Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo
2020; Elsevier BV; Volume: 295; Issue: 39 Linguagem: Inglês
10.1074/jbc.ra120.013728
ISSN1083-351X
AutoresClément Immarigeon, Sandra Bernat-Fabre, Emmanuelle Guillou, Alexis Verger, Elodie Prince, Mohamed A. Benmedjahed, Adeline Payet, Marie Couralet, Didier Monté, Vincent Villeret, Henri-Marc Bourbon, Muriel Boube,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes. The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit–TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes. Transcription, the first stage of gene expression, is a fundamental cellular process governed by the binding of sequence-specific transcription factors (TFs) at gene enhancers, inducing the recruitment/activation of the general RNA Polymerase II (Pol II) machinery at gene promoters. In eukaryotes, TFs do not bind directly the Pol II enzyme but instead contact a multisubunit complex called Mediator (MED), serving as a physical and functional interface between DNA-bound TFs and PolII (for review see Refs. 1Soutourina J. Transcription regulation by the Mediator complex.Nat. Rev. Mol. Cell Biol. 2018; 19 (29209056): 262-27410.1038/nrm.2017.115Crossref PubMed Scopus (166) Google Scholar, 2Jeronimo C. Robert F. The Mediator complex: At the nexus of RNA polymerase II transcription.Trends Cell. Biol. 2017; 27 (28778422): 765-78310.1016/j.tcb.2017.07.001Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 3Verger A. Monté D. Villeret V. Twenty years of Mediator complex structural studies.Biochem. Soc. Trans. 2019; 47 (30733343): 399-41010.1042/BST20180608Crossref PubMed Scopus (18) Google Scholar). Although TF DNA-binding specificity has been largely decoded, how TFs interact with the Mediator complex has been less extensively studied, and it is not clear whether each TF binds a specific MED subunit or whether TF–MED interactions obey more complex rules. Mediator is an evolutionarily conserved complex composed of 25 to 30 distinct proteins distributed in four modules: Head, middle, and tail forming the core MED, and a separable regulatory Cdk8 kinase module (CKM) (1Soutourina J. Transcription regulation by the Mediator complex.Nat. Rev. Mol. Cell Biol. 2018; 19 (29209056): 262-27410.1038/nrm.2017.115Crossref PubMed Scopus (166) Google Scholar). Despite a general role of the Mediator complex in regulating transcription, some MED subunits display striking functional specificities, as exemplified by their differential requirements for cell viability (4El Khattabi L. Zhao H. Kalchschmidt J. Young N. Jung S. Van Blerkom P. Kieffer-Kwon P. Kieffer-Kwon K.R. Park S. Wang X. Krebs J. Tripathi S. Sakabe N. Sobreira D.R. Huang S.C. et al.A pliable mediator acts as a functional rather than an architectural bridge between promoters and enhancers.Cell. 2019; 178 (31402173): 1145-1158.e2010.1016/j.cell.2019.07.011Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 5Boube M. Faucher C. Joulia L. Cribbs D.L. Bourbon H.M. Drosophila homologs of transcriptional Mediator complex subunits are required for adult cell and segment identity specification.Genes Dev. 2000; 14 (11090137): 2906-291710.1101/gad.17900Crossref PubMed Scopus (59) Google Scholar), their involvement in specific human diseases (6Schiano C. Casamassimi A. Rienzo M. de Nigris F. Sommese L. Napoli C. Involvement of Mediator complex in malignancy.Biochim. Biophys. Acta. 2014; 1845 (24342527): 66-8310.1016/j.bbcan.2013.12.001PubMed Google Scholar, 7Berk A.J. Yin and yang of mediator function revealed by human mutants.Proc. Natl. Acad. Sci. U. S. A. 2012; 109 (23184968): 19519-1952010.1073/pnas.1217267109Crossref PubMed Scopus (6) Google Scholar), or their roles in given developmental processes (8Grants J.M. Goh