Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19

Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded “co-factor” that acts together with Hox proteins through their homeodomains in regulated developmental transcription.


Introduction
The finely regulated gene transcription permitting development of pluricellular organisms involves the action of transcription factors (TFs) that bind DNA targets and convey this information to RNA polymerase II (PolII). Hox TFs, discovered through iconic mutations of the Drosophila melanogaster Bithorax and Antennapedia Complexes, play a central role in the development of a wide spectrum of animal species [1,2]. Hox proteins orchestrate the differentiation of morphologically distinct segments by regulating PolII-dependent transcription of complex batteries of downstream target genes whose composition and nature are now emerging [3][4][5][6][7]. The conserved 60 amino acid (a.a.) homeodomain (HD), a motif used for direct binding to DNA target sequences, is central to this activity. Animal orthologs of the Drosophila proteins make use of their homeodomains to play widespread and crucial roles in differentiation programs yielding the very different forms of sea urchins, worms, flies or humans [8]. They do so by binding simple TAAT-based sequences within regulatory DNA of developmental target genes [9][10][11][12][13][14]. One crucial aspect of understanding how Hox proteins transform their versatile but low-specificity DNA binding into an exquisite functional specificity involves the identification of functional partners. Known examples include the TALE HD proteins encoded by extradenticle (exd)/Pbx and homothorax (hth)/ Meis, which assist Hox proteins to form stable ternary DNAprotein complexes with much-enhanced specificity. This involves contacts with the conserved Hox Hexapeptide (HX) motif near the HD N-terminus, or alternatively, with the paralog-specific UBD-A motif detected in Ubx and Abdominal-A (Abd-A) proteins [15,16]. Other TFs that can serve as positional Hox partners include the segment-polarity gene products Engrailed (En) and Sloppy paired, that collaborate with Ubx and Abd-A to repress abdominal expression of Distal-less [17]. Finally, specific a.a. residues in the HX motif, the HD and the linker separating them play a distinctive role in DNA target specificity, allowing one Hox HD region to select paralog-specific targets [18,19].
Contrasting with our knowledge of collaborations involving Hox and partner TFs, virtually nothing is known of what transpires at the interface with the RNA Polymerase II (PolII) machinery itself to generate an appropriate transcriptional response. The lone evidence directly linking Hox TFs to the PolII machine comes from the observation that the Drosophila TFIID component BIP2 binds the Antp HX motif [20].
Another key component of the PolII machinery is the Mediator (MED) complex conserved from amoebae to man that serves as an interface between DNA-bound TFs and PolII. MED possesses a conserved, modular architecture characterized by the presence of head, middle, tail and optional CDK8 modules. Some of the 30 subunits composing MED appear to play a general structural role in the complex while others interact with DNA-bound TFs bridging them to PolII. Together, these subunits and the MED modules they form associate with PolII, TFs and chromatin to regulate PolII-dependent transcription [21][22][23][24][25][26][27][28].
Our analysis of a Drosophila skuld/Med13 mutation isolated by dose-sensitive genetic interactions with homeotic proboscipedia (pb) and Sex combs reduced (Scr) genes led us to view MED as a Hox cofactor [29]. However, how MED might act with Hox TFs in developmental processes has not been explored. The work presented here pursues the hypothesis that Hox TFs modulate PolII activity through direct binding to one or more MED subunits. Starting from molecular assays, we identify Med19 as a subunit that binds to the homeodomain of representative Hox proteins through an animal-specific motif. Loss-of-function (lof) Med19 mutations isolated in this work reveal that Med19 affects Hox developmental activity and target gene regulation. Taken together, our results provide the first molecular link between Hox TFs and the general transcription machinery, showing how Med19 can act as an embedded functional partner, or ''cofactor'', that directly links DNA-bound Hox homeoproteins to the PolII machinery.

