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J. Biol. Chem., Vol. 278, Issue 25, 22367-22373, June 20, 2003

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Oligomerization of Transcriptional Intermediary Factor 1 Regulators and Interaction with ZNF74 Nuclear Matrix Protein Revealed by Bioluminescence Resonance Energy Transfer in Living Cells^*

Delphine Germain-Desprez, Martine Bazinet, Michel Bouvier  and Muriel Aubry 

From the Department of Biochemistry, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Quebec H3C 3J7, Canada

Transcriptional intermediary factor 1 (TIF1) and KAP-1/TIF1, 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 TIF1 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 TIF1 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·TIF1 complexes suggests the existence of cross-talk between KAP-1-interacting KRAB proteins and TIF1-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.

Received for publication, March 4, 2003 , and in revised form, April 7, 2003.

^* This work was supported by grants from the Canadian Institute of Health Research (to M. A. and M. Bouvier), the Heart and Stroke foundation of Canada (to M. A.), and the Natural Sciences and Engineering Research Council of Canada (to M. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Canada Research Chair.

Supported by a scholarship from the Fonds de la Recherche en Santé du Québec. To whom correspondence and reprints should be addressed: Dept. of Biochemistry, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Quebec H3C 3J7, Canada. Tel.: 514-343-6322; Lab: 514-343-6111 (ext: 3747); Fax: 514-343-2210; E-mail: Muriel.Aubry{at}UMontreal.ca.

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