G.Y.S. Taubert S. The Mediator complex of Caenorhabditis elegans: Insights into the developmental and physiological roles of a conserved transcriptional coregulator.Nucleic Acids Res. 2015; 43 (25634893): 2442-245310.1093/nar/gkv037Crossref PubMed Scopus (22) Google Scholar, 9Loncle N. Boube M. Joulia L. Boschiero C. Werner M. Cribbs D.L. Bourbon H.M. Distinct roles for Mediator Cdk8 module subunits in Drosophila development.EMBO J. 2007; 26 (17290221): 1045-105410.1038/sj.emboj.7601566Crossref PubMed Scopus (78) Google Scholar, 10Malik N. Agarwal P. Tyagi A. Emerging functions of multi-protein complex Mediator with special emphasis on plants.Crit. Rev. Biochem. Mol. Biol. 2017; 52 (28524697): 475-50210.1080/10409238.2017.1325830Crossref PubMed Scopus (14) Google Scholar). It has been proposed that MED subunit specificity comes from their ability to contact specific transcription factors and mediate their regulatory activity (11Borggrefe T. Yue X. Interactions between subunits of the Mediator complex with gene-specific transcription factors.Semin. Cell Dev. Biol. 2011; 22 (21839847): 759-76810.1016/j.semcdb.2011.07.022Crossref PubMed Scopus (85) Google Scholar, 12Yin J.W. Wang G. The Mediator complex: A master coordinator of transcription and cell lineage development.Development. 2014; 141 (24550107): 977-98710.1242/dev.098392Crossref PubMed Scopus (126) Google Scholar). For example, specific interactions have been demonstrated between Med15 and SMAD transcription factors in Xenopus (13Kato Y. Habas R. Katsuyama Y. Näär A.M. He X. A component of the ARC/mediator complex required for TGFβ/nodal signalling.Nature. 2002; 418 (12167862): 641-64610.1038/nature00969Crossref PubMed Scopus (126) Google Scholar), Med23 and RUNX2 in mice (14Liu Z. Yao X. Yan G. Xu Y. Yan J. Zou W. Wang G. Mediator MED23 cooperates with RUNX2 to drive osteoblast differentiation and bone development.Nat. Commun. 2016; 7 (27033977): 1-1110.1038/ncomms11149Crossref Scopus (117) Google Scholar), Med12 and Gli3 in mammalian cells (15Zhou H. Kim S. Ishii S. Boyer T.G. Mediator modulates Gli3-dependent sonic hedgehog signaling.Mol. Cell Biol. 2006; 26 (17000779): 8667-868210.1128/MCB.00443-06Crossref PubMed Scopus (81) Google Scholar), Med19 and REST in mammals and Med19 and HOX developmental regulators in Drosophila (16Boube M. Hudry B. Immarigeon C. Carrier Y. Bernat-Fabre S. Merabet S. Graba Y. Bourbon H.M. Cribbs D.L. Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of Mediator subunit Med19.PLoS Genet. 2014; 10 (24786462): e100430310.1371/journal.pgen.1004303Crossref PubMed Scopus (28) Google Scholar, 17Crona F. Holmqvist P.H. Tang M. Singla B. Vakifahmetoglu-Norberg H. Fantur K. Mannervik M. The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos.Dev. Biol. 2015; 407 (26260775): 173-18110.1016/j.ydbio.2015.08.005Crossref PubMed Scopus (2) Google Scholar), or also between Med1 and hormone nuclear receptors or GATA TF families in mammalian cells (18Fondell J.D. The Mediator complex in thyroid hormone receptor action.Biochim. Biophys. Acta. 2013; 1830 (22402254): 3867-387510.1016/j.bbagen.2012.02.012Crossref PubMed Scopus (39) Google Scholar, 19Stumpf M. Waskow C. Krötschel M. Van Essen D. Rodriguez P. Zhang X. Guyot B. Roeder R.G. Borggrefe T. The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220.Proc. Natl. Acad. Sci. U. S. A. 2006; 103 (17132730): 18504-1850910.1073/pnas.0604494103Crossref PubMed Scopus (78) Google Scholar). GATA transcription factors represent a good model to analyze interaction between TFs and Mediator subunits. The mammalian GATA TF family comprises six members (GATA1–6), shown to specifically interact with the Med1 Mediator subunit (20Crawford S.E. Qi C. Misra P. Stellmach V. Sambasiva Rao M. Enge J.D. Zhu Y. Reddy J.K. Defects of the heart, eye, and megakaryocytes in peroxisome proliferator activator receptor-binding protein (PBP) null embryos implicate GATA family of transcription factors.J. Biol. Chem. 2002; 277 (11724781): 3585-359210.1074/jbc.M107995200Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). They have conserved homologs between both vertebrates and invertebrates (21Chlon T.M. Crispino J.D. Combinatorial regulation of tissue specification by GATA and FOG factors.Development. 