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) ␣ 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 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 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 ZNF74⅐KAP-1⅐TIF1␣ 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-transcriptionbased oligomerization detection system to study the interaction of transcriptionally active proteins, including nuclear matrix proteins, in living cells.

TIF1δ, a Novel HP1-interacting Member of the Transcriptional Intermediary Factor 1 (TIF1) Family Expressed by Elongating Spermatids

“…Evidence for TIF1␦ Self-association-Because of the previous demonstration that TIF1s (␣, -␤, and -␥) can form homodimers (12,37) as well heterodimers (TIF1␣-TIF1␥) (12), we examined the dimerization capacities of TIF1␦ using the yeast two-hybrid system. In this assay, DBD-TIF1␦ was coexpressed with AAD fusion proteins between the AAD of VP16 and any one of the family members (AAD-TIF1␣, AAD-TIF1␤, AAD-TIF1␥, and AAD-TIF1␦; Fig.…”

“…Amino acid comparison revealed that, among the three mammalian members of the family, TIF1␥ is closer to TIF1␣ than to TIF1␤ (50% overall identity between TIF1␣ and TIF1␥ and ϳ30% identity among the other TIF1s) (9,36). In vitro, TIF1␣ and TIF1␥ hetero-oligomerize as efficiently as they homo-oligomerize, whereas TIF1␤ does homooligomerize but does not hetero-oligomerize with TIF1␣ or TIF1␥ (12,37). Moreover, it has been shown that an overexpression of TIF1␥ in transiently transfected cells can interfere with the transrepression activity of TIF1␣ (12).…”

An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects

“…Currently, assays to identify and characterize these interactions are primarily in vitro binding assays (4)(5)(6). In the past 5 years, imaging strategies based on yeast two-hybrid assays, reporter complementation assays, and resonance energy transferbased assay methods (7)(8)(9)(10)(11)(12) have been developed. However, all of these approaches have encountered shortcomings, limiting their potential to serve as a single imaging assay for measuring proteinprotein interactions from both single cells and physiologically relevant small living animal models.…”

Sumoylation of the Transcriptional Intermediary Factor 1β (TIF1β), the Co-repressor of the KRAB Multifinger Proteins, Is Required for Its Transcriptional Activity and Is Modulated by the KRAB Domain

“…All TIF1 family members (TIF1␣, ␤, ␥, ␦, and bonus) are characterized by an N-terminal tri-partite motif (named TRIM or RBCC) composed of a RING (really interesting new genes) finger followed by two B-boxes and a coiled-coil domain as well as a C-terminal bi-partite motif encompassing a PHD (Plant HomeoDomain) finger and a bromo domain (2, 8 -11). The RBCC motif of TIF1␤ is known to homodimerize and to specifically interact with the KRAB domain of KRAB multifinger proteins (12)(13)(14). The TIF1␤ PHD finger and bromo domain were reported to cooperate in transcriptional repression via recruitment of repressive enzymatic activities such as histone deacetylase complexes (NurD/Mi2-␣) and a methyltransferase protein (SETDB1) (15,16).…”