Mediator complex subunit Med19 binds directly GATA DNA-binding zinc finger and functions with Med1 in GATA-driven gene regulation in vivo

The evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.


Introduction
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 (1)(2)(3)). Whereas 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) (1). 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 (4,5), their involvement in specific human diseases (6,7), or their roles in given developmental processes (8)(9)(10). It has been proposed that MED subunit specificity comes from their ability to contact specific transcription factors and mediate their regulatory activity (11,12). For example, specific interactions and cofactor activities have been demonstrated between Med15 and SMAD transcription factors in Xenopus (13), Med23 and RUNX2 in mice (14) Med12 and Gli3 in mammalian cells (15), Med19 and REST in mammals or Med19 and HOX developmental regulators in Drosophila (16,17), or also between Med1 and hormone nuclear receptors or GATA TF families in mammalian cells (18,19).
Mammalian GATA TF family comprises 6 members (GATA1-6), with conserved homologs among both vertebrates and invertebrates (20). They generally contain two highly-conserved zinc finger (ZF) domains, of which the C-terminal one (C-ZF) is both necessary and sufficient for sequence-specific DNA binding at WGATAR genomic sites, while the N-terminal ZF (N-ZF) appears only to modulate DNA-binding affinity (21) and has been involved in direct interactions with GATA cofactors (22)(23)(24)(25). Mammalian GATAs are key regulators of developmental processes: GATA1, -2 and -3 are crucial hematopoietic TFs while GATA4, -5 and -6 control cardiac development, among other functions (20). Interestingly, among the 5 GATA TFs encoded by the Drosophila genome, only Serpent (Srp), is a bona fide hematopoietic GATA factor, while another one, Pannier (Pnr), is involved in cardiac development (26). GATA/Pnr activity is also crucial during central thorax patterning and dorso-central (DC) mechanosensory bristle formation, and it has been studied in depth in this context (27)(28)(29) (28). Within the wing imaginal disc, the Pnr TF directly activates proneural genes of the achaete-scute (ac-sc) complex in the dorso-central cluster, which gives rise to the DC bristles (27). In addition, Pnr activates wingless in a strip of cells of the presumptive centro-lateral notum (30).
A genome-wide RNA interference screen in Drosophila cultured cells allowed us identifying a set of MED subunits as modulators of GATA/Serpent-induced transactivation, among which, Med12, Med13, Med1 and Med19 (31). 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. To address the question of evolutionary conservation of MED subunits-TFs partnerships, we recently asked whether the Med1 subunit mediates also GATA TFs function in Drosophila (32). We showed that Drosophila 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 Pnrdriven thorax differentiation and is required for Srp and Pnr target gene expression in the corresponding tissues. These data both established that the Med1 GATA cofactor is evolutionarily conserved and involves the GATA N-and C-zinc finger domains. Nevertheless, we also showed that Drosophila Med1 is critical for achaete-but not for wingless-induced transactivation by Pnr, raising the possibility that other MED subunits could mediate GATA TFs functions.
Here, we reveal that another MED subunit, Med19, also acts as a GATA coactivator.

Drosophila Med19 is required for notum morphogenesis, bristle development and GATA/Pannier target gene activation
Our study of the first-characterised metazoan mutant for Med19 revealed a Med19 function as a specific co-activator of HOX TF family during Drosophila development (16). Our whole-genome dsRNA screen in Drosophila cultured cells previously identified Med19 as one of several MED subunits capable of modulating Srp TF-induced transactivation. (31). 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, giving rise to adult thoracic structures whose proper development depends on GATA/Pnr activity. Flies bearing Med19clones displayed specific phenotypes in the thorax, including thoracic cleft and loss of dorso-central (DC) mechanosensory macrochaetes (Fig.1A, D), typical of pnr loss-of-function (28,29). We observed similar phenotypes upon expression of interfering RNAs (RNAi) against Med19 in the apterous (ap) domain encompassing all the presumptive notum (Fig S1A-B). To investigate functional relationship between Med19 and Pnr, we first examined pnr gene expression in Med19-deficient wing discs by fluorescent in situ hybridization (FISH), and observed that pnr is expressed in Med19depleted wing discs (Fig S1C-F). Thus, Med19 mutant phenotypes cannot be explained by a loss of pnr expression. To further investigate 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 to wild-type cells shown in Fig. 1B-C", we observed that both wingless (wg) and achaete (ac) expression was cell autonomously lost in Med19 -/cells ( Fig.1E-F") indicating that Med19 is required for Pnr target gene expression. 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 (27).
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.

