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. 2014 May 1;10(5):e1004303.
doi: 10.1371/journal.pgen.1004303. eCollection 2014 May.

Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of mediator subunit Med19

Muriel Boube  1 , Bruno Hudry  2 , Clément Immarigeon  1 , Yannick Carrier  1 , Sandra Bernat-Fabre  1 , Samir Merabet  2 , Yacine Graba  2 , Henri-Marc Bourbon  1 , David L Cribbs  1
Affiliations

Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of mediator subunit Med19

Muriel Boube et al. PLoS Genet. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Hox proteins bind Med19 through their homeodomains in vitro and in vivo.
(A) GST-pulldown binding assays of 35S-Med19 to immobilized GST-Hox fusions containing full length or protein fragments (below each lane: rectangles represent the entire protein; portions present in GST-Hox chimeric proteins are black, except the HD, represented in red). (B–D) BiFC assays were carried out co-expressing Med19-VC with VN-Ubx (B), VN-Dfd (C) or VN-AbdA (D), from UAS constructs under engrailed-Gal4 control (en>). Med19-VC accumulation, detected with antibody against the GFP C-terminal region, is similar in all tests (B′–D′). Gal4-driven Hox protein accumulation is comparable to endogenous, as detected with Ubx, Dfd and AbdA specific antibodies (B″–D″). Relative BiFC fluorescent signals were quantified as in . VN-Ubx signal (B) and VN-Dfd (C) yielded serial rows of nuclear fluorescence; VN-AbdA (D) gave no detectable signal. (E) Direct homeodomain binding to Med19. Pulldowns with immobilized GST or GST-Med19 employed 70 aa-long 35S-labelled peptides centered on the HDs of Antp, Dfd, Ubx, AbdB, En and Hth. (F–I) Direct homeodomain binding to Med19 in BiFC assay. Co-expression of Med19-VC with VN-Ubx (F), or with its HD (VN-HDUbx; G), under Ubx-Gal4 control gives indistinguishable BiFC signals. Expression of Med19-VC under abdA-Gal4 control yielded no fluorescence with VN-AbdA (H) but gave a strong signal with VN-HDAbdA alone (I).
Figure 2
Figure 2. Med19 mutations affect cell viability and Hox-related developmental processes.
(A) Mutant alleles were generated by imprecise excision of a viable P element insertion 37 bp upstream of the putative Med19 transcription initiation site (19P). Med191 is a pupal-lethal hypomorph with 14 bp deleted 5′ to the Med19 transcription start. Med192 is an embryonic lethal amorph deleted for 1174 bp of DNA spanning exon 1 with its ATG initiation codon. (B–D) Clonal analyses of Med192. (B) “Twin spot” analysis. Mitotic recombination induced in hsp70-Flp; Med192 FRT-2A/Ub-GFP FRT-2A larvae (30′ heat shock at 38°C) gave +/+ clones (intense green), but no −/− sister clones (GFP-) were observed. (C) Twin spot analysis in rescue conditions. The engrailed-Gal4 driver was used to simultaneously induce mitotic clones (UAS-Flp) and to direct expression of Med19-VC (UAS transgene). Homozygous Med192/2 (−/−) cells (lacking GFP) are now detected. (D) The Med19 − condition is not intrinsically cell-lethal. In this wing imaginal disc (genotype, en-Gal4>UAS-Flp/+; Med192 FRT-2A/Ub-GFP M− FRT-2A), GFP- Med19 −/− clones are observed. (E–J) Med19 function is required for multiple Hox-related developmental processes. (E,F) Med19 is required for eversion of anterior pupal spiracles. Normal anterior spiracles (E) are absent from Med191 /2 hypomorphs (F). (G,H) One maxillary palp (G, arrow) is absent in a surviving Ub-Med19; Med192/2 hypomorph (H, arrow). (I,J) Med19 is required for haltere-specific sensory organs. (I) Wild-type haltere, with zone of interest (dotted box) showing rows of pedicellar sensillae on the wild-type dorsal haltere (enlargement, I′). (J) Haltere harboring Med19 −/− clones (genotype: ap>Flp; Ub-GFP M− FRT-2A/Med192 FRT-2A), with zone of interest in dotted box indicating disorganized sensory organ rows (J′).
Figure 3
Figure 3. Synergistic interactions between Med19 and Hox mutations.
Dose-sensitivity for Med19 was tested relative to Hox gain-of-function mutations of Antp (A–C), Dfd (D–F), and Ubx (G–I). (A) Wild-type antenna, with distal arista (ar) indicated by an arrowhead; (B) AntpNs–directed transformation of antenna toward leg with distal claw (cl, arrowhead); (C) the transformation is attenuated in AntpNs/Med192 trans-heterozygotes, as shown by the presence of a partial arista (ar, arrowhead). (D) Wild-type head, with the maxillary palp (Mx) indicated by arrowhead. (E) Dfd1 provokes head defects including reduced eyes and the appearance of ectopic Mx (arrowhead), here positioned behind the antenna. (F) In Dfd1/Med192 heterozygotes (or here, Dfd1Med192/+ Med19P), no ectopic Mx were observed. (G) Wild-type wing. (H) Homozygote for the UbxCbx1 gof allele that expressed Ubx protein in the posterior compartment of the wing. Note the discrete hemi-haltere induced by Ubx, which is oriented at right-angles relative to the longitudinal wing axis. (I) In UbxCbx1 Med192/UbxCbx1 Med19P wings, the posterior wing is no longer organized as a hemi-haltere, and the cellular trichomes are reoriented toward the long wing axis (arrow).
