Complementary quantitative proteomics reveals that transcription factor AP-4 mediates E-box-dependent complex formation for transcriptional repression of HDM2.
Transcription factor activating enhancer-binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements. AP-4 has received increasing attention for its regulatory role in cell growth and development, including transcriptional repression of the human homolog of murine double minute 2 (HDM2), an important oncoprotein controlling cell growth and survival, by an unknown mechanism. Here we demonstrate that AP-4 binds to an E-box located in the HDM2-P2 promoter and represses HDM2 transcription in a p53-independent manner. Incremental truncations of AP-4 revealed that the C-terminal Gln/Pro-rich domain was essential for transcriptional repression of HDM2. To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we used DNA-affinity purification followed by complementary quantitative proteomics, cICAT and iTRAQ labeling methods, to identify a previously unknown E-box-bound AP-4 protein complex containing 75 putative components. The two labeling methods complementarily quantified differentially AP-4-enriched proteins, including the most significant recruitment of DNA damage response proteins, followed by transcription factors, transcriptional repressors/corepressors, and histone-modifying proteins. Specific interaction of AP-4 with CCCTC binding factor, stimulatory protein 1, and histone deacetylase 1 (an AP-4 corepressor) was validated using AP-4 truncation mutants. Importantly, inclusion of trichostatin A did not alleviate AP-4-mediated repression of HDM2 transcription, suggesting a previously unidentified histone deacetylase-independent repression mechanism. In contrast, the complementary quantitative proteomics study suggested that transcription repression occurs via coordination of AP-4 with other transcription factors, histone methyltransferases, and/or a nucleosome remodeling SWI.SNF complex. In addition to previously known functions of AP-4, our data suggest that AP-4 participates in a transcriptional-regulating complex at the HDM2-P2 promoter in response to DNA damage.
Ku
Wei-Chi
WC
Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan.
Chiu
Sung-Kay
SK
Chen
Yi-Ju
YJ
Huang
Hsin-Hung
HH
Wu
Wen-Guey
WG
Chen
Yu-Ju
YJ
D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024721:E-Box Elements / Q000235:genetics*; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D040901:Proteomics; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D004268:DNA-Binding Proteins; D006877:Hydroxamic Acids; D050505:Mutant Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; C055473:enhancer-binding protein AP-4; C012589:trichostatin A; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D006655:Histone Deacetylases