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Molecular & Cellular Proteomics 8:2034-2050, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Complementary Quantitative Proteomics Reveals that Transcription Factor AP-4 Mediates E-box-dependent Complex Formation for Transcriptional Repression of HDM2^*,

Wei-Chi Ku^,, Sung-Kay Chiu^¶, Yi-Ju Chen^||, Hsin-Hung Huang^||, Wen-Guey Wu^, and Yu-Ju Chen^,**,,

From the Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan,
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan,
¶Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, China,
||Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan,
**Genomics Research Center, Academia Sinica, Taipei 115, Taiwan, and
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan

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.

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