2012; 139 (23048181): 3905-391610.1242/dev.080440Crossref PubMed Scopus (58) Google Scholar) and contain two highly conserved zinc finger (ZF) domains. The C-terminal one (C-ZF) is both necessary and sufficient for sequence-specific DNA binding at WGATAR genomic sites, whereas the N-terminal ZF (N-ZF) appears only to modulate DNA-binding affinity (22Whyatt D.J. deBoer E. Grosveld F. The two zinc finger-like domains of GATA-1 have different DNA binding specificities.EMBO J. 1993; 12 (8262042): 4993-500510.1002/j.1460-2075.1993.tb06193.xCrossref PubMed Scopus (110) Google Scholar) and has been involved in direct interactions with GATA cofactors (23Tsang A.P. Visvader J.E. Turner C.A. Fujiwara Y. Yu C. Weiss M.J. Crossley M. Orkin S.H. FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation.Cell. 1997; 90 (9230307): 109-11910.1016/S0092-8674(00)80318-9Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 24Rodriguez P. Bonte E. Krijgsveld J. Kolodziej K.E. Guyot B. Heck A.J.R. Vyas P. De Boer E. Grosveld F. Strouboulis J. GATA-1 forms distinct activating and repressive complexes in erythroid cells.EMBO J. 2005; 24 (15920471): 2354-236610.1038/sj.emboj.7600702Crossref PubMed Scopus (220) Google Scholar, 25Tripic T. Deng W. Cheng Y. Zhang Y. Vakoc C.R. Gregory G.D. Hardison R.C. Blobel G.A. SCL and associated proteins distinguish active from repressive GATA transcription factor complexes.Blood. 2009; 113 (19011221): 2191-220110.1182/blood-2008-07-169417Crossref PubMed Scopus (142) Google Scholar, 26Wilkinson-White L. Gamsjaeger R. Dastmalchi S. Wienert B. Stokes P.H. Crossley M. Mackay J.P. Matthews J.M. Structural basis of simultaneous recruitment of the transcriptional regulators LMO2 and FOG1/ZFPM1 by the transcription factor GATA1.Proc. Natl. Acad. Sci. U. S. A. 2011; 108 (21844373): 14443-1444810.1073/pnas.1105898108Crossref PubMed Scopus (32) Google Scholar). Mammalian GATAs are key regulators of developmental processes: GATA1, -2, and -3 are crucial hematopoietic TFs whereas GATA4, -5, and -6 control cardiac development, among other functions (21Chlon T.M. Crispino J.D. Combinatorial regulation of tissue specification by GATA and FOG factors.Development. 2012; 139 (23048181): 3905-391610.1242/dev.080440Crossref PubMed Scopus (58) Google Scholar). Interestingly, among the five GATA TFs encoded by the Drosophila genome, only Serpent (Srp), is a bona fide hematopoietic GATA factor, whereas Pannier (Pnr) is involved in cardiac development (27Sorrentino R.P. Gajewski K.M. Schulz R.A. GATA factors in Drosophila heart and blood cell development.Semin. Cell Dev. Biol. 2005; 16 (15659345): 107-11610.1016/j.semcdb.2004.10.005Crossref PubMed Scopus (44) Google Scholar). Pnr activity is also crucial during central thorax patterning and dorsocentral (DC) mechanosensory bristle formation, and it has been studied in depth in this context (28García-García M.J. Ramain P. Simpson P. Modolell J. Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila.Development. 1999; 126 (10409499): 3523-3532PubMed Google Scholar, 29Ramain P. Heitzler P. Haenlin M. Simpson P. Pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1.Development. 