Functional interactions with Pit-1 reorganize co-repressor complexes in the living cell nucleus.
The co-repressor proteins SMRT and NCoR concentrate in specific subnuclear compartments and function with DNA-binding factors to inhibit transcription. To provide detailed mechanistic understanding of these activities, this study tested the hypothesis that functional interactions with transcription factors, such as the pituitary-gland-specific Pit-1 homeodomain protein, direct the subnuclear organization and activity of co-repressor complexes. Both SMRT and NCoR repressed Pit-1-dependent transcription, and NCoR was co-immunoprecipitated with Pit-1. Immunofluorescence experiments confirmed that endogenous NCoR is concentrated in small focal bodies and that incremental increases in fluorescent-protein-tagged NCoR expression lead to progressive increases in the size of these structures. In pituitary cells, the endogenous NCoR localized with endogenous Pit-1 and the co-expression of a fluorescent-protein-labeled Pit-1 redistributed both NCoR and SMRT into diffuse nucleoplasmic compartments that also contained histone deacetylase and chromatin. Automated image-analysis methods were applied to cell populations to characterize the reorganization of co-repressor proteins by Pit-1 and mutation analysis showed that Pit-1 DNA-binding activity was necessary for the reorganization of co-repressor proteins. These data support the hypothesis that spherical foci serve as co-repressor storage compartments, whereas Pit-1/co-repressor complexes interact with target genes in more widely dispersed subnuclear domains. The redistribution of co-repressor complexes by Pit-1 might represent an important mechanism by which transcription factors direct changes in cell-specific gene expression.
Voss
Ty C
TC
Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
Demarco
Ignacio A
IA
Booker
Cynthia F
CF
Day
Richard N
RN
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology* / Q000648:ultrastructure; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000502:physiology*; D056971:Nuclear Receptor Co-Repressor 1; D056985:Nuclear Receptor Co-Repressor 2; D051381:Rats; D012097:Repressor Proteins / Q000096:biosynthesis / Q000502:physiology*; D012680:Sensitivity and Specificity
D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; C539127:NCOR1 protein, human; C539130:NCOR2 protein, human; C539128:Ncor1 protein, mouse; C539129:Ncor1 protein, rat; C539131:Ncor2 protein, mouse; C539132:Ncor2 protein, rat; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D056985:Nuclear Receptor Co-Repressor 2; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D004247:DNA