Hox proteins directly bind Med19 via their homeodomain
To search for MED subunits that contact Hox proteins directly, a Hox/MED binding matrix was established with the in vitro GSTpulldown assay. GST fusions of the eight Drosophila Hox proteins (full-length Labial (Lab), Deformed (Dfd), Scr, Ubx, Abd-A and Abdominal-B (Abd-B), or portions of Pb and Antp) were probed with 35 S-labelled MED proteins in standard conditions for 11 MED subunits that are not required for cell viability in yeast and/ or that are known to interact physically with TFs in mammalian cells (Materials and Methods). The Med19 subunit stood out, binding strongly to multiple GST-Hox proteins in this assay (mean binding, $5% of input in multiple tests, except for Abd-B, 0.8%; Figure 1A).
To provide an independent and in vivo test for direct Med19/ Hox binding, we used the Bimolecular Fluorescence Complementation (BiFC) assay. Auto-fluorescence from the Venus variant of Green Fluorescent Protein (GFP), abolished by truncating Venus protein into N-and C-terminal portions (VN and VC), can be effectively reconstituted when VC and VN fragments are coupled to interacting protein partners that reunite them in cultured cells [30] or living organisms [31,32]. BiFC tests made use of the Gal4/UAS system [33] to direct co-expression of chimeric VN-Hox and Med-VC proteins. The functional validation of VN-AbdA relative to AbdA has been described [32]. VN-Ubx and VN-Dfd were likewise validated by their gain-of-function (gof) transformations in embryos ( Figure S1). Fused Med19-VC was validated by its capacity to rescue lethal Med19 mutants (described below). UAS-directed co-expression of Med19-VC with VN-Ubx, VN-Dfd or VN-AbdA in embryos under engrailed-Gal4 control (en.Med19-VC+VN-Hox) resulted in serial stripes of clear nuclear fluorescent signal for VN-Ubx and VN-Dfd ( Figure 1B-C). VN-AbdA was negative in this test ( Figure 1D, but see Figure 1H, I below). Nevertheless, the concordant results from GST pulldown and BiFC tests indicate direct Med19 binding to Ubx and Dfd.
The observed direct Med19/Dfd or Ubx binding led us to ask whether these interactions utilize the homeodomain (HD) common to all Hox proteins. Consistent with this possibility, GST fusions containing the HX-HD regions of Pb (middle, a.a. 119-327) and Antp (C-ter, aa 279-378) bound Med19 at levels 15to 50-fold superior to Pb N-ter (a.a. 1-158), Pb C-ter (a.a. 267-782) and Antp N-ter (a.a. 1-90) peptides ( Figure 1A). On dissecting the HD-containing C-terminal regions of Ubx, GST-Med19 bound similarly to wild-type and HX-mutated Ubx [15], or to the linker-shortened C-terminal region of crustacean Artemia salina Ubx ( Figure S2). Tests with the Antp C-ter peptide (a.a. 279-378) containing its HX, linker and HD, led to the same conclusion (not shown). Confirmation came from binding experiments using immobilized GST-Med19 and Antp, Ubx, Dfd and Abd-B HD peptides, where strong binding was observed for all four homeodomains despite marked divergence of their primary sequences (60% identity of Abd-B and Ubx HD) ( Figure 1E). Contrary to the four Hox HD, neither Engrailed HD (43% identity with Ubx HD) nor the TALE class Hth HD (21% identity) bound detectably ( Figure 1E). Since Med19 binds Dfd, Antp, Ubx and Abd-B HD but not those of En or Hth, we infer that it is specific and can discriminate among homeodomains.
In BiFC assays for Med19/HD association in vivo, comparable signals were observed on co-expressing Med19-VC with full-length VN-Ubx, or its HD alone (VN-HDUbx), in the normal Ubx expression domain (Ubx-Gal4; Figure 1F,G). Co-expressing

Author Summary
Mutations of Hox developmental genes in the fruit fly Drosophila melanogaster may provoke spectacular changes in form: transformations of one body part into another, or loss of organs. This attribute identifies them as important developmental genes. Insect and vertebrate Hox proteins contain highly related homeodomain motifs used to bind to regulatory DNA and influence expression of developmental target genes. This occurs at the level of transcription of target gene DNA to messenger RNA by RNA polymerase II and its associated protein machinery (.50 proteins). How Hox homeodomain proteins induce finetuned transcription remains an open question. We provide an initial response, finding that Hox proteins also use their homeodomains to bind one machinery protein, Mediator complex subunit 19 (Med19) through a Med19 sequence that is highly conserved in animal phyla. Med19 mutants isolated in this work (the first animal mutants) show that Med19 assists Hox protein functions. Further, they indicate that homeodomain binding to the Med19 motif is required for normal expression of a Hox target gene. Our work provides new clues for understanding how the specific transcriptional inputs of the highly conserved Hox class of transcription factors are integrated at the level of the whole transcription machinery.
VN-AbdA with Med19-VC under abdA-Gal4 did not give rise to fluorescence ( Figure 1D, H). By contrast, AbdA HD (VN-HDAbdA) yielded a strong fluorescent signal ( Figure 1I), confirming that the Hox HD is sufficient for direct Med19 binding. The differing responses obtained for full-length AbdA versus its HD raise the interesting possibility that AbdA sequences outside the HD limit its access to Med19 in vivo. Taken together, these biochemical and in vivo results indicate that the Hox HD is sufficient for direct binding to Med19.