1993; 119 (7916679): 1277-1291PubMed Google Scholar, 30Heitzler P. Haenlin M. Ramain P. Calleja M. Simpson P. A genetic analysis of pannier, a gene necessary for viability of dorsal tissues and bristle positioning in Drosophila.Genetics. 1996; 143 (8807299): 1271-1286Crossref PubMed Google Scholar). Within the wing imaginal disc, the Pnr TF directly activates proneural genes of the achaete-scute complex in the dorsocentral cluster, which gives rise to the DC bristles (28García-García M.J. Ramain P. Simpson P. Modolell J. Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila.Development. 1999; 126 (10409499): 3523-3532PubMed Google Scholar). In addition, Pnr activates the wingless gene in a strip of cells of the presumptive thorax (31Sato M. Saigo K. Involvement of pannier and u-shaped in regulation of Decapentaplegic-dependent wingless expression in developing Drosophila notum.Mech. Dev. 2000; 93 (10781946): 127-13810.1016/S0925-4773(00)00282-3Crossref PubMed Scopus (51) Google Scholar). In a genome-wide RNAi screen in Drosophila cultured cells we identified a set of MED subunits as modulators of GATA/Serpent–induced transactivation, among which were Med12, Med13, Med1, and Med19 (32Gobert V. Osman D. Bras S. Augé B. Boube M. Bourbon H.-M. Horn T. Boutros M. Haenlin M. Waltzer L. A genome-wide RNA interference screen identifies a differential role of the Mediator CDK8 module subunits for GATA/RUNX-activated transcription in Drosophila.Mol. Cell Biol. 2010; 30 (20368357): 2837-284810.1128/MCB.01625-09Crossref PubMed Scopus (27) Google Scholar). This work further showed that Med12 and Med13 subunits are required in vivo for Srp-driven developmental processes, but we were unable to detect direct physical interaction with Srp in vitro, suggesting that GATA/Srp may recruit the Mediator complex by contacting other MED subunits. Indeed, we recently showed that Med1 mediates GATA TFs function in Drosophila (33Immarigeon C. Bernat-Fabre S. Augé B. Faucher C. Gobert V. Haenlin M. Waltzer L. Payet A. Cribbs D.L. Bourbon H.-M.G. Boube M. Drosophila Mediator subunit Med1 is required for GATA-dependent developmental processes: Divergent binding interfaces for conserved coactivator functions.Mol. Cell Biol. 2019; 39 (30670567): 1-1810.1128/MCB.00477-18Crossref Scopus (2) Google Scholar). Med1 does interact physically with both Pnr and Srp GATA TFs, through their conserved zinc finger region. Furthermore, in vivo experiments showed that Med1 is involved in Srp-driven hematopoiesis and Pnr-driven thorax differentiation and is required for Srp and Pnr target gene expression in the corresponding tissues. These data established that the Med1 GATA cofactor activity is evolutionarily conserved and involves the GATA N- and C-zinc finger domains in both mammals and Drosophila. Nevertheless, we also showed that Drosophila Med1 is not critical for wingless-induced transactivation by Pnr, raising the possibility that other MED subunits could mediate some GATA TFs functions. Here, we reveal that another MED subunit, Med19, also acts as a GATA coactivator. Med19 mutants phenocopy pnr loss-of-function and extinguish the expression of both Pnr target genes achaete and wg, whereas Med1 mutants were previously shown to affect only achaete expression. Using immunoprecipitation, pulldown, and bimolecular fluorescence complementation (BiFC) techniques, we establish that Med19 physically interacts with Pnr in cellulo, in vivo, and in vitro through its C-ZF domain. Med19 also interacts physically with GATA/Srp, suggesting that Med19 acts as a generic GATA cofactor. Moreover, we show that both Med1 and Med19 jointly regulate a series of Srp target genes in Drosophila cultured cells. Finally, in vitro experiments revealed that Med1 and Med19 physically interact through the Med1 