Complementary quantitative proteomics reveals that transcription factor AP-4 mediates E-box-dependent complex formation for transcriptional repression of HDM2

Mol Cell Proteomics. 2009 Sep;8(9):2034-50. doi: 10.1074/mcp.M900013-MCP200. Epub 2009 Jun 7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Cell Line, Tumor
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • E-Box Elements / genetics*
  • Histone Deacetylases / metabolism
  • Humans
  • Hydroxamic Acids / pharmacology
  • Models, Biological
  • Molecular Sequence Data
  • Mutant Proteins / metabolism
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic / genetics
  • Protein Binding / drug effects
  • Protein Structure, Tertiary
  • Proteomics*
  • Proto-Oncogene Proteins c-mdm2 / metabolism*
  • Repressor Proteins / metabolism*
  • Reproducibility of Results
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism*
  • Transcription, Genetic* / drug effects
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • DNA-Binding Proteins
  • Hydroxamic Acids
  • Mutant Proteins
  • Nuclear Proteins
  • Repressor Proteins
  • Transcription Factors
  • Tumor Suppressor Protein p53
  • enhancer-binding protein AP-4
  • trichostatin A
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • Histone Deacetylases