Med19 participates in Hox organizing activity
Apart from yeast, no mutants of Med19 have been described. To address Med19 gene function in vivo, we employed imprecise excision of a viable P element insertion mutant, Med19 P to generate two loss-of-function (lof) mutations, Med19 1 (a pupallethal hypomorphic allele harboring a 14 base pair (bp) upstream deletion), and Med19 2 (deleted for much of its protein coding sequence; see Figure 2A). Homozygotes for the presumptive null mutation Med19 2 die at the end of embryogenesis but do not show cuticular defects. To remove maternally contributed protein and/ or mRNA [34] that might mask early requirements for Med19, we used the Dominant Female Sterile technique to generate mitotic germ-line clones. When clones were induced in females heterozygous for Med19 2 and for ovo D1 , egg-laying was observed. Such embryos devoid of maternally contributed Med19 product undertake development (as visualised by nuclear DAPI staining, Figure S3). Though the first nuclear divisions proceed normally ( Figure S3, <1 hr), abnormalities are already visible in pre-cellular blastoderm ( Figure S3, <2 hr), leading to massively disorganised cellular embryos that die soon after ( Figure S3). We conclude that a major maternal contribution to embryonic Med19 activity masks its zygotic roles.
As an alternative approach to examining Med19 function in embryonic development, we made use of Med19-directed RNAi. Ubiquitous RNAi expression under daughterless-Gal4 control gave rise to cuticular defects in the spiracles and head of L2/L3 larvae ( Figure S4) reminiscent of the embryonic consequences of Abd-B lof in the posterior spiracles, or of lab/Dfd/Scr lof in the head. However, the late appearance of these defects, their incomplete penetrance and variable expressivity made it difficult to ascertain a functional link to embryonic Hox activities.
We therefore decided to examine post-embryonic development, making use of partial loss-of-function combinations and of clonal analysis. Med19 1 /Med19 2 animals die as pupae, but adult viability is restored by ubiquitous transgenic expression of Med19 (Ub-Med19 or arm.Med19-VC; Figure S9). These results show that lethality is due solely to loss of Med19 function. They also functionally validate the UAS-Med19-VC element used in BiFC assays. To better characterize the consequences of Med19 loss-offunction, we employed FLP/FRT-mediated mitotic recombination [35] to generate clones of cells homozygous for Med19 2 (2/2). In ''twin spot'' experiments where 2/2 and +/+ cells arise from mitotic recombination in a single 2/+ cell during mitosis, only +/+ cells were subsequently detected ( Figure 2B). This cell lethality is due to loss of Med19, since expressing Med19-VC protein in mutant cells restores viability ( Figure 2C); indeed, large Med19 2 /Med19 2 clones are observed even though Med19-VC accumulation is less than for wild-type protein in adjacent cells ( Figure S5,A-A0). Strikingly, Med19 2 /Med19 2 cells also survived in the presence of a Minute (M) mutation [36] that slows growth of surrounding heterozygous M 2/+ cells ( Figure 2D). Immune staining with anti-Med19 sera confirmed that Med19 2 is a protein-null mutation ( Figure S5, B-B0). Thus the existence of these clones shows that Med19 is not strictly required for cell viability. The influence of cell environment on cell lethality suggests a role for Med19 in the control of cell competition.
If Hox/Med19 binding is functionally relevant to homeotic activity, Med19 mutants might provoke Hox-like phenocopies or modify Hox-induced homeotic defects. In light of the strong maternal contribution of Med19 present in embryos, we turned our attention to later developmental stages. Hypomorphic Med19 1 /Med19 2 animals, or Med19 2 /Med19 2 animals with lowlevel ubiquitous expression from the Ub-Med19 transgene, survive to the pupal stage and show a fully penetrant loss of anterior spiracles ( Figure 2E, F). Rare adult Ub-Med19; Med19 2 /Med19 2 survivors showed defects including loss of maxillary palps ( Figure 2G, H). Tissue-directed induction of Med19 2 /Med19 2 clones in the dorsal compartment of the haltere imaginal disc (apterous-Gal4.UAS-Flp) is associated with disorganization of the distinctive pedicellar sensillae in halteres [37], where these sensory organs are reduced in number and their well-ordered rows disrupted in adult halteres ( Figure 2I-I9 vs. 2J-J9).
All the preceding defects ( Figure 2E-J9) resemble Hox loss-offunction phenotypes: of Antp for the pupal anterior spiracles [38]; of Dfd for the adult maxillary palps [39,40]; and of Ubx for haltere sensory organs [37]. We therefore asked whether Med19 mutants can act as dose-sensitive modifiers of Hox activity in genetic interaction tests. For Antp, the fully penetrant spiracle loss observed in Med19 1 /Med19 2 pupae ( Figure 2F) is absent from Med19 2 or Antp 2 heterozygotes but can be detected in Med19 2 /Antp 2 double heterozygotes ( Figure S6). This synergistic interaction links Med19 to normal Antp function. Conversely, ectopic expression from the gain-of-function (gof) Antp Ns allele directs a fully penetrant transformation of antenna toward leg ( Figure 3A, B) that is partially suppressed in Med19 2 heterozygotes, as shown by the replacement of distal claws by antennal aristae ( Figure 3C, Figure  S6). Adult maxillary (Mx) palps ( Figure 3D, arrowhead) require Dfd function in a territory abutting the antennal primordium of the eye-antennal imaginal disc [40], and the palp loss noted above ( Figure 2H) suggested a link to Dfd. In interaction tests, palp loss provoked by Dfd-specific RNAi (patched.dsRNADfd) was enhanced in Med19 2 heterozygotes ( Figure S6). Conversely, ectopic Mx organs observed in heterozygotes for the gof allele Dfd 1 (20% of adult heads) were fully suppressed in Dfd 1 /Med19 2 double heterozygotes ( Figure 3E, F and Figure S6). Similarly, the transformation of the posterior wing ( Figure 3G) to a hemi-haltere in Ubx Cbx1 homozygotes ( Figure 3H) was synergistically modified on reducing Med19 activity ( Figure 3I). These dose-sensitive modifications of Antp, Dfd and Ubx-dependent phenotypes by Med19 lof mutants support a functional Hox/Med19 link in vivo.