domain which is conserved throughout eukaryotes. Taken together, our results show that GATA-driven regulatory functions in Drosophila require two MED complex subunits, Med19 in all tested cases and Med1 in a majority. The evolutionary conservation of Med19 and GATA interacting domains suggests that Med19 may play a conserved GATA cofactor function in mammals. Our whole-genome dsRNA screen in Drosophila cultured cells identified Med19 as one MED subunit capable of modulating Srp TF-induced transactivation ex vivo (32Gobert V. Osman D. Bras S. Augé B. Boube M. Bourbon H.-M. Horn T. Boutros M. Haenlin M. Waltzer L. A genome-wide RNA interference screen identifies a differential role of the Mediator CDK8 module subunits for GATA/RUNX-activated transcription in Drosophila.Mol. Cell Biol. 2010; 30 (20368357): 2837-284810.1128/MCB.01625-09Crossref PubMed Scopus (27) Google Scholar). This led us to ask whether and how Med19 could interact with GATA TFs in vivo. To this end, we generated Med19 mutant clones in the larval wing imaginal disc, which gives rise to adult thoracic structures whose proper development depends on GATA/Pnr activity. Flies bearing Med19- clones displayed specific phenotypes in the thorax, including thoracic cleft and loss of DC mechanosensory macrochaetes (Fig. 1, A and D), typical of pnr loss-of-function (29Ramain P. Heitzler P. Haenlin M. Simpson P. Pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1.Development. 1993; 119 (7916679): 1277-1291PubMed Google Scholar, 30Heitzler P. Haenlin M. Ramain P. Calleja M. Simpson P. A genetic analysis of pannier, a gene necessary for viability of dorsal tissues and bristle positioning in Drosophila.Genetics. 1996; 143 (8807299): 1271-1286Crossref PubMed Google Scholar). We observed similar phenotypes upon expression of RNAi against Med19 in the apterous (ap) domain encompassing all the presumptive notum (Fig. S1, A and B). To investigate the functional relationship between Med19 and Pnr, we first examined pnr gene expression in Med19-deficient wing discs by FISH and observed that pnr is expressed in Med19-depleted wing discs (Fig. S1, C–F). Thus, Med19 mutant phenotypes cannot be explained by a loss of pnr expression. To further analyze the functional relationship between Med19 and Pnr, we then examined GATA/Pnr TF activity in Med19 loss-of-function clones by analyzing the expression of known Pnr target genes. Compared with WT cells shown in Fig. 1, B–C′′, we observed that both wingless (wg) and achaete (ac) expression was cell autonomously lost in Med19−/− cells (Fig. 1, E–F′′), indicating that Med19 is required for the expression of both Pnr target genes. Note that ac expression has been visualized by a DC-ac-lacZ reporter gene which is directly activated upon Pnr binding to the DC ac enhancer (28García-García M.J. Ramain P. Simpson P. Modolell J. Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila.Development. 1999; 126 (10409499): 3523-3532PubMed Google Scholar). These data show that Med19 is cell autonomously required for Pnr activity but not for Pnr expression, suggesting that it could act as a GATA/Pnr cofactor. We investigated whether GATA/Pnr transcription factor and Med19 physically interact by using three independent experimental approaches: Co-immunoprecipitation from cultured cells, in vitro pulldown and in vivo Bimolecular Fluorescence Complementation (BiFC) interaction tests. We first tested whether Pnr-MED complexes actually form within Drosophila cells by performing co-immunoprecipitations experiments on total protein extracts from cultured cells expressing a functional Myc-tagged Pnr form. We observed that Pnr co-precipitated with endogenous Drosophila Med19 (Fig. 2A). In the reverse experiment, endogenous