Col direct control of <i>ap</i> expression in Ap neurons.

<p>(A) Annotation of the Col peak in <i>ap</i>, same representation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133387#pone.0133387.g002" target="_blank">Fig 2A</a>; 35.8 kb of the <i>ap</i> genomic region are shown (Chr2R: 1.593.000–1.628.800); the previously described apC enhancer is represented by a blue box. (B) <i>ap</i>_Col (GFP) expression in the dAp (yellow arrow) and Tv1-Tv4 neurons (white arrow) in stage 15 embryos, ventral view. (C) <i>ap</i>_Col^mut expression is severely reduced in dAP neurons and Tv neurons. (D,D’) Close up view of 4 segments of stage 16 embryos, showing the specific overlap between Col (red) and <i>ap</i>_Col (green) in the Tv1 and dAp neurons. (E) all Tv neurons express <i>ap</i>_Col and Eya. (F,G) <i>ap_</i>Col^mut expression is lost in dAp and strongly reduced in Tv neurons.</p

Cross-regulation between <i>eya</i> and <i>col</i> is required to specify dorso-lateral muscles.

<p>(A) <i>eya_</i>Col (GFP) expression in the DA3 muscle (arrows) in stage 15 embryos, is not detected for <i>eya_</i>Col^mut (B). (C-E) Triple staining of st11 wt (C), <i>+;col</i>^<i>eCRM</i><i>-lacZ</i> (D) and <i>col</i>^<i>1</i><i>;col</i>^<i>eCRM</i><i>-lacZ</i> (E) embryos for Nau (blue), <i>eya</i> transcripts (red), and either Col (C) or β-galactosidase (D,E) (green), shows co-expression of Col, <i>col</i>^e-CRM-LacZ, Nau and <i>eya</i> in DL muscle PCs (white arrow). (C’-E’) only Nau and <i>eya</i> stainings are shown. <i>eya</i> transcription is specifically lost in DL PCs in <i>col</i>^<i>1</i> mutant embryos (E,E’); dorso-lateral view of the T2 and T3 segments is shown. (F) Schematic drawing of the dorsal, dorso-lateral and lateral transverse muscles in a stage 16 wt embryo, with the DA3 muscle in red and the LL1 muscle indicated by an arrow. (G, H) Staining of stage 16 wt (G) and (H), <i>eya</i>^{<i>cIi-IID</i>}<i>/Df(2L)BSC354</i> (null) mutant embryos for Col (red) and β3-tubuliin (green). Lateral view of 3 segments. In absence of <i>eya</i>, Col expression is lost in most segments, the LL1 muscle is missing (white arrow) and the DA3 muscle (asterisk) malformed. White brackets indicate dorsal, unaffected muscles while the lateral transverse and ventral muscles (yellow brackets) are moderately affected. (I-J) Col immunostaining of st.16 wt (I), and (J) <i>eya</i> mutant embryos, showing the loss of Col muscle expression in most segments (see also G, H), in <i>eya</i> mutants. (K, L) promuscular Col expression, early stage 11, is reduced in <i>eya</i> mutant embryos (L), compared to wt (K).</p

Scheme for Col direct regulation of <i>ap</i> and <i>eya</i> in specific neuron and muscle lineages.

<p>Col directly controls <i>ap</i>_Col and <i>eya</i>_Col CRM activity in the Tv and dAP neurons and <i>eya</i>_Col and <i>col</i>_Col CRM in the DA3 muscle lineage. The two Col binding sites in <i>eya</i>_Col CRM are not functionally equivalent (double arrow). <i>eya</i> and <i>col</i> cross-regulate each other in the DA3 PC. Control of <i>DopR</i> and <i>Nplp1</i> expression in Tv and dAP neurons (Baumgardt et al., 2007) could be indirect.</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

Col direct control of <i>eya</i> expression in Ap neurons.

<p>(A) Annotation of the Col peaks in <i>eya</i>, same representation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133387#pone.0133387.g002" target="_blank">Fig 2A</a>; 24 kb of the <i>eya</i> genomic region are shown (Chr2L: 6.524.500–6.548.500); the summits of the two ChIP-Col peaks are numbered 1 and 2. (B, C) <i>eya_</i>Col (GFP) expression in the dAp (yellow arrow) and Tv1-Tv4 neurons (white arrow) in stage 16 embryos, ventral view. (C) Close up view of abdominal segments, showing the specific overlap between Col (red) and <i>eya_</i>Col (green) in the dAp (yellow arrow) and Tv1 neurons (white arrow). (D, E) <i>eya_</i>Col^mut expression is lost in dAP neurons and reduced in TV neurons. (F, G) Mutation of the Col binding site 2 (G), but not site 1 (F) eliminates <i>eya_</i>Col (RFP) expression in dAp neurons (yellow arrow).</p

Col control of <i>cnc</i> and <i>Ama</i> expression in the head.

<p>(A) Annotation of the Col peak in <i>cnc</i>, adapted from <i>Gene Browser</i> (GEO submission GSE67805). 39,5 kb of <i>cnc</i> genomic region are shown (Chr3R: 19.009.000–19.048.500) with the Flybase gene annotation indicated by bars (transcribed regions) and intervening blue lines (introns). Black arrows indicate the direction of transcription of <i>cnc</i> and <i>fuzzy onions</i> (<i>fzo</i>), <i>inwardly rectifying potassium channel 1</i> (<i>Irk1</i>). The <i>cnc</i> transcripts coding for the protein isoforms CncA and CncB are indicated. ChIP-seq data for Col (green) substracted from HA (mock) data (red) are shown on the bottom. The Col Dam-ID binding regions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133387#pone.0133387.ref059" target="_blank">59</a>] are indicated by yellow bars, top line. The summit of the ChIP-Col peak identified by SISSRs and position of the Col binding site(s) identified by MEME are indicated by blue and violet lines, respectively; the position of <i>cnc_</i>Col is represented by a black box; scale is indicated. (B-D) Ventral anterior views of stage 11 embryos. (B) Overlap between <i>cnc_</i>Col (GFP, green) and Col (red) expression in the HL (white arrow). (C) <i>cnc_</i>Col and (D) <i>cnc_</i>Col^mut mRNA expression, showing down-regulation of <i>cnc_</i>Col^mut in the mandibular segment (open arrow). (E,F) <i>Ama</i>_Col, (G,H) <i>Ama</i>_Col^mut expression in stage 10 (E,G) and 11 (F,H) embryos. HL <i>Ama</i>_Col expression (arrow) is lost in <i>Ama</i>_Col^mut (open arrow). (I, J) Overlap between <i>Ama</i> (red), Col (blue), and <i>col</i>2.6–0.9moeGFP (green) expression in the HL (white arrow) in stage 11 wt (I), and <i>col</i>^<i>1</i> mutant embryos (J). Separate signals for <i>Ama</i>, GFP (I, J) and Col (inset in I, left panel) are shown in black and white. <i>Ama</i> expression is specifically lost in the HL in <i>col</i>^<i>1</i> mutants. The asterisk in C, D, F, H, J indicates <i>Ama</i> and <i>cnc</i> expression independent on Col.</p

Candidate Col direct targets and linked CRMs.

<p>n.d.: not detected</p><p>Candidate Col direct targets and linked CRMs.</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

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