Drosophila Med19 interacts physically with the Pannier GATA TF
We investigated whether GATA/Pnr transcription factor and Med19 physically interact by using three independent experimental approaches: coimmunoprecipitation (co-IP) from cultured cells, in vitro pull-down 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 (co-IP) 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). Altogether, 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 with Glutathione Stransferase (GST) fusion proteins. In vitroproduced 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 factor, especially other MED subunits.
We then used Bimolecular Fluorescence complementation (BiFC) technique (16,33) to tackle the question of Med19-GATA molecular 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 (34) 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 and suggest that the Pnr TF functionally interacts with the entire MED complex via a direct molecular contact with its Med19 subunit.

Med19 Core and HIM domains bind the Czinc finger domain of GATA/Pnr
We previously showed that Med1 directly interacts with the dual Zinc Finger domains of Pnr and that HOX TFs bind so-called Med19 HIM (Homeodomain-interacting motif) domain. We therefore decided to further investigate two aspects: whether or not Med19 and Med1 interact with the same Pnr domain and whether or not Med19 binds GATA and HOX TFs through the same 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 stronglyconserved central region spanning the 2 zinc fingers, N-and C-ZF, and the divergent Cterminal region containing two amphipathic alpha helices, H1 and H2. Only the ZFcontaining 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 zincchelating 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 GATA C-ZF domains indicates that the basic tail region has been strongly conserved during evolution, especially at positions which have been shown to participate in DNA binding (open circles in Fig. 3B, (35,36)). Together, these experiments indicate that within the Pnr C-ZF domain, the proper zinc-finger subdomain and its adjacent basic tail are both necessary for optimal Med19 binding.
In the reciprocal experiment, we examined the GATA/Pnr interacting domain within the Med19 protein. Our prior analysis of Drosophila Med19 function and evolutionary conservation within the eukaryotic kingdom (16,37), allowed 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 Pnr 1-291 HA tagged form to bind a series of GST-Med19 truncated forms ( Fig.3C-G). A Med19 protein deleted for its evolutionarily-conserved CORE domain (ΔCORE) still bound Pnr 1-291 (Fig.3D). Binding was also retained after truncating both C-ter and HIM domains, but was abolished if the deletion included the Cterminal end of the CORE domain (aa 126 to 165) (Fig.3E). Deletions starting from the Med19 N-terminus indicated that a truncated protein containing HIM and Cter domains also interacts with Pnr 1-291 (Fig.3F). Further deletions revealed that one fragment of HIM from aa 206 to 220 was critical for Pnr binding in the absence of the CORE domain. Taken together, our data suggest the presence of two Pnr binding sites within Med19, aa 126 to 165 of the CORE (BS1) and aa 206 to 220 of the HIM domain (BS2) (Fig 3C). To further assess their implication, we deleted both BS1 and BS2 in an otherwise full-length Med19 protein.
As shown in Fig.3G, the ΔBS1-BS2 mutant no longer bound GATA/Pnr, indicating that at least one of the two binding sites is necessary.
In conclusion, our in vitro binding assays indicate that the GATA/Pnr C-zinc finger domain, including its basic tail, binds two separate domains within the evolutionarilyconserved Med19 CORE and HIM regions. Whereas Med19 appears only to bind the C-ZF, Med1 has been shown to bind both GATA/Pnr C-ZF or N-ZF domains in vitro (32).

Med19 physically interacts with GATA/Srp
To investigate whether Med19 is a general GATA cofactor, we asked whether it is able to interact with Serpent (Srp), another Drosophila GATA TF family member, as it does with Pnr. First, we used similar GST-pull-down assays (Fig.4A). They showed that recombinant GST-Med19 protein bound in vitro-translated fulllength GATA/Srp protein. As previously shown for GATA/Pnr, when assaying Srp truncated forms, binding was only retained with the ZF-containing middle part. Splitting the Srp protein in two halves and separating both zinc finger domains indicated that only the C-ZF one is involved in binding Med19 (Fig 4A), as it is also the case for GATA/Pnr (Fig 3E).
To test whether this Med19-Srp interaction also occurs in vivo, we used again the BiFC experimental approach. Upon overexpression of Med19-VC with VN-tagged Srp in the dpp expression domain, we observed a strong BiFC signal, similar to what we obtained with Pnr-VN (Fig. 4B), indicating that Med19 and GATA Srp indeed interact in vivo.
Altogether, these results show that, Med19 interacts both with Pnr and Srp, in vivo and, in vitro via the GATA family-defining C-zinc finger domain, indicating that Med19 is a general GATA cofactor.

