Dynamic interactions between Pit-1 and C/EBPalpha in the pituitary cell nucleus.
The homeodomain (HD) transcription factors are a structurally conserved family of proteins that, through networks of interactions with other nuclear proteins, control patterns of gene expression during development. For example, the network interactions of the pituitary-specific HD protein Pit-1 control the development of anterior pituitary cells and regulate the expression of the hormone products in the adult cells. Inactivating mutations in Pit-1 disrupt these processes, giving rise to the syndrome of combined pituitary hormone deficiency. Pit-1 interacts with CCAAT/enhancer-binding protein alpha (C/EBPalpha) to regulate prolactin transcription. Here, we used the combination of biochemical analysis and live-cell microscopy to show that two different point mutations in Pit-1, which disrupted distinct activities, affected the dynamic interactions between Pit-1 and C/EBPalpha in different ways. The results showed that the first alpha-helix of the POU-S domain is critical for the assembly of Pit-1 with C/EBPalpha, and they showed that DNA-binding activity conferred by the HD is critical for the final intranuclear positioning of the metastable complex. This likely reflects more general mechanisms that govern cell-type-specific transcriptional control, and the results from the analysis of the point mutations could indicate an important link between the mislocalization of transcriptional complexes and disease processes.
Demarco
Ignacio A
IA
Department of Medicine, University of Virginia Health Services, Charlottesville, VA 22908-0578, USA.
Voss
Ty C
TC
Booker
Cynthia F
CF
Day
Richard N
RN
D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050817:Transcription Factor Pit-1 / Q000235:genetics / Q000378:metabolism*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1