Med19 is required for Ubx target gene activation
The intensive attention given to Ubx target gene regulation in the haltere imaginal disc [3][4][5]41,42] makes it an excellent paradigm for understanding Hox interplay with Med19 in a developmental program. We therefore examined the effect of removing Med19 function on Ubx activity towards selected target genes in the haltere imaginal disc ( Figure 4A). Clones of 2/2 cells induced in haltere discs in the presence of a Minute mutation showed Ubx levels comparable to their +/2 neighbors ( Figure 4B-B90). The presence of normal nuclear Ubx signal in mutant cells after several mitotic divisions shows that the Med19 2 condition has not generally affected transcription. We next examined the effects of Med19 2/2 mitotic clones on several Ubx target genes in haltere development [41]. Ubx is known for its role in suppressing wing development, and acts to repress a number of prominent wing developmental genes in the haltere imaginal disc [41]. With the combined use of ectopic Hox expression and analysis of the transcriptome, additional Ubx targets have emerged. The first example of a Ubx-activated target was CG13222, which is regulated through an autonomous cis-regulatory region called ''edge'' for its expression in a band of cells along the posterior border of the haltere disc [42]. Expression of the edge-GFP reporter construct, that recapitulates Ubx-dependent CG13222 expression [42] is cell-autonomously abolished in Med19 2/2 haltere clones ( Figure 4C,D; identified with anti-Med19 sera). This shows that Med19 is required for activation of the direct Ubx target CG13222 in the haltere disc. A second positively-regulated target identified in recent whole-genome analyses, bric-à-brac2 (bab2), is induced by ectopic Ubx [5] and correlates with direct Ubx binding to regulatory DNA in vivo [3,4]. Using mitotic recombination, we examined the expression of bab2 in Ubx 2/2 haltere cells and found that Bab2 accumulation is cell-autonomously abolished ( Figure 4E-E90). On examining bab2 expression with respect to Med19 activity, Bab2 accumulation was cell-autonomously downregulated in Med19 2/2 cells ( Figure 4F-F90). This shows that Med19 activity is required in the haltere disc for normal Ubxmediated activation of bab2. However, contrary to edge, residual low-level bab2 expression is present in some Med19 2 cells ( Figure 4F-F90). The responses of these two regulatory sequences to Med19 lof indicate that Med19 is required for Ubx target gene activation.
Several targets whose expression is repressed by Ubx were tested for a requirement for Med19 ( Figure 4A). For example, the broad central band of spalt (sal) expression in the wing pouch is absent from Ubx-expressing cells of the haltere pouch. sal is derepressed in Ubx 2/2 haltere disc cells [41], as shown by the appearance of Sal protein in Ubx mutant cells ( Figure 4G-G90). By contrast, no new expression of Sal is detected in equivalently placed Med19 2/2 clones ( Figure 4H-H90). De-repression was likewise not observed for other tested Ubx-repressed targets (not shown), lending molecular support to the interpretation that Med19 is not involved in Ubx repressive activities. While further examples will be required to determine whether this illustrates a general property of Med19 action in transcriptional regulation, these results suggest that Med19 collaboration with Ubx is limited to gene activation.

Med19 binds the Hox homeodomain through a conserved motif
Binding of Hox proteins to Med19 specifically involves their conserved homeodomain. To identify Med19 sequences involved in HD binding, we used GST-pulldown to test full-length or deleted versions of Med19 with GST-HDAntp. Binding was retained on truncating the terminal regions of Med19 ( Figure 5A, constructs 1-2), but was abolished on deleting an internal 70 aa region ( Figure 5A, constructs 3-4). Smaller deletions confirmed that this region contains sequences required for full binding ( Figure 5A, constructs 5-6). This 70 a.a. Med19 peptide is not only required but also proved sufficient to bind the HD ( Figure 5A, construct 7), leading us to call it Homeodomain Interacting Motif (HIM).
The HIM interval of Med19 was then compared with Med19 orthologs from a spectrum of eukaryotes. Sequence alignments reveal a lysine/arginine-rich sequence that is strongly conserved in Med19 orthologs from six vertebrate or insect species ( Figure S7). This striking conservation suggests that the contribution of HIM to Med19 function is subject to strong selective pressure.
Med19 is required for Ubx-mediated activation of specific target genes in vivo, and directly binds Hox HDs in vitro through its conserved HIM motif. This suggested that Ubx function passes through Med19, potentially via its HIM sequence. We therefore sought evidence for Med19/Hox binding in cellulo. Cultured Drosophila cells expressing UAS-Med19-VC or UAS-Med19-DHIM-VC were used for co-immunoprecipitations with anti-GFP (VC). As shown in the Western blot of Figure S8, the three endogenous Med1 isoforms were associated with both Med19-VC and Med19DHIM-VC. This is consistent with the incorporation of full-length and HIM-deleted forms into the MED complex.
We next co-expressed these proteins with Ubx-HA and tested for their association in cellulo. As seen in Figure 5B, Ubx-HA coprecipitates with Med19-VC, indicating the association of Ubx transcription factor with Med19. By contrast, less Ubx-HA was detected on co-precipitating with Med19DHIM-VC (relative to a non-specific band that serves as a de facto internal loading control, * in Figure 5B). These results indicate that the HIM domain contributes to Ubx-Med19 interaction.
To further investigate the contribution of the HIM domain to HD binding in vivo, we generated transgenic lines containing the same UAS-Med19-VC, -Med19DHIM-VC and -HIM-VC constructs used above, and tested each protein's ability to bind to VN-HDUbx in the BiFC assay. In control experiments, Med19-VC, HIM-VC and Med19DHIM-VC accumulated at comparable levels in wing imaginal discs ( Figure S8). Med19-VC and HIM-VC are fully nuclear. The Med19DHIM-VC protein (lacking the highly basic HIM element) is seen to accumulate in both the cytoplasm and the nucleus ( Figure S8). This indicates that the HIM element contributes to nuclear localisation, together with other Med19 sequences. As noted in embryos ( Figure 1G), coexpressing Med19-VC with VN-HDUbx under dpp-Gal4 control in wing imaginal discs resulted in clear fluorescent signal ( Figure 5C). When HIM-VC was tested for its ability to interact with the Ubx HD, it gave rise to a fluorescent signal stronger than for intact Med19; by contrast, the Med19DHIM-VC fusion yielded only a background-level signal with VN-HDUbx ( Figure 5C). Taken together, these results of biochemical and BiFC experiments indicate that Med19 HIM is necessary and sufficient for full HD binding.

