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.
Mesh Terms:
Base Sequence, Binding Sites, Cell Line, Tumor, DNA-Binding Proteins, E-Box Elements, Histone Deacetylases, Humans, Hydroxamic Acids, Models, Biological, Molecular Sequence Data, Mutant Proteins, Nuclear Proteins, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Proteomics, Proto-Oncogene Proteins c-mdm2, Repressor Proteins, Reproducibility of Results, Transcription Factors, Transcription, Genetic, Tumor Suppressor Protein p53
Base Sequence, Binding Sites, Cell Line, Tumor, DNA-Binding Proteins, E-Box Elements, Histone Deacetylases, Humans, Hydroxamic Acids, Models, Biological, Molecular Sequence Data, Mutant Proteins, Nuclear Proteins, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Proteomics, Proto-Oncogene Proteins c-mdm2, Repressor Proteins, Reproducibility of Results, Transcription Factors, Transcription, Genetic, Tumor Suppressor Protein p53
Mol. Cell Proteomics
Date: Sep. 01, 2009
PubMed ID: 19505873
View in: Pubmed Google Scholar
Download Curated Data For This Publication
141317
Switch View:
- Interactions 85