Distinct molecular strategies for Hox-mediated limb suppression in Drosophila: from cooperativity to dispensability/antagonism in TALE partnership

International audienceThe emergence following gene duplication of a large repertoire of Hox paralogue proteins underlies the importance taken by Hox proteins in controlling animal body plans in development and evolution. Sequence divergence of paralogous proteins accounts for functional specialization, promoting axial morphological diversification in bilaterian animals. Yet functionally specialized paralogous Hox proteins also continue performing ancient common functions. In this study, we investigate how highly divergent Hox proteins perform an identical function. This was achieved by comparing in Drosophila the mode of limb suppression by the central (Ultrabithorax and AbdominalA) and posterior class (AbdominalB) Hox proteins. Results highlight that Hox-mediated limb suppression relies on distinct modes of DNA binding and a distinct use of TALE cofactors. Control of common functions by divergent Hox proteins, at least in the case studied, relies on evolving novel molecular properties. Thus, changes in protein sequences not only provide the driving force for functional specialization of Hox paralogue proteins, but also provide means to perform common ancient functions in distinct ways

A study into the regulation of the transcriptional activity of Abdominal-A

Les gènes Hox codent des facteurs de transcription à homéodomain (HD). Bien que ce dernier reconnaisse des séquences similaires in vitro, les protéines Hox achèvent des fonctions hautement spécifiques in vivo. Des séquences protéiques en dehors de l’HD influencent la spécificité d’action des protéines Hox par le recrutement de cofacteurs, dont le mieux caractérisé est Extradenticle (Exd) chez la drosophile. Des travaux récents au sein de notre équipe ont démontré la contribution fonctionnelle de trois motifs de AbdA, aussi bien dans des fonctions Exd-dépendantes qu’à des fonctions Exd-indépendantes. Mon travail de thèse a porté sur la caractérisation de la contribution des motifs protéiques de AbdA dans la sélection puis dans la régulation des gènes cibles en utilisant une approche combinée ChIPseq/RNAseq, dans un contexte Exd-indépendant. Le code ADN identifié nous a renseigné sur la présence d’inputs transcriptionnels additionnels. Ces derniers correspondant à des facteurs de transcription déjà connus, leur présence dans un complexe protéique avec AbdA a été démontrée par des analyses de spectrométrie de masse. Un second volet de mon travail de thèse a été l’identification de modifications post-traductionnelles pouvant rendre compte d’un mécanisme de régulation de l’activité des protéines Hox. Des analyses prédictives in silico, confirmées par des approches biochimiques et des analyses in vivo ont démontré la SUMOylation de AbdA. Ces résultats préliminaires posent les bases pour des travaux futures qui auront pour objectif d’identifier les résidus d’AbdA SUMOylés et d’élucider le rôle de cette modification dans la régulation de l’activité de la protéine AbdA.Hox genes encode homeodomain-containing transcription factors (HD). Although the HD binds to similar DNA sequences in vitro, Hox proteins display a high functional specificity in vivo. Protein motifs outside of the HD influence Hox specificity through recruiting additional cofactors, with the best characterized being Extradenticle (Exd in Drosophila). Recent evidence from our group has uncovered the functional contribution of AbdA intrinsic motifs to AbdA Exd-dependent functions as well as AbdA Exd-independent functions. My PhD work has aimed to characterize the contribution of AbdA motifs to target gene selection and regulation using a combined approach of ChIPseq/RNAseq in an Exd-independent context. The DNA code identified provides us with new insights about additional transcriptional inputs from additional DNA-binding proteins lying in the vicinity of AbdA recognition sites. Mass spectrometry analysis establishes the occurrence of these additional DNA binding proteins in a multi-protein complex with AbdA. Deciphering the involvement of post-translational modifications in the regulation of Hox protein activity was another aspect of my PhD work. In silico predictive analysis, followed by biochemical approaches and in vivo assays reveal the potential for SUMOylation of AbdA as a potentially important regulatory component of AbdA activity. These preliminary results set the bases for further work aimed at identifying SUMOylated residues on AbdA and the functional relevance of such post-translational modification on AbdA activity regulation

Imaging of native transcription and transcriptional dynamics in vivo using a tagged Argonaute protein