HIM is required for Ubx target gene activation
While Med19 bereft of its conserved HIM element can be incorporated into MED, as shown above, its functional requirements in vivo remained an open question. Accordingly, we tested whether Drosophila HIM is relevant to Med19 developmental functions. In a genetic rescue test, ubiquitous Med19-VC expression restored adult viability to pupal-lethal Med19 1 / Med19 2 hypomorphs, whereas Med19DHIM-VC did not. Med19-DHIM-VC also showed a reduced aptitude to rescue pupal spiracles, adult maxillary palps and haltere sensillae compared with Med19-VC ( Figure S6). These results indicate a requirement for Med19 HIM in several Hox-dependent developmental processes.
We therefore sought to test the influence of the Med19 HIM peptide on Ubx-dependent transcriptional activation of the direct target CG13222/edge. To this end, (i) FLP/FRT-mediated mitotic recombination was used to generate cells devoid of wild-type protein, while (ii) UAS/Gal4-directed expression supplied normal or HIM-deleted Med19-VC, and (iii) the edge-GFP reporter was employed to assess Ubx-mediated activation of CG13222. En-Gal4-directed UAS-Flp expression in the posterior haltere disc compartment served to induce mitotic recombination there, while en-Gal4 simultaneously directed expression of Med19-VC or Med19DHIM-VC in the posterior compartment ( Figure 6A-B, stained with anti-VC, blue).
These twin-spot experiments provided two important observations. Firstly, large 2/2 clones (RFP-) were observed not only in Med19-VC but also in Med19DHIM-VC expressing discs ( Figure 6A9, B9). The existence of 2/2 clones is in marked contrast with their complete absence in Figure 2B. This shows that both Med19-VC and Med19DHIM-VC restore cell viability. We conclude that HIM is not necessary for cell viability.  Figure 6A0, C). By contrast, edge-GFP expression was entirely absent from most clones expressing Med19DHIM-VC (7 of 11 clones; Figure 6B0, C). The existence of HIM-independent cell proliferation/survival shows that this cellular function of Med19 can be uncoupled from its Hox-related role. These results also provide clear functional evidence that Ubx-dependent activation of its edge-GFP target requires HIM-endowed Med19.

Med19, a MED regulatory subunit that binds Hox transcription factors
Hox homeodomain proteins are well-known for their roles in the control of transcription during development. Further, much is known about the composition and action of the PolII transcription machine. However, virtually nothing is known of how the information of DNA-bound Hox factors is conveyed to PolII in gene transcription. The Drosophila Ultrabithorax-like mutant affecting the large subunit of RNA PolII provokes phenotypes reminiscent of Ubx mutants [43], but the molecular basis of this remains unknown. The lone direct evidence linking Hox TFs to the PolII machine is binding of the Antp HX motif to the TFIID component BIP2 [20]. Here, we undertook to identify physical and functional links between Drosophila Hox developmental TFs and the MED transcription complex. Our results unveil a novel aspect of the evolutionary Hox gene success story, extending the large repertory of proteins able to interact with the HD [44] to include the Drosophila MED subunit Med19. HD binding to Med19 via the conserved HIM suggests this subunit is an ancient Hox collaborator. Accordingly, our loss-of-function mutants reveal that Med19 contributes to normal Hox developmental function and does so at least in part via its HIM element. Thus this analysis reveals a previously unsuspected importance for Med19 in Hoxaffiliated developmental functions.
A fundamental property of the modular MED complex is its great flexibility that allows it to wrap around PolII and to change form substantially in response to contact with specific TFs [45]. Recent work in the yeast S. cerevisiae places Med19 at the interfaces of the head, middle and CDK8 kinase modules [46,47]. Med19 is thus well-positioned to play a pivotal regulatory role in governing MED conformation (Figure 7). Our results raise the intriguing possibility that MED structural regulation and physical contacts with DNA-bound TFs can pass through the same subunit. In agreement with this idea, recent work identified direct binding between mouse Med19 (and Med26) and RE1 Silencing Transcription Factor (REST) [48]. This binding involves a 460 a.a. region of REST encompassing its DNA-binding Zn fingers [48]. The present work goes further, in identifying a direct link between the conserved Hox homeodomain and Med19 HIM (Figure 7) that is, to our knowledge, the first instance for a direct, functionally relevant contact of MED with a DNA-binding motif rather than an activation domain.

