Cloning And Functional Studies Of Human Transcription Factor Ap The-2delta The Protein

AP-2 transcription factors are sequence-specific DNA-binding proteinsexpressed in neural crest and other tissues during mammalian development.The mammalian AP-2 family of transcription factors consists of five members,AP-2α,AP-2β,AP-2γ,AP-2δand AP-2ε, which play an important role inregulating gene expression during development and differentiation of multipleorgans and tissues. A new AP-2 gene, which we named Ap-2δ, was identifiedin tandem with AP-2βon human chromosome 6p12-p21．1．Ap-2δproteincomprises 452 amino acids and is highly similar to other AP-2 proteins acrossthe DNA-binding and dimerization domains. The PY motif and critical residuesin the transactivation domain, which are highly conserved in the AP-2 familyand believed necessary for transactivation, were divergent in Ap-2δ. Theunique protein sequence and functional features of Ap-2δsuggestmechanisms, besides tissue-specific AP-2 gene expression, for specificcontrol of target gene activation. RT-PCR analysisi shows its relatively highexpression level in adult thymus, prostate, small intestine, skeletal muscle,placenta, brain and testis tissues. In an effort to better understand thetransactivation function of AP-2δ, we used cDNA microarray to evaluate therelative expression of human genes in AD293 cells and real-time PCR analysisto confirm our cDNA microarray results, our study demonstrated that AP-2δcould significantly regulate some gene, including ACE2, EIF4B, Cep290, MSN,RPL29, CAPNS1, HMOX1, ITSN1, LDOC1L, PDGFC and RPL29．Dual-Luciferase reporter assay system analyses indicate that exogenousexpression of AP-2δrobustly transactivated heme oxygenase1 (HMOX1) promoter activities in AD293 that express AP-2δand NIH3T3 cell lines that donot express AP-2δ. In this study, transcriptional regulation of hemeoxygenasel (HMOX1) by AP-2δwas investigated.Deletional analyses of the HMOX1 promoter for its transactivation byAP-2δindicate that sequence at-2O6bp--1bp upstream of the transcriptionstart site exit AP-2δbinding site. Our analysis of protein/DNA inreactionssupposed that AP-2δinteract with residing at-161bp--104bp upstream of the transcription. AP-2δprotein bound an optimized AP-2 consensus DNAsequence 5'-GCCTGAGGC-3' in vitro, and transactivated gene expression ineukaryotic cells. It bound poorly to a suboptimal AP-2 sequence5'-GCCN3GGC-3' to which all other AP-2 proteins bind in vitro, providing thefirst example of DNA target specificity. The transactivation domain of Ap-2δdiffers notably from those in the other AP-2 proteins because it lacks the PYmotif and several others conserved residues that are important for thetranscriptional activity of AP-2 proteins, yet functions as an equally strongactivator.