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Submitted on April 8, 2002
Accepted on November 25, 2002
) in the living pituitary cell nucleus
1 Departments of Medicine and Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908, USA; W.M. Keck Center for Cellular Imaging, Department of Biology, University of Virginia, Charlottesville, VA 22904; Metabolic Research Unit and Department of Medicine, Box 0540, University of California, San Francisco, California, 94143-0540, USA; Present address: Department of Biology, Austin College, Suite 61582, Sherman, TX 75090
* To whom correspondence should be addressed.
The homeodomain (HD) protein Pit-1 cooperates with the basic-leucine zipper (b-ZIP) protein CCAAT/enhancer binding protein alpha (C/EBP
) to control pituitary-specific PRL (PRL) gene transcription. We previously observed that C/EBP was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that co-expressed Pit-1 re-distributed C/EBP to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer (FRET) microscopy to show 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. FRET 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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