Hox-independent Med19 roles in cell proliferation/ survival
Med19 contributes to developmental processes with Antp (spiracle eversion), Dfd (Mx palp), and Ubx (haltere differentiation). Other phenotypes identified with our mutants indicate further, non-Hox related roles for Med19. As shown here, complete loss of Med19 function leads to cell lethality that can be conditionally alleviated when surrounded by weakened, Minute mutation-bearing cells. These observations, that uncouple HIMdependent functions from the role of Med19 in cell survival/ proliferation ( Figure 6B), are compatible with reports correlating over-expression of human Med19/Lung Cancer Metastasis-Related Protein 1 (LCMR1) in lung cancer cells with clinical outcome [49]. Further, RNAi-mediated knock-down of Med19 in cultured human tumor cells can reduce proliferation, and tumorigenicity when injected into nude mice [50][51][52][53][54][55][56][57][58]. A recent whole-genome, RNAi-based screen identified Med19 as an important element of Androgen Receptor activity in prostate cancer cells where gene expression levels also correlated with clinical outcome [59]. It will be of clear interest to examine how, and with what partners, Med19 carries out its roles in cell proliferation/survival.

Transcriptional activation versus repression
The role played by mammalian Med19 and Med26 in binding the REST TF, involved in inhibiting neuronal gene expression in non-neuronal cells [48,60], provides an instance of repressive Med19 regulatory function. We found that Med19 activity is required in the Drosophila haltere disc for transcriptional activation of CG13222/edge and bab2, but is dispensable for Ubx-mediated repression of five negatively-regulated target genes ( Figure 4). Ubx can choose to activate or it can repress, at least in part through an identified repression domain at the C-terminus just outside its homeodomain [61]. Conversely Med19, which binds the Ubx homeodomain, appears to have much to do with activation.
Concerning the mechanisms of Ubx-mediated repression, one illuminating example comes from analyses of regulated embryonic Distal-less expression [17]. Ubx can associate combinatorially with Exd and Hth, plus the spatially restricted co-factors Engrailed or Sloppy-paired in repressing Distal-less [17]. Engrailed in turn is able to recruit Groucho co-repressor [62], suggesting that localized repression involves DNA-bound Ubx/Exd/Hth/Engrailed, plus Engrailed-bound Groucho. Groucho has been proposed to function as a co-repressor that actively associates with regulatory proteins and organizes chromatin to block transcription. Wong and Struhl [63] demonstrated that the yeast Groucho homolog Tup1 interacts with DNA-binding factors to mask their activation domains, thereby preventing recruitment of co-activators (including MED) necessary for activated transcription. The number of targets remains too small to be sure Med19 is consecrated to activation. Nonetheless, it will be of interest to determine whether Groucho can play a role in blocking MED/Ubx interactions that could provide an economical means for distinguishing gene activation from repression.

The Hox-MED interface in evolution and development
The conserved Hox proteins and the gene complexes that encode them are well-known and widely used to study development and evolution. As to the evolutionary conservation of the Mediator transcription complex, the presence of MED constituents in far-flung eukaryotic species from unicellular parasites to humans [21] indicates that this complex existed well before the emergence of the modern animal Hox protein complexes. The DNA-binding domains are often the most conserved elements of TF primary sequence, and in the case of the Hox HD, recent forays into ''synthetic biology'' agree that this was the functional heart of the ancestral proto-Hox proteins [64][65][66]. Indeed, Scr, Antp and Ubx mini-Hox peptides containing HX, linker and HD motifs behave to a good approximation like the full-length forms, directing appropriate gene activation and repression resulting in genetic transformations [64][65][66]. Our results showing direct HD binding to Med19 HIM, and thus access to the PolII machinery, allow the activity of these mini-Hox proteins to be rationalized. We surmise that at the time when the Hox HD emerged to become a major developmental transcription player, its capacity to connect with MED through specific existing sequences was a prerequisite for functional success. One expected consequence of this presumed initial encounter with Med19 -a selective pressure on both partners and subsequent refinement of binding sequences -is in agreement with the well-known conservation of Hox homeodomains, and with the observed conservation of the newlyidentified HIM element in Hox-containing eumetazoans. We imagine that subsequent evolution over the several hundred million years separating flies and mammals will have allowed this initial contact to be consolidated through subsequent binding to other MED subunits, ensuring versatile but reliable interactions at the MED-TF interface (Figure 7).

