Ludwig-Maximilians-University Munich, 80337 Munich, Germany;
authors.forename: Caroline C
authors.initials: CC
authors.lastname: Friedel
authors.affiliation: §§Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Gunter
authors.initials: G
authors.lastname: Meister
authors.affiliation: ‡‡Institute for Informatics, Ludwig-Maximilians-University Munich, 80337 Munich, Germany;
authors.forename: Ralf
authors.initials: R
authors.lastname: Zimmer
authors.affiliation: ‖Department of Biochemistry and Functional Proteomics, Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany; **Center for Biological Systems Analysis (ZBSA), University of Freiburg, 79104 Freiburg, Germany;
authors.forename: Bettina
authors.initials: B
authors.lastname: Warscheid
authors.affiliation: From the ‡Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-University Munich, Thalkirchner Straβe 36, 80337 Munich, Germany; §German Cancer Consortium (DKTK), D-69120 Heidelberg, Germany; ¶German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany; heiko.hermeking@med.uni-muenchen.de.
authors.forename: Heiko
authors.initials: H
authors.lastname: Hermeking
mesh_terms: D001120:Arginine; D002247:Carbon Isotopes; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008239:Lysine; D035683:MicroRNAs / Q000378:metabolism*; D009587:Nitrogen Isotopes; D062085:RNA, Long Noncoding / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D035683:MicroRNAs; D009587:Nitrogen Isotopes; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D004247:DNA; D001120:Arginine; D008239:Lysine