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 in vitro and in vivo as demonstrated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Luciferase assay further revealed that AP-4 represses HDM2 transcription in a p53-independent manner. In addition, incremental truncations of AP-4 showed that the C-terminal glutamine/proline-rich domain is essential for transcriptional repression of HDM2. To further delineate the molecular mechanism(s) of AP-4 transcriptional control and its potential implications, we performed single-step 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. The two labeling methods complementarily quantified 75 putative components in AP-4 protein complex, including the most significant recruitment of DNA damage–responsive proteins, followed by transcription factors, transcription repressors/corepressors, and histone-modifying proteins. Using AP-4 truncation mutants and DNA pull-down assay, specific interaction of AP-4 with CTCF, SP1, and histone deacetylase 1 (an AP-4 corepressor) was validated. Although AP-4 may repress HDM2 transcripion by recruiting HDAC, inclusion of HDAC specific inhibitor, trichostatin A, did not alleviate AP-4-mediated repression of HDM2 transcription. Taken together the data suggest a previously unidentified histone deacetylase-independent repression mechanism. Alternatively, the complementary quantitative proteomics study suggests 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 promoter in response to DNA damage. In conclusion, we successfully demonstrate how AP-4 regulates HDM2 transcription via binding to the previously unknown AP-4 binding site. By taking the advantage of the complementary quantitative proteomics, we identify a DNA bound AP-4 protein complex from single-step DNA afftinity purification from crude nuclear extracts. By analyzing the components and functions of the AP-4 protein complex, we are able to deduce the possible repressive mechanisms on HDM2 transcription and the potential role of AP-4 in DNA damaging response. Our findling may shed a light in better understanding the physiological role of AP-4. Finally, we also develop a strategy combining single-step purification and complementary quantitative proteomics for target proteomics study, which provides an alternative MS-based way to study protein complex in the future.