Med19 shares GATA-cofactor functions with the Med1 Mediator subunit
We previously showed that Med1, another subunit of the MED middle module, is required for Pnr and Srp TF activities in vivo and interacts directly with Srp and Pnr, in this case through both their N-and C-zinc finger domains (32). Our new data showing that Med19 can also act as GATA cofactor thus raises the question: do Med1 and Med19 regulate the exact same GATA target genes or do they play distinct roles? Respective to Pnrdependent transcriptional activity, we have shown that like Med1, Med19 is cell autonomously required for DC-ac-lacZ reporter expression whereas wg expression requires Med19, but not Med1. This prompted us to consider each GATA target gene as a particular case that could involve interaction with different MED subunits. Kuuluvainen and collaborators (38) identified a set of Srp target genes in Drosophila S2 cells, which could be used to test the impact of Med1-or Med19 mRNA depletion. Here, we quantified the expression of 6 Srp target genes: SrCl, CG14629, CG8157, arg, and CG34417 which are activated (positive targets), and CG13252 which is repressed by Srp, using Real Time quantitative PCR (qRT-PCR) on control, Med1-or Med19-mRNA depleted S2 cells. Quantification of mRNAs coding for the Myosin light chain (Mlc2) served as a control for housekeeping transcription. As shown in Fig. 5A, in cells depleted for 85% of Med19 mRNA, expression of the 5 activated Srp target genes SrCl, CG14629, CG8157, arg and CG34417 was significantly down-regulated and the Srp-repressed target gene CG13252 was instead up regulated, indicating that Med19 is required in cellulo for GATA/Srp transcriptional activity. In cells depleted for about 75% of the Med1 transcript (Fig. 5B), expression of Srp target genes followed the same trend than after Med19 mRNA depletion, although less efficiently. Several conclusions can be drawn from these experiments: (1) they show that contrary to other MED components, Med1 and Med19 are not generally required for PolII-dependent transcription given that some genes are unchanged or even upregulated. (2) Med19 and Med1 are both required for Srp-mediated gene regulation in cultured cells, seemingly on the same target genes, both for activation and repression (although it is not known whether CG13252 repression by Srp is direct or not).

Med19 and Med1 can interact directly
Given the shared functional implication of both Mediator subunits in mediating GATA activity, we lastly asked whether Med1 and Med19 proteins are able to interact physically using GST pulldown assays. As shown in Fig.5C, we observed that a GST fusion of Med1 largest isoform A (Med1 A ) bound in vitro-translated full-length Med19. In the reverse experiment, we showed that purified GST-Med19 also bound in vitro translated Med1 A (Fig.5D), showing that both proteins indeed bind to each other in vitro, in absence of other MED subunits. We next sought to identify Med19-interacting domain(s) within the Med1 isoforms by analysing truncated proteins. As shown in Fig.5D, the Med19 interacting domain lies within the evolutionarily-conserved Med1 Nterminal part, which has been proposed to be required for its incorporation within the MED complex (39) and is shared by the three Med1 isoforms. Conversely, Med1 isoform-specific parts are not required for Med19 interaction. Taken together, GST-pulldown data reveal a direct interaction between Med1 and Med19 which could not be anticipated from structural data (see Fig.6) given that Med1 and Med19 are supposed to lie in opposite parts of the Mediator complex middle module (40).

Discussion
In this work, we establish using molecular, cellular and genetic analyses that Drosophila GATA factors' transcriptional activity depends on the activity of two Mediator complex subunits, Med19 and Med1. Whereas Med1 could bind both GATA zinc-finger domains (32), we show that Med19 only interacts with the C-ZF domain which also serves as the GATA DNA-binding domain (Fig. 6A). Our analysis of both Pnr and Srp target gene expression in Med19-or Med1-depleted cells indicate that both subunits are critical for multiple GATA target genes regulation, suggesting a close cooperation of Med19 and Med1 to mediate GATA-driven signals. However, at least the Pnr target wg relies on Med19 but not Med1, suggesting that the use of MED SU by GATAs can vary depending on the target gene.

