cAMP-dependent transactivation involving the homeodomain protein Pbx1.
Pbx1 is a DNA-binding homeodomain protein originally discovered in the t(1;19) chromosomal translocation associated with pediatric pre-B acute lymphoblastic leukemia. Previously we reported a cAMP-regulatory sequence (CRS1) in the promoter region of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase cytochrome P450 (P450c17) to be the first endogenous Pbx1 binding site and that overexpression of Pbx1 in mouse adrenal Y1 tumor cells enhances cAMP-dependent transcription mediated by this element. Here we report further characterization of Pbx1 binding site in CRS1 and role of Pbx1 in cAMP-dependent, CRS1-mediated transcription. By gel shift analysis utilizing nuclear extracts from Y1 cells, a high-affinity Pbx-binding sequence has been determined to be TTGAT(T/G)GA(T/C)A which represents the 5' portion of CRS1. An artificial Pbx-binding sequence (PRS), previously determined by random PCR analysis, is similar to the Pbx1-binding sequence in CRS1 and by both gel shift analysis and transfection studies shows characteristics very similar to CRS1. Upon overexpression, Pbx1 is found capable of enhancing CRS1-mediated transcription in both steroidogenic (Y1, JEG3) and nonsteroidogenic (HepG2 and S194) cells when coexpressed with the catalytic subunit of cAMP-dependent protein kinase A. Thus even though Pbx1 has been found to be involved only in cAMP-dependent transcription of a gene involved in steroidogenesis (CYP17), Pbx1 is capable of participating in cAMP-dependent transcription of target genes without complex formation with steroidogenic tissue-specific nuclear factors.
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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232-0146, USA.
Waterman
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MR
Kagawa
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D000311:Adrenal Glands / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000502:physiology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology*; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; C083400:PBX1 protein, human; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase