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.
Highlights
Transcription factor activating enhancer-binding protein 4 (AP-4) is a basic helix-loop-helix protein that binds to E-box elements
Identification of an homolog of murine double minute 2 (HDM2)-P2 Promoter E-box Responsible for p53-independent Transcriptional Repression by AP-4 —It is well established that transcription of HDM2 is mediated by a constitutive P1 promoter and an inducible P2 promoter [44]
1.78 Ϯ 0.17 1.67 Ϯ 0.17 a Mascot protein score. b The number of nondegenerate peptide used for quantification. c cICAT ratios, WT and MU indicate proteins enriched on WT and MU DNA beads, respectively. d iTRAQ ratios, WT1 and WT2, indicate proteins enriched on WT DNA beads, whereas MU1 and MU2 indicate proteins enriched on MU
Summary
Chemicals—Monomeric acrylamide/bisacrylamide solution (40%, 29:1) was purchased from Bio-Rad. Nuclear extracts from 1 ϫ 107 HCT116 p53ϩ/ϩ cells (ϳ200 g) were incubated at a 250:1 (w/w) ratio with poly(deoxyinosinic-deoxycytidylic) acid in binding buffer containing 20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 100 mM NaCl, 0.1% (v/v) Nonidet P-40, and protease inhibitor mixture (Calbiochem) at room temperature for 10 min, followed by addition of 10-g MagnaBind streptavidin beads conjugated with either WT or MU oligonucleotides (ϳ60 fmol DNA) at room temperature for 30 min. For cICAT and iTRAQ analyses, large-scale DNA pull-downs were performed as above except nuclear extracts from ϳ4 ϫ 109 HCT116 p53ϩ/ϩ cells (ϳ140 mg of total protein) were harvested, and equal amounts (ϳ70 mg each) were incubated with 25 nmol of bead-bound WT or MU oligonucleotide. The resulting membranes were washed four times in TTBS at room temperature for 5 min, and immunopositive bands were visualized using the ECL detection system
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