Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha (C/EBPα) in the living pituitary cell nucleus

The homeodomain (HD) protein Pit-1 cooperates with the basic-leucine zipper (b-ZIP) protein CCAAT/enhancer binding protein alpha (C/EBPα) to control pituitary-specific prolactin (PRL) gene transcription. We previously observed that C/EBPα was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that co-expressed Pit-1 redistributed C/EBPα to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer (FRET) microscopy to show when C/EBPα 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/EBPα transactivation domain disrupted the redistribution of C/EBPα by Pit-1. FRET analysis revealed that the mutant Pit-1 still associated with C/EBPα, and the truncated C/EBPα still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPα that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPα 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/EBPα 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/EBPα at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression. Day et al.