Model for the role of Med19 at the interface of Hox and MED.

<p>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.</p

Hox proteins bind Med19 through their homeodomains <i>in vitro</i> and <i>in vivo</i>.

<p>(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 <i>engrailed</i>-Gal4 control (<i>en</i>>). 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004303#pgen.1004303-Hudry1" target="_blank">[32]</a>. 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 <i>Ubx</i>-Gal4 control gives indistinguishable BiFC signals. Expression of Med19-VC under <i>abdA</i>-Gal4 control yielded no fluorescence with VN-AbdA (H) but gave a strong signal with VN-HDAbdA alone (I).</p

Direct HD binding through a conserved 70 a.a. Med19 homeodomain-interacting motif (HIM).

<p>(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 <i>dpp</i>-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.</p

Synergistic interactions between <i>Med19</i> and Hox mutations.

<p>Dose-sensitivity for <i>Med19</i> was tested relative to Hox gain-of-function mutations of <i>Antp</i> (A–C), <i>Dfd</i> (D–F), and <i>Ubx</i> (G–I). (A) Wild-type antenna, with distal arista (ar) indicated by an arrowhead; (B) <i>Antp^Ns</i>–directed transformation of antenna toward leg with distal claw (cl, arrowhead); (C) the transformation is attenuated in <i>Antp^Ns</i>/<i>Med19²</i> 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) <i>Dfd¹</i> provokes head defects including reduced eyes and the appearance of ectopic Mx (arrowhead), here positioned behind the antenna. (F) In <i>Dfd¹</i>/<i>Med19²</i> heterozygotes (or here, <i>Dfd¹Med19²</i>/+ <i>Med19^P</i>), no ectopic Mx were observed. (G) Wild-type wing. (H) Homozygote for the <i>Ubx^Cbx1</i> 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 <i>Ubx^Cbx1 Med19²</i>/<i>Ubx^Cbx1 Med19^P</i> wings, the posterior wing is no longer organized as a hemi-haltere, and the cellular trichomes are reoriented toward the long wing axis (arrow).</p

<i>Med19</i> mutations affect cell viability and Hox-related developmental processes.

<p>(A) Mutant alleles were generated by imprecise excision of a viable <i>P</i> element insertion 37 bp upstream of the putative <i>Med19</i> transcription initiation site (<i>19P</i>). <i>Med19¹</i> is a pupal-lethal hypomorph with 14 bp deleted 5′ to the <i>Med19</i> transcription start. <i>Med19²</i> is an embryonic lethal amorph deleted for 1174 bp of DNA spanning exon 1 with its ATG initiation codon. (B–D) Clonal analyses of <i>Med19²</i>. (B) “Twin spot” analysis. Mitotic recombination induced in <i>hsp70</i>-Flp; <i>Med19²</i> 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 <i>Med19^2/2</i> (−/−) cells (lacking GFP) are now detected. (D) The <i>Med19</i>⁻ condition is not intrinsically cell-lethal. In this wing imaginal disc (genotype, <i>en-</i>Gal4>UAS-Flp/+; <i>Med19²</i> FRT-2A/Ub-GFP <i>M⁻</i> FRT-2A), GFP- <i>Med19</i>^−/− clones are observed. (E–J) <i>Med19</i> function is required for multiple Hox-related developmental processes. (E,F) <i>Med19</i> is required for eversion of anterior pupal spiracles. Normal anterior spiracles (E) are absent from <i>Med19¹</i>^/<i>2</i> hypomorphs (F). (G,H) One maxillary palp (G, arrow) is absent in a surviving Ub-Med19; <i>Med19^2/2</i> hypomorph (H, arrow). (I,J) <i>Med19</i> 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 <i>Med19</i>^−/− clones (genotype: <i>ap</i>>Flp; Ub-GFP <i>M⁻</i> FRT-2A/<i>Med19²</i> FRT-2A), with zone of interest in dotted box indicating disorganized sensory organ rows (J′).</p