International audienceA flexible method to image unmodified transcripts and transcription in vivo would be a valuable tool to understand the regulation and dynamics of transcription. Here, we present a novel approach to follow native transcription, with fluorescence microscopy, in live C. elegans. By using the fluorescently tagged Argonaute protein NRDE-3, programmed by exposure to defined dsRNA to bind to nascent transcripts of the gene of interest, we demonstrate transcript labelling of multiple genes, at the transcription site and in the cytoplasm. This flexible approach does not require genetic manipulation, and can be easily scaled up by relying on whole-genome dsRNA libraries. We apply this method to image the transcriptional dynamics of the heat-shock inducible gene hsp-4 (a member of the hsp70 family), as well as two transcription factors: ttx-3 (a LHX2/9 orthologue) in embryos, and hlh-1 (a MyoD orthologue) in larvae, respectively involved in neuronal and muscle development

Cell-cycle regulation of non-enzymatic functions of the Drosophila methyltransferase PR-Set7

International audienceTight cell-cycle regulation of the histone H4-K20 methyltransferase PR-Set7 is essential for the maintenance of genome integrity. In mammals, this mainly involves the interaction of PR-Set7 with the replication factor PCNA, which triggers the degradation of the enzyme by the CRL4CDT2 E3 ubiquitin ligase. PR-Set7 is also targeted by the SCFβ-TRCP ligase, but the role of this additional regulatory pathway remains unclear. Here, we show that Drosophila PR-Set7 undergoes a cell-cycle proteolytic regulation, independently of its interaction with PCNA. Instead, Slimb, the ortholog of β-TRCP, is specifically required for the degradation of the nuclear pool of PR-Set7 prior to S phase. Consequently, inactivation of Slimb leads to nuclear accumulation of PR-Set7, which triggers aberrant chromatin compaction and G1/S arrest. Strikingly, these phenotypes result from non-enzymatic PR-Set7 functions that prevent proper histone H4 acetylation independently of H4K20 methylation. Altogether, these results identify the Slimb-mediated PR-Set7 proteolysis as a new critical regulatory mechanism required for proper interphase chromatin organization at G1/S transition

DMX-R cis sequence requirements for repression by Ubx/AbdA and AbdB.

<p>Schematic representation of cis sequence requirements for DMX repressive activity in A1 (Ubx/AbdA- mediated, blue) and A8 (AbdB-mediated, red) segments. 100% derepresion was defined by the level of abdominal DMX derepression in embryos fully deficient for Ubx, AbdA and AbdB m and r isoforms (<i>Df P9</i>). Cis sequence requirement was evaluated by quantifying the levels of derepression of 18 mutated forms of DMX-R (see Figures S5 and S6). These scanning mutations (altering simultaneously two to 5 nucleotide positions) cover 42 of the 57 nucleotide positions of the DMX-R element. Sequence is annotated according to transcription factor binding site (Slp, Exd, Hth, En and Hox) allocation from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307-Gebelein2" target="_blank">[20]</a>.</p

Dispensability of Exd/Hth cofactors for AbdB-mediated DMX posterior repression in posterior segments.

<p>A) Embryos co-stained for Hth (red) and AbdB (green). Arrowheads point to segments A1, A3 and A8. wild type embryo, decrease of Hth expression is seen from segment A3 reaching almost undetectable levels in A8. B) EMSA of DMX-R (containing binding sites Slp,Hox1, Exd, En, Hth and Hox2) with AbdB and increasing amounts of Exd, Hth, En, or En and with combined increasing amounts of Exd, Hth and En. The amount of AbdB remains constant whenever present, except in the last lane (depicted by a thin pink line) where 1/3 of this quantity was used. Exd-Hth-En/DNA and AbdB/DNA complexes are highlighted by arrows. C) Quantification of AbdB binding in EMSA to DIIR (containing binding sites Hox1, Exd, En, and Hth) mutated in the Exd (DIIR<i>^exd</i>) or Hth (DIIR<i>^Hth</i>) binding sites in the presence of AbdB alone, with Exd or Exd and Hth (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307.s004" target="_blank">Figure S4</a>). Note that mutation of the Exd binding site affect the formation of AbdB/DNA complexes. For the ease of comparison, AbdB binding to DIIR<i>^exd</i> and DIIR<i>^Hth</i> have been arbitrarily set to 100%, allowing assessing the effect of Hth and Hth/Exd inhibitory effects independently off the effect of binding site mutations on AbdB/DNA complex assembly. D) Quantification of AbdB binding in EMSA to DIIR with various combinations of AbdB, Exd, Hth and truncated HM (HD less) form of Hth (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307.s004" target="_blank">Figure S4</a>). Note that the Exd-mediated release of inhibitory effect seen for full length Hth is lost with the truncated Hth HM protein.</p

Protein sequence requirements for AbdB-mediated DME repression.

<p>Sequence conservation in the AbdB HD (shown only for the N-terminal arm and helix 3) and HD flanking regions HX/LR and C-ter region. Sequences upstream and downstream of these regions display progressively weaker conservation. Web logo was obtained using sequences the following AbdB sequences (Drosophila (AAA84402), Tribolium (AAF36721.1), Anophela (XM311628), Sacculine (AAQ49317.1), Folsomia (AAK52499.1) and human (BCO10023)). The <i>Drosophila melanogaster</i> sequence is shown below the web logo. The position and the nature of the mutations generated are represented below the web logo. Effects of the mutations on the repressive activity of AbdB on DME are displayed in a box plot representation. While mutations in the HX/LR region have little effect on AbdB repressive activity, mutations within the HD, including the N-terminal arm, helix 3 and the Cter alter to different extent AbdB repressive potential. Illustration of data is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307.s010" target="_blank">Figure S10</a>.</p

Requirement of protein domains in AbdB/Ubx chimeric proteins for DME repression.

<p>Left part of the figure depicts wild type and mutated variants of Ubx (blue), including mutations (indicated by crosses) in the HX, and UbdA (UA) domains as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307-Merabet2" target="_blank">[15]</a> and in helix 3 (Q50 to K50) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307-Chan1" target="_blank">[55]</a>. AbdB protein sequences, including or not the QR domain, either with a wild type or mutated helix 3, is represented in red. Effects of the mutations on the repressive activity of AbdB on DME are displayed in a box plot representation. Switching the Ubx HD by that of AbdB endows the chimera with a posterior AbdB like dependent mode of DME repression. Illustration of data is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307.s011" target="_blank">Figure S11</a>.</p

AbdB m and r isoforms repress <i>Dll</i> in the posterior abdominal segments A8 and A9.

<p>(A) Embryo stained for β-gal driven by the DMX enhancer (red) showing restricted thoracic activity. (B–C) DMX(X2X5) embryos co-stained for β-gal (red) and AbdB (green). This modified DMX enhancer is derepressed in all abdominal segments, including A8 and A9. Co-localization of β-gal and AbdB in A8 and A9 cells normally subject to enhancer activity repression is highlighted in the magnified view. (D–E) Embryo lacking Ubx and AbdA function (<i>Df(109)</i>) shows DMX abdominal de-repression (red) up to A7 (arrowhead) and remains repressed in A8 and A9 segments where AbdB is expressed at high levels (green). (F–G) Embryo lacking Ubx, AbdA and the AbdBm isoform, but retaining the AbdB r isoform, shows derepression of DMX (red) till A8 (arrowhead). (H–I) Embryos lacking Ubx, AbdA and the AbdB m and r isoforms (<i>Df P9</i>) show DMX de-repression (red) in all abdominal segments, including in A9 (arrowhead). (J–K) <i>prd-Gal4</i> driven ectopic expression of the AbdBm isoform represses DMX activity (arrow in T2). (L–M) <i>prd-Gal4</i> driven ectopic expression of the AbdBr isoform represses DMX activity (arrow in T2).</p

Models for distinct Hox cofactor partnership for <i>Dll</i> repression.

<p>(A) Model for repression of <i>Dll</i> by Ubx/AbdA in anterior abdominal segments A1-7. Repression relies on the assembly of a Hox/Exd/Hth protein complex <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen.1003307-Gebelein2" target="_blank">[20]</a>. DNA binding by Hox proteins is not essential (depicted by a dashed delineated pink zone of contact between the Hox protein and the DNA), as supported by the limited loss of repressive activity of a DNA binding deficient Ubx protein (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003307#pgen-1003307-g007" target="_blank">Figure 7</a>), and by the limited dereprepression associated to mutation in Hox binding sites. The non-essential character of Hox DNA binding may result from acting in a context of a multiprotein complex containing two additional DNA binding proteins (Exd and Hth). (B) Model for repression of <i>Dll</i> by AbdB in posterior abdominal segments in A8-9. AbdB represses Exd and Hth, and consequently act without the aid of Exd and Hth to repress <i>Dll</i>. This difference likely imposes a strict requirement for AbdB DNA binding for efficient repression.</p