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A PIT-1 Homeodomain Mutant Blocks the Intranuclear Recruitment Of the CCAAT/Enhancer Binding Protein Required for Prolactin Gene Transcription

Departments of Medicine and Cell Biology (J.F.E., M.A.K.-C., T.C.V., R.N.D.), University of Virginia Health System, Charlottesville, Virginia 22908-0578; and Metabolic Research Unit (F.S.), University of California, San Francisco, California 94143-0540

Address all correspondence and requests for reprints to: Richard N. Day, Ph.D., Department of Medicine, Box 800578, University of Virginia Health System, Charlottesville, Virginia 22908-0578. E-mail: rnd2v{at}virginia.edu.

The pituitary-specific homeodomain protein Pit-1 cooperates with other transcription factors, including CCAAT/enhancer binding protein (C/EBP), in the regulation of pituitary lactotrope gene transcription. Here, we correlate cooperative activation of prolactin (PRL) gene transcription by Pit-1 and C/EBP with changes in the subnuclear localization of these factors in living pituitary cells. Transiently expressed C/EBP induced PRL gene transcription in pituitary GHFT1–5 cells, whereas the coexpression of Pit-1 and C/EBP in HeLa cells demonstrated their cooperativity at the PRL promoter. Individually expressed Pit-1 or C/EBP, fused to color variants of fluorescent proteins, occupied different subnuclear compartments in living pituitary cells. When coexpressed, Pit-1 recruited C/EBP 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/EBP. A point mutation in the Pit-1 homeodomain associated with the syndrome of combined pituitary hormone deficiency in humans also failed to recruit C/EBP. This Pit-1 mutant functioned as a dominant inhibitor of PRL gene transcription and, instead of recruiting C/EBP, was itself recruited by C/EBP to centromeric heterochromatin. Together our results suggest that the intranuclear positioning of these factors determines whether they activate or silence PRL promoter activity.

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