A functional Hox-MED interface
Hox homeodomain proteins are traditionally referred to as selector or ''master'' genes that determine developmental transcription programs. The low sequence specificity of Hox HD transcription factors is enhanced by their joint action with other TFs, of which prominent examples, the TALE homeodomain proteins Extradenticle/Pbx and Homothorax/Meis are considered to be Hox co-factors. However, a Hox TF in the company of Exd and Hth could still not be expected to shoulder all the regulatory tasks necessary to make a segment with all the coordinated celltypes it is made up of, and collaboration with cell-type specific TFs appears to be requisite. A useful alternative conception visualizes Hox proteins not as ''master-selectors'' that act with co-factors, but as highly versatile co-factors in their own right that can act with diverse cell-specific identity factors to generate the cell types of a functional segment [67]. We envisage a model where a Hox protein would be central to assembling cell-specific transcription factors into TF complexes that interface with MED ( Figure 7).
Such Hox-anchored TF complexes could make use of selective HD binding to Med19 as a beach-head for more extensive access to MED, such that loss of the Hox protein would incapacitate the complex: in the case of Ubx 2 cells, inactivating bab2 or derepressing sal. Accordingly, three observations suggest that binding of Hox-centered TF complexes involves additional MED subunits surrounding Med19 (Figure 7) : (i) bab2 target gene expression is entirely lost in Ubx-deficient cells but can persist in some Med19 2 cells; (ii) edge-GFP in Med19 2 cells expressing Med19DHIM-VC was not altogether refractory to Ubx-activated edge-GFP expression ( Figure 6); and (iii) Med19DHIM-VC is not entirely impaired for Ubx binding, as seen in co-immunoprecipitations ( Figure 5). Thus Hox protein input conveyed through Med19-HIM at the headmiddle-Cdk8 module hinge might provide an economical contribution toward organizing TF complexes that influence overall MED conformation [45] and hence transcriptional output. Decoding how the information-rich MED interface including Med19 accomplishes this will be an important part of understanding transcriptional specificity in evolution, development and pathology.

Ethics statement
Our work using Drosophila was performed in conditions in conformity with French and international standards.

Co-immunoprecipitations
Cultured Drosophila S2 cells were transfected using FuGENE HD transfection reagent (Roche) with pActin-V5 (negative control; pActin-GAL4 driver with either pUAS-Med19-VC or pUAS-Med19DHIM-VC (MED co-IP); or adding pUAS-Ubx-HA (Med19-Ubx co-IP). 10 7 cells were transfected with driver plasmid plus the UAS responder plasmid(s). After 72 hr, cells were harvested by scraping and pooled, collected by centrifuging then washed with 1x PBS. All subsequent steps until Western blotting were carried out at 4uC. Cell pellets were resuspended in IP buffer (50 mM TrisHCl, pH = 8, 150 mM NaCl, 0.5% NP40, 1 mM EGTA and Roche complete protease inhibitor cocktail), lysed by four-fold passage through a 27G needle, then centrifuged for 10 min at 14,500 rpm. Immunoprecipitation from 1.5 mg of total protein extract (5 mg/ml in IP buffer) was performed with mouse anti-GFP (ROCHE 4 mg/IP), with gentle agitation overnight. 15 ml of G-protein-coupled Sepharose beads (SIGMA, P3296) were added, then gently agitated for 2 hr. The non-bound fraction was discarded. Beads were washed 4 times with fresh IP buffer, taken up in 2X Laemmli buffer containing DTT and SDS, heated to 95uC, and centrifuged. Supernatants were then submitted to polyacrylamide gel electrophoresis. Med1 and Med19 were revealed using polyclonal sera from guinea-pig (diluted 1:500), while Ubx-HA was detected with rabbit anti-HA (SIGMA) diluted 1:1000.

Bimolecular Fluorescence Complementation (BiFC)
Constructions corresponding to UAS-VN-Ubx, UAS-VN-AbdA and UAS-VN-HDAbdA transgenic lines are described in [32]. UAS-VN-Dfd, UAS-VN-HDUbx: Dfd and Ubx HD sequences were cloned into XhoI-XbaI sites downstream of the Venus VN fragment into the pUAST or pUASTattB plasmids described in [32]. UAS-Med19-VC, UAS-HIM-VC: Full-length Med19 coding sequences, or the internal HIM sequence generated from Med19 cDNA by PCR, were introduced as EcoRI-XhoI fragments to replace Hth coding sequences of pUaHth-VC [32]. For UAS-Med19DHIM-VC, internally deleted Med19 was generated from the full-length construct by double PCR, using the overlap extension method. The PCR-derived internal deletion product was cleaved by RsrII and XhoI, then cloned in place of the equivalent fragment of UAS-Med19-VC. All constructs were sequence-verified before fly transformation. Transgenic lines were established by classical P-element mediated germ line transformation or by site-specific integration using the WC-31 integrase. Embryos were analysed as described in [32]. For BiFC in imaginal discs, late third-instar larvae of appropriate genotypes were cultured in parallel in the same environmental conditions of temperature and larval density, then were dissected at the same time and fixed in the same solution (209; 4% para-formaldehyde, 0.5M EGTA, 1X PBS). Wing and haltere imaginal discs were dissected and mounted in Vectashield (Vector Labs). Image acquisition was performed on a Leica SP5 using the same laser excitation, brightness/contrast and z settings. Confocal projections from at least 10 distinct wing discs per genotype were analyzed with ImageJ software.