Figure 4
Figure 4. Med19 acts as a “co-factor” for Ubx-mediated gene activation.
(A) Summary of Ubx target genes analyzed. Ubx can repress (red bars) or activate (green arrows) direct target genes. (B–H) Med19 function in Ubx target gene expression. Images show whole haltere imaginal discs that are wild-type (C), or bear mitotic clones of Med192 in a Minute background (B, D, F, H) or of Ubx1 (E,G). The other columns contain enlargements of boxed images in the first column, showing genotypic markers to identify clones (B′–H′); expression of gene of interest (B″–H″); and merged images (B′″–H′″). (B–B′″) Ubx protein (red) accumulates normally in a Med19 −/− clone (GFP-). (C–C′″) edge-GFP reporter gene expression (green) is localized in a row of cells at the posterior border of the disc; ubiquitous expression of wild-type Med19 is revealed by anti-Med19 (red). (D–D′″) edge-GFP expression (green) is absent in a Med19 −/− clone (absence of anti-Med19, red; circled) crossing the line of edge-expressing cells. (E–E′″) Bab2 protein (anti-Bab2, red) is absent from Ubx1/1 cells (GFP-). (F–F′″) Bab2 expression (red) is cell-autonomously down-regulated in Med19 −/− cells (GFP-). (G–G′″) spalt (sal) expression (anti-Sal, red) appears in centrally positioned cells of Ubx−/− clones (GFP-) in the haltere pouch. (H) Spalt (red) is not de-repressed in Med19 mutant cells (GFP-) positioned as for the Ubx clone (G′).
Figure 5
Figure 5. Direct HD binding through a conserved 70 a.a. Med19 homeodomain-interacting motif (HIM).
(A) HD binding involves a 70 a.a. region of Med19. 35S-labelled full-length (construct #1) or deleted versions (#2–7) of Med19 were used to probe immobilized GST or GST-HDAntp. Proteins containing a.a. 159–229 bound GST-HD (#1, 2, 7). Deleting the entire interval (#4) or of a 40 a.a. interval from 190–229 (#6) abolished binding. Deleting the N-terminal 14 aa of this region (160–173) resulted in reduced binding (#5). The 70 a.a. HIM peptide (160–229) bound GST-HD (#7). (B) Co-immunoprecipitations. Transfected S2 cells contained pActin-Gal4 driver with pUAS-Ubx-HA and either pUAS-Med19-VC or pUAS-Med19ΔHIM-VC. Negative controls were cells transfected with pAct5C-V5. Inputs represent 2% of extracts used for the IP. Cell extracts were immunoprecipitated with mouse anti-GFP sera that recognises the VC tag, then analysed by Western blots. In the upper portion, bands were revealed with guinea pig anti-Med19, while in the lower portion, a duplicate blot was stained using rabbit anti-HA sera. Solid arrowheads indicate identified proteins of interest and “*”, a non-specific signal serving as an internal loading control. As the HIM motif and VC tag are of equal size, endogenous Med19 and ΔHIM-VC migrate at the same position. (C,D) BiFC test, co-expressing VN-HDUbx with Med19-VC, Med19ΔHIM-VC or HIM-VC in the wing imaginal disc from UAS constructs under dpp-Gal4 control. (C) The BiFC signal observed for VN-HDUbx with Med19-VC was higher for HIM-VC while it was reduced to background levels with Med19ΔHIM-VC. (D) Quantification of BiFC fluorescent signals.
Figure 6
Figure 6. Med19 HIM is required for Ubx target gene activation, but not for cell proliferation/survival.
Med19-VC (A) or Med19ΔHIM-VC (B) proteins were expressed (UAS constructs, en-Gal4) in posterior haltere imaginal discs harboring Med19 mutant clones. Med19-VC or Med19ΔHIM-VC were detected with antisera directed against C-terminal GFP (αVC, blue). (A–A′″, B–B′″) Med19 clones were identified using a ubiquitous RFP marker: −/− (no red), +/− (red), +/+ (intense red). Activation of the Ubx target edge-GFP at the posterior haltere edge was visualized by GFP (green). Regions containing Med19− /− clones of interest are enlarged (A′–A′″, B′–B′″). (A–A′″) Med19-VC restored expression of edge-GFP. (B–B′″) Med19ΔHIM-VC failed to rescue edge-GFP activation here. GFP-expression here is limited to a single wild-type cell that abuts the −/− clone (B′, B″, arrow). (C) Three levels of edge-GFP expression could be discerned: normal, present but reduced, or none. All correctly positioned −/− clones with Med19-VC showed GFP expression (11 of 11) of which 9/11 were normal. Most clones possessing Med19ΔHIM-VC showed no GFP (7 of 11), and only two of 11 clones showed normal expression.
Figure 7
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 α-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.
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Grants and funding

This work was supported by recurring support from Centre National de Recherche Scientifique (CNRS) and from the Université Paul Sabatier, a graduate fellowship from the Ligue Nationale contre le Cancer (attributed to CI), and by grants from the Association pour la Recherche sur le Cancer (ARC; http://www.arc-cancer.net/) and the Agence Nationale de Recherche (ANR NT05-3-42540; http://www.agence-nationale-recherche.fr/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.