New models for GATA -MED interactions
Several models can be envisioned respective to Med19 and Med1 regulatory activities. In the first one (Fig. 6B), we propose that enhancerbound GATAs use their evolutionarilyconserved ZF-containing domain to directly contact not only the previously identified cofactor Med1 but also the Med19 subunit to recruit the Mediator complex and thus the PolII machinery at some GATA target genes (such as the Pnr target gene ac), both contacts being absolutely required. In some cases such as wg (Fig. 6C), Med19 is critical but Med1 is dispensable, so we propose that the presence of other TFs might help recruiting the MED via other subunits.
Previous models of core MED structurefunction analysis suggest that the middle and head modules contact the PolII enzyme and associated general transcription factors (GTFs) while the tail module interacts with sequencespecific TFs (2,3). Our data show that two different MED subunits, Med1 and Med19, are able to bind GATA factors and required for their function and both belong to the middle module. They emphasize that MED should be viewed as a much more complex interface using diverse MED module subunit combinations to contact different TF combinations thus mediating specific transcriptional responses.

Evolutionarily-conserved GATAcoactivator functions of Med19?
While Med1 is a known GATA cofactor both in mammals and in Drosophila (19,32), the role of Med19 in mediating GATA transcription regulatory properties had never been investigated until now. Here we show that Drosophila Med19 binds GATA factors, via motifs lying within the evolutionary-conserved Med19 CORE and HIM domains. Both of these domains bind to the C-ZF domain of GATAs, which is a hallmark of GATA TF family suggesting that interaction with Med19 is likely to be conserved in mammals. Yet, Med1 depletion experiments in mammalian cultured cells induces defects in only a subset of GATA1-activated genes and does not prevent GATA1-dependent repression (41,42). Furthermore, in studies of the different blood cell types produced by conditional Med1 knock-out mice, Med1 has been shown to be critical for erythroid lineages which depend upon GATA1-function but is dispensable for hematopoietic stem cell production and T-cell development which require GATA2 and GATA3, respectively (43). Thus, despite being capable of binding all GATA factors in vitro, Med1 is not critical for all GATA functions, which suggests that (an) other MED subunit(s) also bind(s) GATAs to relay their regulatory signals to the PolII machinery. Considering the evolutionary-conservation of interaction motifs within both GATAs and Med19 (this work), we argue that Med19 is a strong candidate as a GATA cofactor in mammals.

Overlapping DNA-Binding and Activation domains of GATA TFs
TFs minimally contain two domains: the DNA binding domains (DBD), which have been extensively studied and allowed to define different TF families, and transcriptional activation domains (TAD), which often link TFs to the RNA polymerase II machinery, and whose structure and characteristics are less well defined. GATA TFs are characterised by the presence of two ZFs which where, so far, thought to play distinct roles. While the C-ZF appeared to be dedicated to DNA binding, coactivators such as dLMO (44) and FOG (45) were shown to bind the N-ZF. Another Pnr interactor, Chip binds to the C-terminal αhelices H1 and H2 (46). Our present data show that Med19 interacts specifically with the Pnr C-ZF. Full interaction requires both the zinc finger proper with the four cysteines responsible for the "finger" structure and the conserved basic tail which contributes to DNA binding (35,36). It is the first evidence that the Drosophila GATA C-ZF may play a dual role, in DNA binding and as an interface with MED subunit(s). Interestingly, the analysis of GATA ZF evolutionary conservation indicates that Nand C-ZF domains comes from a duplication event and that only C-ZF with its basic tail has been conserved in plants, nematode, some insects and echinoderms. Thus, this transactivation function of GATAs'DBD might represent an ancestral GATA function allowing minimal primitive GATAs, essentially composed of the DBD, to connect the MED complex and thus recruit the transcriptional machinery to regulate its target genes.
It seems that this TF property is not restricted to GATA factors. We have previously shown that the Med19 Mediator complex subunit acts in vivo as a HOX coactivator and binds through direct contacts with their evolutionarily-conserved helix-turn-helix homeodomain serving as sequence-specific DNA-binding domain (16). These data also corroborate results from a recent highthroughput approach, looking for transactivation domains of Drosophila transcription factors. This work shows that trans-activation domains of several zinc-finger-(ZF-) and basic Helix-Loop-Helix-(bHLH-) TFs overlap structured DNA-binding domains (47). Altogether, these results identify a novel class of TF characterized by overlapping activation and DNA-binding domains and suggest an emerging Med19 property as a dedicated cofactor directly connecting these TFs DNAbinding domains to the general PolII transcriptional machinery.