Fly strains
Stocks and crosses were maintained at 25uC on standard yeastagar-cornmeal medium. Mutant stocks harboring Antp Ns , Dfd 1 , Figure 7. Model for the role of Med19 at the interface of Hox and MED. The Mediator complex, composed of four modules -tail, middle, head and CDK8 -, binds physically to PolII, principally through its head module. Hox transcription factors (HD in blue, its three a-helices indicated as cylinders) bind to regulatory DNA sequences distant from the transcription start site (grey arrow), together with unknown numbers of other TFs (here, Hox co-factors Exd and Hth plus cell-specific factors TF1 and TF2). We propose that the DNA-bound Hox homeodomain serves to recruit MED directly through Med19 HIM (green hook). This Hox-MED association then permits the general PolII transcription machinery (PolII+GTF) to be recruited to the Hox target promoter. This link to a MED subunit situated at the interface of the head, middle and CDK8 modules could modify overall MED conformation, favoring additional contacts between the TF complex and MED that modulate transcriptional activity. doi:10.1371/journal.pgen.1004303.g007 Ubx Cbx1 and Df(3L)BSC8 were from the Bloomington Drosophila Stock Collection. Antp Ns+Rc3 was provided by R. Mann. Edge-GFP and vgQ-lacZ originate from the S. Carroll lab. The Ub-Med19 transgenic line expressing full-length Med19 cDNA under ubi73 control is a homozygous-viable insertion on the X chromosome at attP site ZH 2A. These transgenic elements carry the visible marker miniwhite. UAS-RNAi against Dfd is from a non-directed insertion on chromosome 2 (Vienna Drosophila Research Collection stock 50110). UAS-RNAi lines against Med19 are stocks 27559 and 33710 from the Bloomington collection. Gal4-expressing driver lines used were: dpp-Gal4 (imaginal disc-specific, AP boundary, anterior compartment), arm-Gal4 (ubiquitous), ptc-Gal4 (AP boundary, anterior compartment), en-Gal4 (posterior compartment), ap-Gal4 (dorsal compartment of wing and haltere discs), Ubx-Gal4 and abdA-Gal4 (abdominal expression under Ubx or abdA control, respectively).

Rescue experiments
The following stocks were employed for rescue tests:

Clonal analyses
Mitotic clones were induced by Flp recombinase expressed from a hsp70-Flp transgene on heat induction (309 at 38uC), or from a UAS-Flp element under Gal4 control as indicated above (en.Flp, ap.Flp). Clones were generated and identified in marked progeny from crosses using the following stocks: Embryos resulting from germline clones were collected on egg lay plates, then analysed by confocal microscopy after mounting in DAPI-containing Vectashield medium. In positive controls where Med19 + replaced Med19 2 , all expected zygotic classes were obtained as viable, fertile adults. In the absence of heat shock, no eggs were laid.

Phenotypic analyses
Adult phenotypes were analyzed by light microscopy (Zeiss Axiophot) of dissected samples mounted in Hoyer's medium or by scanning electron microscopy (Hitachi TM-1000 Tabletop model) of frozen adults

Sequence alignments
These were generated with the T-Coffee Program, employing the methodology described by [21]. Figure S1 Venus fusion proteins VN-Ubx and VN-Dfd are functional in the Drosophila embryo. Cuticles of wild-type embryos, or of embryos ectopically expressing Hox proteins. First line: left, wild-type embryo showing anterior cuticle from the head to abdominal segment 1 (A1). The three thoracic belts of fine denticles and the first band of denser abdominal denticles are indicated (T1, T2, T3 and A1, respectively); middle and right, similar transformations of T1, T2 and T3 denticle belts to A1 are induced by the ubiquitous expression of Ubx or of chimeric VN-Ubx, respectively, from UAS enhancers under arm-Gal4 control (arm.). Second line: left, wild-type embryonic head with cephalopharyngeal cuticle. Middle and right: arm-Gal4 driver-directed expression of Dfd or VN-Dfd from a UAS enhancer (arm.) results in similar, major defects of normal head structures, accompanied by the appearance of ectopic maxillary cirri (arrows and inset) typical of Dfd function. (TIF) Figure S2 Hexapeptide and linker region are dispensable for interaction with Med19. Bar drawings on the left represent (top) the HD region of Drosophila Ubx, with its hexapeptide (HX), linker region and HD; (middle) the HD region of Drosophila Ubx, but with its hexapeptide mutated (HXm) as described in Hudry et al (34); (right) the HD region of crustacean Artemia Ubx, whose HD is identical to the Drosophila sequence but whose linker region is much shorter. On the right, GST pulldowns show similar binding to wild-type Ubx (7% of input), Ubx whose HX is mutated (5%), or Artemia Ubx with shortened linker (5%).