title: Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells.
abstract: Transcriptional intermediary factor 1 (TIF1) alpha and KAP-1/TIF1beta, two members of the TIF1 family of nuclear cofactors, are ubiquitous co-regulators of nuclear receptors and KRAB motif-containing zinc finger transcription factors, respectively. Despite the functional evidence suggesting a role for TIF1 proteins as modulators of transcription, the study of their interactions with transcriptional machineries in physiologically relevant systems has been difficult. Here, we have developed a bioluminescence resonance energy transfer (BRET) biophysical approach to study protein-protein interactions in the nuclear compartment of living mammalian cells. We report that TIF1alpha and KAP-1 form homo- and hetero-oligomers in intact mammalian cells. BRET titration experiments indicate that both homo- and hetero-oligomers occur with relatively high affinity suggesting that they could co-exist in cells. Furthermore, we demonstrate that KAP-1 but not TIF1alpha interacts with the KRAB multifinger ZNF74 in the nuclear matrix. Splice variants and point mutants of ZNF74 that lack transcriptional activity were found not to interact with KAP-1 confirming the physiological importance of this interaction in living cells. The interaction of ZNF74 with KAP-1 did not prevent KAP-1 homomerization indicating that the oligomers most likely represent the transcriptionally active species. Furthermore, the detection of ternary ZNF74.KAP-1.TIF1alpha complexes suggests the existence of cross-talk between KAP-1-interacting KRAB proteins and TIF1alpha-interacting nuclear receptors. In addition to providing new insights into the molecular interactions involved in the transcriptional activities of these proteins, this study shows that BRET can be advantageously used as a non-transcription-based oligomerization detection system to study the interaction of transcriptionally active proteins, including nuclear matrix proteins, in living cells.
authors.affiliation: Department of Biochemistry, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Quebec H3C 3J7, Canada.
authors.forename: Delphine
authors.initials: D
authors.lastname: Germain-Desprez
authors.forename: Martine
authors.initials: M
authors.lastname: Bazinet
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.forename: Muriel
authors.initials: M
authors.lastname: Aubry
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins; D051741:Kruppel-Like Transcription Factors; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C100388:ZNF74 protein, human; C093517:transcriptional intermediary factor 1; D049452:Green Fluorescent Proteins