Med19 protein co-precipitated with Myc-tagged Pnr protein (Fig. 2B). These data provide complementary evidence for the formation of Med19-GATA complexes in Drosophila cells. To investigate whether Med19–Pnr interaction is direct, we tested the ability of Med19 and Pnr proteins to bind each other physically in vitro through pulldown assays with GSH S-transferase (GST) fusion proteins. In vitro–produced Med19 readily bound full-length recombinant GST-Pnr (Fig. 2C), and vice versa (Fig. 2D). These results show that Med19 and Pnr can interact physically in the absence of any other Drosophila MED subunits. We then used BiFC (16Boube M. Hudry B. Immarigeon C. Carrier Y. Bernat-Fabre S. Merabet S. Graba Y. Bourbon H.M. Cribbs D.L. Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of Mediator subunit Med19.PLoS Genet. 2014; 10 (24786462): e100430310.1371/journal.pgen.1004303Crossref PubMed Scopus (28) Google Scholar, 34Hu C.-D. Grinberg A.V. Kerppola T.K. Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC).Anal. Curr. Protoc. Cell Biol. 2005; 29 (18228482): 21.3.1-21.3.2110.1002/0471143030.cb2103s29Crossref Google Scholar) to analyze Med19–GATA interaction in vivo. Based on fusing N- and C-terminal portions (VN and VC) of the GFP-variant Venus protein with two proteins of interest respectively, this technique allows the reconstitution of a fluorescent Venus protein if the two candidate proteins are close enough within the cell. We used the dppGAL4 driver (Gal4/UAS system (35Brand A.H. Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.Development. 1993; 118 (8223268): 401-415Crossref PubMed Google Scholar)) to co-express VN-Pnr with either Med19-VC or another MED subunit fusion, CycC-VC, along the antero/posterior frontier of the wing imaginal disc (Fig. 2, E and F). The co-expression of VN-Pnr and Med19-VC resulted in a clear BiFC signal, whereas the control VN-Pnr/CycC-VC combination gave a very low signal (Fig. 2, E′ and F′), even though CycC-VC and Med19-VC proteins were expressed at similar levels (Fig. 2, E and F). These data indicate that the BiFC technique discriminates specific interactions between different subunits within the MED complex and that Med19 and Pnr are in close proximity in the nucleus of living cells. Of note, the BiFC signal was observed in the entire dppGAL4 expression domain, including the wing pouch where endogenous GATA/Pnr is not expressed, showing that the Pnr-Med19 interaction can occur at ectopic locations independently of tissue-specific Pnr partners, thus providing further support for a direct molecular interaction in vivo. Collectively, in cellulo, in vitro, and in vivo data support a direct physical interaction between the GATA/Pnr transcription factor and the Med19 Mediator subunit. Together with our previous results (33Immarigeon C. Bernat-Fabre S. Augé B. Faucher C. Gobert V. Haenlin M. Waltzer L. Payet A. Cribbs D.L. Bourbon H.-M.G. Boube M. Drosophila Mediator subunit Med1 is required for GATA-dependent developmental processes: Divergent binding interfaces for conserved coactivator functions.Mol. Cell Biol. 2019; 39 (30670567): 1-1810.1128/MCB.00477-18Crossref Scopus (2) Google Scholar), these data suggest that the Pnr TF can interact with the entire MED complex via a direct molecular contact with the Med19 subunit in addition to or in place of the Med1 subunit. We previously showed that Med1 directly interacts with the dual zinc finger domains of Pnr (33Immarigeon C. Bernat-Fabre S. Augé B. Faucher C. Gobert V. Haenlin M. Waltzer L. Payet A. Cribbs D.L. Bourbon H.