title: Quantitative methods to analyze subnuclear protein organization in cell populations with varying degrees of protein expression.
abstract: The control of gene transcription is dependent on DNA-binding and coregulatory proteins that assemble in distinct regions of the cell nucleus. We use multispectral wide-field microscopy of cells expressing transcriptional coregulators labeled with fluorescent proteins (FP) to study the subnuclear localization and function of these factors in living cells. In coexpression studies, the glucocorticoid receptor interacting protein (GRIP) coactivator protein and the silencing mediator of retinoid and thyroid (SMRT) corepressor protein form spherical subnuclear focal bodies that are spatially distinct, suggesting that specific protein interactions concentrate these divergent proteins in separate subnuclear regions. However, the variability of these subnuclear bodies between cells within the population makes analysis based on "representative images" difficult, if not impossible. To address this issue, we develop a protocol for unbiased selection of cells from the population, followed by the automated quantification of the subnuclear organization of the labeled proteins. Statistical methods identify a significant linear correlation between the FP-coregulator expression level and subnuclear focal body formation for both FP-GRIP and FP-SMRT. Importantly, we confirm that these changes in subnuclear organization could be statistically normalized for differences in coregulator expression level. This integrated quantitative image analysis method will allow the rigorous comparison of different experimental cell populations that express variable levels of FP fusion proteins.
authors.affiliation: University of Virginia Health Sciences Center, Departments of Medicine and Cell Biology, Charlottesville, Virginia 22908, USA. rnd2v@virginia.edu
authors.forename: Ty C
authors.initials: TC
authors.lastname: Voss
authors.forename: Ignacio A
authors.initials: IA
authors.lastname: Demarco
authors.forename: Cynthia F
authors.initials: CF
authors.lastname: Booker
authors.forename: Richard N
authors.initials: RN
authors.lastname: Day
mesh_terms: D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D042581:Intranuclear Space / Q000378:metabolism*; D049408:Luminescent Agents; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009687:Nuclear Proteins / Q000378:metabolism*; D056985:Nuclear Receptor Co-Repressor 2; D051958:Nuclear Receptor Coactivator 2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D049408:Luminescent Agents; D008164:Luminescent Proteins; C539131:Ncor2 protein, mouse; D009687:Nuclear Proteins; D056985:Nuclear Receptor Co-Repressor 2; D051958:Nuclear Receptor Coactivator 2; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins