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

Transcriptional intermediary factor 1 (TIF1) α and KAP-1/TIF1β, 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 TIF1α 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 TIF1α 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·TIF1α complexes suggests the existence of cross-talk between KAP-1-interacting KRAB proteins and TIF1α-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. Transcriptional intermediary factor 1 (TIF1) α and KAP-1/TIF1β, 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 TIF1α 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 TIF1α 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·TIF1α complexes suggests the existence of cross-talk between KAP-1-interacting KRAB proteins and TIF1α-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. TIF1α and KAP-1 (TIF1β) are ubiquitously expressed members of the transcriptional intermediary factor 1 (TIF1) 1The abbreviations used are: TIF1, transcriptional intermediary factor 1; FRET, fluorescence resonance energy transfer; BRET, bioluminescence resonance energy transfer; aa, amino acid(s); GFP, green fluorescent protein; MBP, maltose-binding protein; NLS, nuclear localization signal; HA, hemagglutinin; RBCC, ring finger, B boxes, and a coiled-coil domain. family. TIF1α was described as a modulator of ligand-activated transcription mediated by the retinoid nuclear receptors RXR and RAR (1Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (575) Google Scholar, 2Le Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (468) Google Scholar, 3vom B.E. Zechel C. Heery D. Heine M.J. Garnier J.M. Vivat V. Le Douarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: 110-124Crossref PubMed Scopus (350) Google Scholar, 4Nielsen A.L. Ortiz J.A. You J. Oulad-Abdelghani M. Khechumian R. Gansmuller A. Chambon P. Losson R. EMBO J. 1999; 18: 6385-6395Crossref PubMed Scopus (294) Google Scholar, 5Venturini L. You J. Stadler M. Galien R. Lallemand V. Koken M.H. Mattei M.G. Ganser A. Chambon P. Losson R. de The H. Oncogene. 1999; 18: 1209-1217Crossref PubMed Scopus (135) Google Scholar). KAP-1, for its part, has been proposed to act as a co-repressor for several KRAB motif-containing zinc finger transcription factors (6Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. et al.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17821) Google Scholar) such as human KOX1 (7Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2978Crossref PubMed Scopus (539) Google Scholar, 8Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (248) Google Scholar), ZNF133 and ZNF140 (7Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2978Crossref PubMed Scopus (539) Google Scholar), rat Kid1 (9Kim S.-S. Chen Y.-M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (247) Google Scholar), and mouse KRAZ1 and KRAZ2 (10Agata Y. Matsuda E. Shimizu A. J. Biol. Chem. 1999; 274: 16412-16422Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Although largely distributed in vertebrates, the absence of KRAB motif-containing proteins and TIF1 family members in yeast Saccharomyces cerevisiae suggests a late evolutionary apparition and expansion of these two protein families (8Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (248) Google Scholar, 11Le Douarin B. You J. Nielsen A.L. Chambon P. Losson R. J. Steroid Biochem. Mol. Biol. 1998; 65: 43-50Crossref PubMed Scopus (36) Google Scholar). Whereas a few studies have suggested that TIF1 family members can act as homo- or hetero-oligomers (12Peng H. Begg G.E. Harper S.L. Friedman J.R. Speicher D.W. Rauscher III, F.J. J. Biol. Chem. 2000; 275: 18000-18010Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), the nature of the complexes in which these proteins are engaged remain largely unknown. Indeed, despite the evidence suggesting an important role for TIF1 proteins as modulator of transcription, the study of their interactions with the transcriptional machinery in physiologically relevant systems has been difficult. Studies addressing their physical interactions included in vitro assays carried out mainly with soluble fragments (to minimize aggregation) of recombinant proteins (1Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (575) Google Scholar, 3vom B.E. Zechel C. Heery D. Heine M.J. Garnier J.M. Vivat V. Le Douarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: 110-124Crossref PubMed Scopus (350) Google Scholar, 4Nielsen A.L. Ortiz J.A. You J. Oulad-Abdelghani M. Khechumian R. Gansmuller A. Chambon P. Losson R. EMBO J. 1999; 18: 6385-6395Crossref PubMed Scopus (294) Google Scholar, 12Peng H. Begg G.E. Harper S.L. Friedman J.R. Speicher D.W. Rauscher III, F.J. J. Biol. Chem. 2000; 275: 18000-18010Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar, 14Peng H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar, Begg G.E. Speicher D.W. Rauscher III, F.J. Mol. Biol. 2000; PubMed Scopus Google Scholar, Schultz D.C. Friedman J.R. Fredericks W.J. Rauscher III, F.J. Mol. Biol. 1999; PubMed Scopus Google Scholar), yeast assays (1Le Douarin B. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat P. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (575) Google Scholar, 3vom B.E. Zechel C. Heery D. Heine M.J. Garnier J.M. Vivat V. Le Douarin B. Gronemeyer H. Chambon P. Losson R. EMBO J. 1996; 15: 110-124Crossref PubMed Scopus (350) Google Scholar, 5Venturini L. You J. Stadler M. Galien R. Lallemand V. Koken M.H. Mattei M.G. Ganser A. Chambon P. Losson R. de The H. Oncogene. 1999; 18: 1209-1217Crossref PubMed Scopus (135) Google Scholar, 8Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (248) Google Scholar, S.-S. Chen Y.-M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (247) Google Scholar, M. Ortiz J.A. C. C. C. L. Chambon P. Losson R. Proc. Natl. Acad. Sci. U. S. A. 2001; Google Scholar) and in systems A.L. Ortiz J.A. You J. Oulad-Abdelghani M. Khechumian R. Gansmuller A. Chambon P. Losson R. EMBO J. 1999; 18: 6385-6395Crossref PubMed Scopus (294) Google Scholar, 14Peng H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar, E. Y. M. H. Shimizu A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). with these to in vitro and studies suggested that TIF1α and KAP-1 can H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar) such interaction was in yeast interaction assays (8Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (248) Google Scholar). of TIF1α with KAP-1 was by yeast L. You J. Stadler M. Galien R. Lallemand V. Koken M.H. Mattei M.G. Ganser A. Chambon P. Losson R. de The H. Oncogene. 1999; 18: 1209-1217Crossref PubMed Scopus (135) Google Scholar), was not in vitro and H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar). The of the interactions TIF1 family members that the transcriptional activities of TIF1 and proteins between these proteins are not and mammalian cells. In to found in mammalian KAP-1, KRAB proteins have transcriptional activity in yeast (8Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (248) Google Scholar) with the that relevant interactions not be found in this system Douarin B. Nielsen A.L. Garnier J.M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (468) Google Scholar). In a physiologically mammalian system has been used to demonstrate the interaction of a KAP-1 with the KRAB of KRAZ1 and KRAZ2 (10Agata Y. Matsuda E. Shimizu A. J. Biol. Chem. 1999; 274: 16412-16422Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). such be used with proteins that have transcriptional its Although could this this approach has the of complexes or Furthermore, could not be used with proteins to the nuclear matrix such as the proteins that are to in the the for the of new to study protein-protein interactions transcriptional nuclear matrix and bioluminescence resonance energy transfer or have been used to protein-protein interactions in living 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R. Sci. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In BRET was used to study homomerization of the transcription factors, in the Y. D.W. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), and the oligomerization of the receptors in mammalian S. A. E. S. D. M. M. Proc. Natl. Acad. Sci. U. S. A. 2000; Google Scholar, M. 2001; PubMed Scopus Google Scholar). Here, nuclear and as BRET was used for the to study protein interactions in the nuclear of living mammalian cells. We report that two TIF1 family TIF1α and KAP-1, can form homo- and hetero-oligomers in intact mammalian and that KAP-1, interacts with the KRAB multifinger protein ZNF74 in the nuclear matrix. In addition to providing new insights into the molecular interactions involved in the transcriptional activities of the TIF1 and proteins, study that BRET can advantageously be used as a non-transcription-based detection system to study the interactions of transcriptional including nuclear matrix proteins, in living cells. KAP-1 and TIF1α KAP-1 was J. V. (9Kim S.-S. Chen Y.-M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (247) Google Scholar, R. O'Leary E. Bonventre J.V. Biochem. PubMed Scopus Google Scholar) and as an into the and of and TIF1α was R. A.L. Ortiz J.A. You J. Oulad-Abdelghani M. Khechumian R. Gansmuller A. Chambon P. Losson R. EMBO J. 1999; 18: 6385-6395Crossref PubMed Scopus (294) Google Scholar) and as a into and an KAP-1 and TIF1α were into the or of M. W. Full Text PDF PubMed Scopus Google Scholar), respectively. ZNF74 for B. M. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), F. B. M. M. Biol. 2001; PubMed Scopus Google Scholar), and the zinc finger of ZNF74 to or to were and in B. M. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, F. B. M. M. Biol. 2001; PubMed Scopus Google Scholar). the KRAB of of the KRAB amino Friedman J.R. H. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) to was by J. Scholar) and the of was by the the BRET ZNF74 were as fragments into the and the of and was used to maltose-binding protein with ZNF74 proteins as described B. M. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). and to a of the to was a protein into the a nuclear localization J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) was the in with and as as were in D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) in by and 1 were a of 1 of were the by the J. Scholar). with or were in as described The of used for was to by for including TIF1α were with and with 1 were in 1 and were distributed in the addition of of the the bioluminescence its was a The energy to was a were with a that of the and The BRET was by the as A. S. A. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The BRET was found to be several addition of the in a S. A. E. S. D. M. M. Proc. Natl. Acad. Sci. U. S. A. 2000; Google Scholar). of was by of fluorescence and of The fluorescence was a with an an and the 1; the fluorescence the were for with a of and the of was a with the 1; In to not to energy transfer to and to activity The BRET were as a of the protein both proteins with the as described to into the in the of to BRET titration were a were with 1 and and for and for The were protein for 1 of the were carried out with protein and mouse were in and the proteins were by and for with and were as described by and B. R. Mol. Biol. 2001; PubMed Scopus Google Scholar). with were for in for the of was with an of or protein for to and with an for 1 by the affinity were by and for the the was and with an to the of of proteins not The of and proteins was by the mouse J.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), to and to of and mouse to Chen Mol. Biol. 1998; 18: PubMed Google Scholar). addition of the a or a was used of TIF1 in the of interaction such as a ring finger, B boxes, and a coiled-coil suggests that members of the TIF1 family could studies such interactions to L. You J. Stadler M. Galien R. Lallemand V. Koken M.H. Mattei M.G. Ganser A. Chambon P. Losson R. de The H. Oncogene. 1999; 18: 1209-1217Crossref PubMed Scopus (135) Google Scholar, 8Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (248) Google Scholar, 12Peng H. Begg G.E. Harper S.L. Friedman J.R. Speicher D.W. Rauscher III, F.J. J. Biol. Chem. 2000; 275: 18000-18010Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar, 14Peng H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar). Here, a developed BRET Y. D.W. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, S. A. E. S. D. M. M. Proc. Natl. Acad. Sci. U. S. A. 2000; Google Scholar, A. S. A. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), homo- and of KAP-1 and TIF1α were in living mammalian cells. the transfer of energy between a bioluminescence and a with a of and that transfer be for Y. D.W. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), BRET was used to interactions between these this KAP-1 and TIF1α were their amino with or green and were in and the of BRET was by the of the by the and the addition of the The BRET was as a of the protein protein in as described to into the in the of to in BRET of protein were for the and In a of was the BRET as a of the and a the of expressed was with of a the were for the two BRET and not as most BRET between and the oligomers used to the interactions with the that of the complexes be the of the KAP-1 and TIF1α oligomers BRET was in or and a to the by to a nuclear localization The nuclear of and was by BRET, most likely BRET A. S. A. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google was with the and The and of KAP-1 oligomerization and with TIF1α was in Indeed, the addition of the BRET with and TIF1α expressed KAP-1 in with could not be for the as the of and for the of the for the cells. The for the can be used to the affinity of the for A. S. A. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). this as the of in to the BRET were these that the to have a affinity the In an to the of to the a the and the was The for this which by to an of and was that this can be used to the of the and and was to the BRET for TIF1α KAP-1 and respectively. indicate that the between the are relatively high and that not for the interactions to Furthermore, the affinity of the homo- and are in the of these complexes can co-exist in living cells. experiments carried in the KAP-1 proteins, and that KAP-1 oligomers represent complexes that and Indeed, as in of the could be with the The of TIF1α did not experiments to be carried out to 1 of with of of et H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar) were to TIF1α homomerization by but to interaction between TIF1α and KAP-1 H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar). The detection by BRET of both TIF1α homo- and with KAP-1, as as of the high of the energy transfer the to the homo- and of TIF1 family members yeast in vitro and out in the BRET a to these interactions and that TIF1α and KAP-1 form homo- and in intact mammalian cells. The functional of TIF1 family oligomerization to be was found that fragments of the KAP-1 can and that such oligomerization for interaction with the KRAB of in vitro H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar). that proteins KAP-1 oligomerization for this suggests that the KAP-1 co-repressor oligomerization be a for KRAB proteins to their a study that with TIF1α and TIF1α of RXR nuclear transcription, was suggested that the of TIF1 family members a transcription H. Feldman I. Rauscher III, F.J. J. Mol. Biol. 2002; 320: 629-644Crossref PubMed Scopus Google Scholar). was suggested that nuclear of KAP-1 to transcriptionally its oligomerization E. Y. M. H. Shimizu A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Indeed, such was by the of KAP-1 but by the with which has the to the in this study localization of these TIF1 family or their transcriptional to be between KAP-1, and TIF1α in a nuclear matrix protein B. M. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) that to the KRAB multifinger family. a a KRAB and a with an KRAB are by and F. B. M. M. Biol. 2001; PubMed Scopus Google Scholar). Whereas to nuclear in factors and transcriptionally has a nuclear localization and its KRAB has been to transcription F. B. M. M. Biol. 2001; PubMed Scopus Google Scholar). that the activity of several KRAB multifinger proteins has been to an interaction with the co-repressor KAP-1 (7Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2978Crossref PubMed Scopus (539) Google Scholar), could that a interaction with of its to the nuclear matrix and its the in interactions of ZNF74 with KAP-1 protein could not be in mammalian by or by a interaction BRET was used to the interaction between and KAP-1 in living mammalian cells. in BRET of and KAP-1 were for the BRET were the were in the In between and the TIF1α was Indeed, the BRET between and the not was not the with the interaction of KAP-1 with ZNF74 and BRET titration in living mammalian cells. of ZNF74 and ZNF74 mutants were was with to of or to the zinc finger of ZNF74 to GFP, was used as a The of shows that this to the nuclear matrix as and not in the soluble of the or the The BRET as a of the the the and are and of experiments were of ZNF74 and mutants by were with an The and the BRET for these are point to ZNF74 and and mutants and and of the molecular in are in vitro and an were with were by and with an The of was by the in the BRET of was to the and the of BRET of KAP-1 not by the interaction of with that can to oligomers of KAP-1 in intact mammalian and suggests that the oligomers most likely represent the transcriptionally active species. interaction between ZNF74 and oligomers of KAP-1 with the in vitro that fragments KAP-1 as with a KRAB KOX1 (12Peng H. Begg G.E. Harper S.L. Friedman J.R. Speicher D.W. Rauscher III, F.J. J. Biol. Chem. 2000; 275: 18000-18010Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Peng H. Begg G.E. Schultz D.C. Friedman J.R. Jensen D.E. Speicher D.W. Rauscher III, F.J. J. Mol. Biol. 2000; 295: 1139-1162Crossref PubMed Scopus (162) Google Scholar). The interaction of KRAB zinc finger with TIF1α L. You J. Stadler M. Galien R. Lallemand V. Koken M.H. Mattei M.G. Ganser A. Chambon P. Losson R. de The H. Oncogene. 