Unexpected direct interaction between Med1 and Med19 middle module subunits
Recently, owing to improvement of electronic microscopy and mass spectrometry techniques, important advances have been made in understanding Mediator 3D architecture. It provides a rather precise structural view of MED subunits' respective position within the yeast and the mammalian complexes (4, 48-50).
Although not yet performed for any insect MED complex, Drosophila MED spatial organization can be modelled given the strong structural similarity observed between yeast and human complexes and primary sequence conservation among animals (37). These data indicate that Drosophila Med1 and Med19 are most likely situated at two opposite ends of the middle module, Med1 near the tail module and Med19 within the so called "hook" domain proposed to anchor the separable CKM MED module (Fig 6.A). How, then, reconcile the proposed MED architecture with our results showing a direct interaction between Med1 and Med19 subunits in vitro? All the more so as our data suggest an evolutionary conservation of this interaction given that Drosophila Med1 binds Med19 through its highly-conserved, N-terminal, MED-addressing domain which is ancestrally present in yeast, as opposed to the C-terminal extensions which are essentially disorganized and fast evolving. We propose two non-exclusive hypotheses: First, MED complexes could adopt different conformations, which would differ from the "canonical" architecture of the MED complex in isolation. This is supported by observations that the MED complex changes its overall shape when engaged in interactions with either TF, CKM or PolII (49). Perhaps when MED is recruited by GATA, Med1 -Med19 contacts within the MED complex could stabilize one of these "alternative" conformations. Second, one could envisage that Med1-Med19 interactions do not occur within but between MED complexes and could thus stabilize "multi-MED" structures. It has been shown that master TFs control gene expression programs by establishing clusters of enhancers called super-enhancers, at genes with prominent roles in cell identity (51). Recent studies have revealed that, at super-enhancers, master TFs and the Mediator coactivator form phase-separated condensates, which compartmentalize and concentrate the PolII machinery to specific nuclear foci, to ensure high level of transcription (52)(53)(54). Interestingly, mammalian Med1 can form such phase-separated droplets that concentrate the transcription machinery at super-enhancers . Bringing together several MED complexes associated with TFs via Med1-Med19 trans-interaction might thus help phase droplets formation at clustered gene enhancers and ensure high transcriptional level (Fig 6.D). Given the more drastic effects of Med19-compared to Med1 depletion on GATA target genes, one could imagine a model where Med19 is absolutely required to recruit MED complex at GATA-bound regulatory sequences whereas Med1 would be preferentially involved in boosting the transcriptional response by favouring the formation of phaseseparated MED complex condensates.
In conclusion, our work shows that 2 MED subunits physically bind GATAs and are required to relay the regulatory signals from common TFs. This argues against the generally admitted view of binary interactions between one MED subunit and one (class of) TFs, which appears as an oversimplified model for MED action. The Mediator should be viewed as a complex interface allowing fine-tuned gene regulation by TFs through specific contacts with different MED subunit combinations. This study highlights the unexpected role of Drosophila Med19 as a GATA cofactor and Med1 interactor. This work sheds new light on the GATA-MED paradigm and suggests novel means by which several MED subunits might collaborate to regulate gene transcription.

BiFC assay:
This technique is based on expressing in vivo two candidate partner proteins with the N-and C-terminal portions (VN and VC) of the Venus protein in order to test the reconstitution of a functional fluorescent protein. UAS-Pnr-VN, UAS-VN-Pnr, and UAS CycC-VC lines were generated by inserting Pnr A or CycC ORF in phase with VN173 (aa 1-172) or VC155 (aa 155-238) ORF in a recipient pUAST-attB plasmid, allowing site specific insertion. attPcarrying embryos expressing PhiC31 integrase in the presumptive germline were injected with these plasmids: Pnr constructions were inserted on the X chromosome (attP ZH-2A), and CycC constructions on the 2 nd chromosome (attP 51D) to ensure identical expression of the different VN lines, and easy combination of VN and VC lines. Note that the BiFC constructions were functionally validated for their ability to rescue mutant lethality for Med19-VC, Med1-VC and CycC-VC or to produce typical gain-of-function phenotypes for VN-Pnr. Crosses were carried out at 22°C for interaction tests to express candidate proteins at homogenous and relatively low levels in order to avoid non-specific signal. We used the dppGAL4 driver (Gal4/UAS system) to direct co-expression of fusion proteins at the anteroposterior frontier of the wing imaginal disc both in the thorax where pnr is normally expressed and in the wing pouch region that does not express pnr.

