A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein alpha required for prolactin gene transcription.
The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein alpha (C/EBPalpha), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBPalpha with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBPalpha induced PRL gene transcription in pituitary GHFT1-5 cells, whereas the coexpression of Pit-1 and C/EBPalpha in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBPalpha, fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBPalpha from regions of transcriptionally quiescent centromeric heterochromatin to the nuclear regions occupied by Pit-1. The homeodomain region of Pit-1 was necessary for the recruitment of C/EBPalpha. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBPalpha. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBPalpha, was itself recruited by C/EBPalpha to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity.
Enwright
John F
JF
Department of Medicine, University of Virginia Health System, Charlottesville, Virginia 22908-0578, USA.
Kawecki-Crook
Margaret A
MA
Voss
Ty C
TC
Schaufele
Fred
F
Day
Richard N
RN
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D002503:Centromere / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D006570:Heterochromatin / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010902:Pituitary Gland / Q000166:cytology / Q000502:physiology; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D022763:CCAAT-Enhancer-Binding Protein-alpha; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D006570:Heterochromatin; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D011388:Prolactin; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494187:POU1F1 protein, human; C494185:Pou1f1 protein, mouse; C494186:Pou1f1 protein, rat