Conformation of CCAAT/enhancer-binding protein alpha dimers varies with intranuclear location in living cells.
The structure of a protein defines its biochemical properties, but the impact of intracellular location and environment on protein structure remains poorly defined. CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a master regulator of transcription and cellular proliferation that concentrates and is kept inactive at transcriptionally quiescent, pericentromeric regions in mouse cell nuclei. C/EBPalpha dimer structure was measured in living cells from the amounts of fluorescence energy transferred between derivatives of the green fluorescent protein attached to different C/EBPalpha domains. Comparing the levels of fluorescence resonance energy transfer at pericentromeric and nonpericentromeric regions of the nucleus indicated that the DNA binding domains of C/EBPalpha dimers were further apart and interacted more poorly at pericentromeric heterochromatin than in the more euchromatic regions of the nucleus. In contrast, the position and interactions of the transcriptional activation domains were similar throughout the nucleus. Phorbol ester treatment caused a shift in the position of the transcriptional activation domain relative to the DNA binding domain. Thus, C/EBPalpha conformation varies with intranuclear location and with cellular environment. These "fluorescence resonance energy transfer nanoscopy" techniques will be broadly applicable for associating conformational and kinetic variations to subcompartment-specific actions of C/EBPalpha or any protein in the dynamic intracellular environment.
Schaufele
Fred
F
Diabetes Center, Metabolic Research Unit and Department of Medicine, University of California, San Francisco 94143-0540, USA. freds@diabetes.ucsf.edu
Wang
Xia
X
Liu
Xiaowei
X
Day
Richard N
RN
D016475:3T3 Cells; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000737:chemistry*; D002467:Cell Nucleus / Q000737:chemistry*; D002503:Centromere / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D051790:G-Box Binding Factors; D006570:Heterochromatin / Q000737:chemistry; D051379:Mice; D011487:Protein Conformation; D012399:Rotation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D022763:CCAAT-Enhancer-Binding Protein-alpha; D004268:DNA-Binding Proteins; D051790:G-Box Binding Factors; D006570:Heterochromatin; D014157:Transcription Factors; D013755:Tetradecanoylphorbol Acetate