-M.G. Boube M. Drosophila Mediator subunit Med1 is required for GATA-dependent developmental processes: Divergent binding interfaces for conserved coactivator functions.Mol. Cell Biol. 2019; 39 (30670567): 1-1810.1128/MCB.00477-18Crossref Scopus (2) Google Scholar). We therefore decided to characterize interacting domains within Pnr and Med19 to determine whether Med19 and Med1 interact with the same Pnr domain. We first looked for the Med19-interacting domain(s) within the GATA/Pnr protein using full-length GST-Med19 as a bait (Fig. 3A). Pnr was split into three parts: The poorly evolutionarily conserved N-terminal region (amino acids (aa) 1–137), the strongly conserved central region spanning the two zinc fingers, N- and C-ZF, and the divergent C-terminal region containing two amphipathic α helices, H1 and H2. Only the ZF-containing region (aa 130–278) displayed significant binding. When cutting full-length Pnr into two halves separating the two zinc finger domains, binding was observed only with the C-ZF–containing part (Fig. 3A), suggesting that C-ZF mediates binding of Pnr to Med19. Consistently, the ability of the N-ZF–containing half of Pnr to bind Med19 was recovered when we added back the C-ZF proper (aa 220–253) containing the four zinc-chelating cysteines forming the finger structure. Interestingly, binding was increased when the C-ZF proper was extended by its neighboring C-terminal 25 amino acids (basic tail motif, aa 253–278) (Fig. 3B). Sequence alignment of Drosophila and mammalian GATAs indicates that the C-ZF basic tail has been strongly conserved during evolution, especially at positions shown to participate in DNA binding (open circles in Fig. 3B) (36Ghirlando R. Trainor C.D. Determinants of GATA-1 binding to DNA: The role of non-finger residues.J. Biol. Chem. 2003; 278 (12941967): 45620-4562810.1074/jbc.M306410200Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 37Newton A. Mackay J. Crossley M. The N-terminal zinc finger of the erythroid transcription factor GATA-1 binds GATC motifs in DNA.J. Biol. Chem. 2001; 276 (11445591): 35794-3580110.1074/jbc.M106256200Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Together, these experiments indicate that the entire Pnr C-ZF domain, zinc-finger proper and adjacent basic tail, is necessary for optimal Med19 binding. In the reciprocal experiment, we identified the GATA/Pnr interacting domain within Med19. Our prior analysis of Drosophila Med19 function and evolutionary conservation within the eukaryotic kingdom (16Boube M. Hudry B. Immarigeon C. Carrier Y. Bernat-Fabre S. Merabet S. Graba Y. Bourbon H.M. Cribbs D.L. Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of Mediator subunit Med19.PLoS Genet. 2014; 10 (24786462): e100430310.1371/journal.pgen.1004303Crossref PubMed Scopus (28) Google Scholar, 38Bourbon H.M. Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex.Nucleic Acids Res. 2008; 36 (18515835): 3993-400810.1093/nar/gkn349Crossref PubMed Scopus (237) Google Scholar) allowed us to define four structural domains: A conserved MED-anchoring “CORE” region, an animal-specific basic HOX homeodomain–interacting motif (HIM) and two less well-conserved N- and C-terminal regions. To investigate which protein domain(s) is (are) required for Pnr binding, we tested the ability of in vitro translated Pnr1–291 to bind a series of GST-Med19 truncated forms (Fig. 3, C–G). A Med19 protein deleted for its evolutionarily conserved CORE domain (ΔCORE) still bound Pnr1–291 (Fig. 3D). Binding was also retained after truncating both C-terminal and HIM domains but was abolished if the deletion included the C-terminal end of the CORE domain (aa 126–165) (Fig. 3E). Deletions starting from the Med19 N terminus indicated that a truncated protein containing HIM and C-terminal domains also interacts with Pnr 1-291 (Fig. 3F). Further deletions revealed that one fragment of HIM from aa 206–220 was
Referência(s)