Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells
Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells
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DOI：
10.1074/jbc.m302234200
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发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Aubry, M
中科院分区：
文献类型：
--
作者：
Germain-Desprez, D;Bazinet, M;Aubry, M
转录中间因子 1 (TIF1) α 和 KAP-1/TIF1β 是核辅因子 TIF1 家族的两个成员，分别是核受体和含 KRAB 基序的锌指转录因子的普遍共调节因子。尽管功能证据表明 TIF1 蛋白作为转录调节剂的作用，但研究它们与生理相关系统中转录机制的相互作用一直很困难。在这里，我们开发了一种生物发光共振能量转移（BRET）生物物理方法来研究活哺乳动物细胞核区室中蛋白质-蛋白质相互作用。我们报道 TIF1α 和 KAP-1 在完整的哺乳动物细胞中形成同源和异源寡聚体。 BRET 滴定实验表明，同源寡聚体和异源寡聚体均具有相对较高的亲和力，表明它们可以在细胞中共存。此外，我们证明 KAP-1 而不是 TIF1alpha 与核基质中的 KRAB 多指 ZNF74 相互作用。缺乏转录活性的 ZNF74 剪接变体和点突变体被发现不与 KAP-1 相互作用，证实了这种相互作用在活细胞中的生理重要性。 ZNF74 与 KAP-1 的相互作用不会阻止 KAP-1 均聚化，表明寡聚体很可能代表转录活性物质。此外，三元 ZNF74.KAP-1.TIF1α 复合物的检测表明 KAP-1 相互作用的 KRAB 蛋白和 TIF1α 相互作用的核受体之间存在串扰。除了提供有关这些蛋白质转录活性的分子相互作用的新见解外，这项研究还表明，BRET 可以有利地用作基于非转录的寡聚化检测系统，以研究活细胞中转录活性蛋白质（包括核基质蛋白）的相互作用。
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 heterooligomers 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.