Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus.
The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPalpha) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPalpha was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPalpha to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPalpha 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/EBPalpha transactivation domain disrupted the redistribution of C/EBPalpha by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPalpha, and the truncated C/EBPalpha still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPalpha that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPalpha 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/EBPalpha 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/EBPalpha at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.
Day
Richard N
RN
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908, USA. rnd2v@virginia.edu
Voss
Ty C
TC
Enwright
John F
JF
Booker
Cynthia F
CF
Periasamy
Ammasi
A
Schaufele
Fred
F
D015153:Blotting, Western; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002467:Cell Nucleus / Q000502:physiology; D002843:Chromatin / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics / Q000502:physiology; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000502:physiology; D006801:Humans; D009154:Mutation; D010902:Pituitary Gland / Q000378:metabolism / Q000502:physiology*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology
D022763:CCAAT-Enhancer-Binding Protein-alpha; D002843:Chromatin; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C494187:POU1F1 protein, human; D050817:Transcription Factor Pit-1; D014157:Transcription Factors