The Pit-1beta domain dictates active repression and alteration of histone acetylation of the proximal prolactin promoter.
A critical problem in current molecular biology is to gain a detailed understanding of the molecular mechanisms by which related transcription factor isoforms with identical DNA sequence specificity mediate distinct transcription responses. Pit-1 and Pit-1beta constitute such a pair of transcription factor isoforms. Pit-1 enhances the Ras signaling pathway to the prolactin promoter, and Pit-1beta represses basal prolactin promoter activity as well as Ras signaling to the prolactin promoter in pituitary cells. We have previously demonstrated that the beta-domain amino acid sequence dictates the transcriptional properties of Pit-1beta. Here, we show that five hydrophobic beta-domain residues are required for Pit-1 isoform-specific repression of Ras signaling, and we demonstrate that sodium butyrate and trichostatin A, pharmacological inhibitors of histone deacetylation, as well as viral Ski protein, a dominant-negative inhibitor of recruitment of N-CoR/mSin3 histone deacetylase complexes, specifically reverse beta isoform-specific repression of Ras signaling. Moreover, we directly demonstrate, with a chromatin immunoprecipitation assay, that the Pit-1beta isoform alters the histone acetylation state of the proximal prolactin promoter. This differential analysis of Pit-1/Pit-1beta isoform function provides significant insights into the structural determinants that govern how different transcription factors with identical DNA sequence specificity can display opposite effects on target gene activity.
Department of Medicine and Department of Biochemistry and Molecular Genetics, Program in Molecular Biology and Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
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D000107:Acetylation; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism
D002087:Butyrates; D002843:Chromatin; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C060614:SKI protein, human; C012589:trichostatin A; D011388:Prolactin; D008156:Luciferases; D018631:ras Proteins; D000409:Alanine