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Imaging the Localized Protein Interactions Between Pit-1 and the CCAAT/Enhancer Binding Protein in the Living Pituitary Cell Nucleus

Departments of Medicine and Cell Biology (R.N.D., T.C.V., J.F.E., C.F.B.), University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908; W.M. Keck Center for Cellular Imaging (A.P.), Department of Biology, University of Virginia, Charlottesville, Virginia 22904; and Metabolic Research Unit and Department of Medicine (F.S.), University of California, San Francisco, California 94143-0540

Address all correspondence and requests for reprints to: Dr. Richard N. Day, Departments of Medicine and Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: rnd2v{at}virginia.edu.

The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein (C/EBP) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBP was concentrated in regions of centromeric heterochromatin in pituitary GHFT1–5 cells and that coexpressed Pit-1 redistributed C/EBP to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBP was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBP transactivation domain disrupted the redistribution of C/EBP by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBP, and the truncated C/EBP still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBP that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBP to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBP are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBP at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.

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