1999; 18: 1209-1217Crossref PubMed Scopus (135) Google Scholar, 8Moosmann P. Georgiev O. Le Douarin B. Bourquin J.P. Schaffner W. Nucleic Acids Res. 1996; 24: 4859-4867Crossref PubMed Scopus (248) Google Scholar, M. Ortiz J.A. C. C. C. L. Chambon P. Losson R. Proc. Natl. Acad. Sci. U. S. A. 2001; Google Scholar) to the that cross-talk could between KRAB multifinger proteins and nuclear receptors to interact with TIF1α Douarin B. You J. Nielsen A.L. Chambon P. Losson R. J. Steroid Biochem. Mol. Biol. 1998; 65: 43-50Crossref PubMed Scopus (36) Google Scholar, R. Biol. Chem. Google Scholar). proteins in living mammalian that ZNF74 not interact with study to demonstrate TIF1α interaction with the KRAB of KRAB zinc finger proteins yeast systems M. Ortiz J.A. C. C. C. L. Chambon P. Losson R. Proc. Natl. Acad. Sci. U. S. A. 2001; Google Scholar). for these KRAB zinc finger proteins as as for a cross-talk with nuclear receptors not likely to occur a interaction with the that a cross-talk between TIF1α and ZNF74 could the of a ternary between and KAP-1, the of KAP-1 to a BRET interaction between TIF1α and was in the BRET between TIF1α and for titration was by the addition of the in the BRET was for the was not was used of not that KAP-1, and ZNF74 are of a protein TIF1α and ZNF74 in for BRET to we that of ternary complexes between the co-regulators KAP-1, and KRAB multifinger proteins such as ZNF74 cross-talk of KRAB multifinger proteins with nuclear receptors. of with ZNF74 of an KRAB the BRET between and ZNF74 was of the Indeed, as in the BRET with the was that with the in for ZNF74 B. M. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), the was to the nuclear matrix and was not in the soluble of the including the or in the as a a nuclear matrix B. F. M. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to was the was in the nuclear matrix and a was for the out that the BRET between and could represent BRET in the nuclear matrix. in vitro and yeast studies that the KRAB of a few proteins and for interaction with KAP-1 co-repressor (7Friedman J.R. Fredericks W.J. Jensen D.E. Speicher D.W. Huang X.P. Neilson E.G. Rauscher III, F.J. Genes Dev. 1996; 10: 2067-2978Crossref PubMed Scopus (539) Google Scholar, S.-S. Chen Y.-M. O'Leary E. Witzgall R. Vidal M. Bonventre J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15299-15304Crossref PubMed Scopus (247) Google Scholar, M. Ortiz J.A. C. C. C. L. Chambon P. Losson R. Proc. Natl. Acad. Sci. U. S. A. 2001; Google Scholar). the an KRAB its amino we its interaction with KAP-1 in living mammalian cells. In with the for the BRET between and the not was not that with the nuclear matrix The importance of the KRAB for of the interaction was by two amino and proposed to be important for the interaction of KRAB motif-containing proteins with KAP-1 Friedman J.R. H. Rauscher III, F.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of and to the KRAB the interaction with KAP-1 as by the absence of BRET between and The absence of BRET was not of of activity were by the The of the protein was by that the molecular for assays expressed ZNF74 and the were in with the BRET in intact The that the transcriptionally active interacts with the KAP-1, the transcriptionally F. B. M. M. Biol. 2001; PubMed Scopus Google Scholar) suggests that the activity of mediated by its interaction with the proposed role of KAP-1 as a co-repressor for KRAB zinc finger proteins D.C. D. Rauscher III, F.J. Genes Dev. 2002; PubMed Scopus Google Scholar). In this BRET for the to represent a and to the interaction of transcriptionally active nuclear proteins in living cells. We of this non-transcription-based to that two members of the TIF1 TIF1α and KAP-1 co-regulators of transcription can homo- and in living mammalian cells. Furthermore, we that the ZNF74 nuclear matrix protein the KRAB multifinger family interacts with of the KAP-1 but with of suggests that such cross-talk between KRAB multifinger proteins and nuclear receptors to interact with TIF1α BRET studies to be in living be to such interactions and transcriptional

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