Quantitative analysis of BiFC:
Image acquisition was performed on a Leica SP5 using the same settings and number of z slices for the different genetic contexts. BiFC fluorescence was quantified using ImageJ software. Region of interest (ROI) was hand-drawn following the contours of VC tagged protein expression as visualized by antiVC staining. The pixel value sum in the ROI of the BiFC channel was used for comparative quantification. The mean value was calculated from at least 20 wing discs from wandering L3 larvae of each genotype. In each disc, the same area located in the adjacent wing disc tissue was used to estimate tissue auto-fluorescence and remove this noise from the quantification.

Co-immunoprecipitation experiments:
Cultured Drosophila S2 cells were grown in 10% serum containing Schneider's medium at 25°C, and transfected using FuGENE HD transfection reagent (Roche) following manufacturer recommendations. Transfections of 18 x 10 6 cells per plate were carried out with pActin-Myc-Pnr (3µg) and pActin-GAL4 (2µg) plasmids. The cell harvest, protein extraction and IP were performed as described in (32) with the following modifications: the buffer used for protein extraction and subsequent IP contained 0,1% NP40 instead of 0,5% to increase purification of large complexes such as MED; 1mg of total protein extract was used for each IP instead of 1,5mg. Anti-Med19 and non-relevant (NR) IPs were performed with 5µl of decomplemented serum from a Med19immunized guinea pig (16) or 5µl of the same animal's pre-immune serum, respectively. We used 10µl G-protein coupled Sepharose beads per IP (SIGMA, P3296). Anti-Myc IPs were performed with 10µl Anti-Myc-Agarose bead (SIGMA, A7470).

GST-pulldown experiments:
Preparation of GST fusion proteins, 35 S-Methionine-labeled proteins and pulldown were performed essentially as described in (Mojica et al 2017). Med1, Pannier and Serpent proteins or sub-fragments have been produced from cDNA corresponding to Pnr A (Ramain et al 1993), Srp B (Waltzer et al 2002) and Med1 A (Immarigeon et al 2019) by in vitro transcription/translation coupled reactions using rabbit reticulocyte extracts (TnT-Promega) isoforms labeled. cDNA encoding full-length dMed19 and deletion derivatives were amplified by PCR using appropriated oligonucleotides and inserted into the BamHI/NotI site of the pGEX-6P1 vector (GE Healthcare). Pnr aa 1-291 fragment-encoding was amplified by PCR and cloned into pcDNA3 vector, with an HA-tag at the Cterminus and a Flag-tag at the N-terminus. All clones were verified by sequencing. Primers sequences and complete clone sequences are available upon request. Bacterial expression vectors pGEX-6P1 were transformed in competent E. coli strain BL21 (DE3). The transformed cells were plated in LB agar containing 50 µg/ml of ampicillin. A single colony was grown overnight in 25 ml of LB medium containing ampicillin on a rotary shaker (180 rpm) at 37˚C. Overnight starter culture was diluted 1:30 and bacteria were grown in 150 mL of LB medium containing ampicillin at 37˚C to an optical density of 0.8-0.9 at 600 nm and expression was induced with 0.5 mM IPTG for 2 h at 37˚C. Bacteria were pelleted by centrifugation and pellets were stored overnight at -20˚C. Pellet was resuspended in 15 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 % w/v glycerol, 0.1 % Nonidet-P40) including one Complete™ EDTA-free protease inhibitor tablet and sonicated on ice. After centrifugation at 10 000 x g 45 minutes at 4˚C, the supernatant was mixed 2 h at 4˚C on a rotating platform with 2 ml Glutathione Sepharose 4B resin. Beads were washed four times with lysis buffer and stored at 4˚C. 6µl of tagged Pnr 1-291-HA in vitro translation product was mixed with 50 µl of glutathioneagarose bead-GST Med19 derivatives in 200 µl of pull-down buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 % w/v glycerol, 0.1 % Nonidet-P40, 10 µM ZnSO4). The mixture was incubated for 2 h at 4˚C, washed four times with 500 µl pull-down buffer. Protein complexes were eluted from the beads with 2X Laemmli sample buffer, boiled 5 min and separated by SDS-PAGE on Mini-PROTEAN® TGX™ precast gels (Bio-Rad). Bound Pnr 1-291 was identified by Westernblot (1:5000 rabbit anti-HA polyclonal) using an ECL kit (Amersham GE Healthcare Life Sciences) based on the manufacturer's recommendations. HRP-conjugated secondary antibodies were used at 1:5000 and were purchased from Amersham GE Healthcare Life Sciences. All the membranes were scanned on an ImageQuant LAS 500 (GE Healthcare Life Sciences).

RT-qPCR:
Two different dsRNA were used for Med1-or Med19 mRNA depletion, only one for control GFP mRNA. The indicated dsRNAs (see Table  I is added at 2µg/ml to exponentially growing S2 cells, in an orbital shaker, at 2 10 6 cell/ml in serum-free medium. After 40min, serum is added. 24h later a second addition of dsRNA is done at 1µg/ml. Cells are collected 5 days after the first dsRNA treatment. For mRNA quantification, mRNAs were purified by RNeasy kit (Qiagen). Reverse transcription was done using SuperScript TM II Reverse Transcriptase (Thermo Fisher Scientific) and cDNA were quantified by real-Time qPCR (CFX Bio-Rad) with specific oligonucleotides (Table  I). Absolute quantification of each mRNA was normalized to GAPDH mRNA quantity in the same sample. mRNA measured in cells treated with a control dsRNA GFP was set at 100% to compare with cells treated with a dsRNA against Med1 or Med19.     GST pulldown assays allows delimitating interacting domains within Med19 and Pnr proteins. Schematic representation of GATA-Pnr and the multiple Pnr fragments generated to probe for binding to full-length GST-Med19 (A). N and C show proper zinc fingers (orange boxes) and their respective basic tails (hatched green and orange boxes). H1 and H2 show amphipathic alpha helices. Autoradiographs from GST pulldown experiments are shown on the right side for each fragment. The critical domain for strong binding is narrowed down to Pnr aminoacids 220-278. This domain comprising C-ZF proper zinc finger and its basic tail is highly conserved in Drosophila GATA factors (Srp, Grn, GATAd and GATAe) as well as human GATA factors (GATA1 to 6) as shown in panel B.

DNA binding-----------------------------------------
Open circles denote residues participating in DNA binding (34,35). Level of amino acid conservation is represented in beneath. Schematic representation of Med19 and the multiple Med19 fragments generated as GST fusions (C). For each truncated construct, the solid bar represents the portion of Med19 that is included. Numbers correspond to amino-acids. + and -summarize experimental results for Pnr binding, deduced from HA-Pnr 1-291 detections after GST-pulldowns (Western blots are shown in panels D to G). 2 distinct domains of Med19 are sufficient for binding Pnr (BS1 and BS2 in A). Schematic representation of GATA/Srp and the multiple Srp fragments generated to probe for binding to full-length GST-Med19 (A). Autoradiographs from GST pulldown experiments are shown on the right side for each fragment. Again, the critical binding domain is restricted to the GATA C-ZF domain containing the zinc-finger proper (orange square) and its basic tail (hatched green and orange boxes).
(B) Bimolecular Fluorescence Complementation (BiFC) assays using the dppGAL4 driver to express Med19-VC together with VN-Pnr or VN-Srp. Immunostaining shows similar expression of VC constructs (pink). Autoradiograph from GST pulldown experiments between GST-Med1 A and full length Med19 reveal direct interaction between the two MED subunits (C).
(D) Schematic representation of the three Med1 protein isoforms (Med1-A, -B and -C) and the multiple Med1 fragments generated to probe for binding to full-length GST-Med19 are schematized. The N-ter darker grey rectangles correspond essentially to the yeast Med1 orthologue and comprise 15 short evolutionarily-conserved motifs (black boxes (36)). The white and C-ter light grey regions emphasizes the divergent long metazoan-specific extensions. Autoradiograph are shown on the right side, and narrow the Med19-binding domain to the highly conserved N-terminal portion of Med1 proteins.