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Hox proteins display a common and ancestral ability to diversify their interaction mode with the PBC class cofactors.

Hudry B, Remacle S, Delfini MC, Rezsohazy R, Graba Y, Merabet S - PLoS Biol. (2012)

Bottom Line: This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale.We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis.Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie du Développement de Marseille Luminy, IBDML, UMR7288, CNRS, AMU, Parc Scientifique de Luminy, Case 907, Marseille, France.

ABSTRACT
Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro analyses have established that the formation of Hox/PBC complexes relies on a short conserved Hox protein motif called the hexapeptide (HX). This paradigm is at the basis of the vast majority of experimental approaches dedicated to the study of Hox protein function. Here we questioned the unique and general use of the HX for PBC recruitment by using the Bimolecular Fluorescence Complementation (BiFC) assay. This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale. We found that the HX is dispensable for PBC recruitment in the majority of investigated Drosophila and mouse Hox proteins. We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis. Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins. Altogether, our results highlight that flexibility in Hox-PBC interactions is an ancestral and evolutionary conserved character, which has strong implications for the understanding of Hox protein functions during normal development and pathologic processes.

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Meis class proteins cannot reveal HX-independent interaction modes in the absence of DNA-binding.(A) Scheme of the short HD-less isoform of Hth, which contains only the Exd interaction domain (HM). This form was fused to the HA tag (not schematized). (B) EMSAs with wild type or HX-mutated forms of Drosophila Hox proteins and Exd and the HD-less isoform of Hth on the Dllcon probe, as indicated. For Hox proteins making a dimeric complex with Exd (Lab, Scr and Antp), an anti-HA raised against Hth-HM was also used to validate the presence of the trimeric complex. In all cases, the HX mutation abolishes trimeric complex formation with Exd and Hth-HM. (C) EMSAs with wild type or HX-mutated forms of central mouse Hox proteins and Pbx1 and the HD-mutated form of Meis1a on the ant/cent nucleotide probe, as indicated. The HD mutation abolishes DNA-binding [48], as exemplified by the absence of DNA-bound Pbx/Meis complexes (red asterisk). This mutation also abolishes (for Hoxb6 and Hoxb7) or drastically affects (for Hoxb8) trimeric complex formation with HX-mutated Hox proteins. (D) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS nucleotide probe, as indicated. On this probe, the HX-mutated form of Hoxb8 is not able to form any dimeric or trimeric complex with Pbx1 or Pbx1/Meis1, respectively. (E) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS probe containing a Meis binding site, as indicated. On this probe, the HX-mutated form of Hoxb8 is able to form trimeric complexes with Pbx1/Meis1. An anti-HA against the HA tag of Hoxb8 was added in the last reaction to confirm the presence of trimeric complexes. Note that Pbx1 is able to bind the two TGAT binding sites of PRS (gray arrowheads). For all gels, colored bars and marks are symbolized as in Figure 3. See also Figure S9.
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pbio-1001351-g005: Meis class proteins cannot reveal HX-independent interaction modes in the absence of DNA-binding.(A) Scheme of the short HD-less isoform of Hth, which contains only the Exd interaction domain (HM). This form was fused to the HA tag (not schematized). (B) EMSAs with wild type or HX-mutated forms of Drosophila Hox proteins and Exd and the HD-less isoform of Hth on the Dllcon probe, as indicated. For Hox proteins making a dimeric complex with Exd (Lab, Scr and Antp), an anti-HA raised against Hth-HM was also used to validate the presence of the trimeric complex. In all cases, the HX mutation abolishes trimeric complex formation with Exd and Hth-HM. (C) EMSAs with wild type or HX-mutated forms of central mouse Hox proteins and Pbx1 and the HD-mutated form of Meis1a on the ant/cent nucleotide probe, as indicated. The HD mutation abolishes DNA-binding [48], as exemplified by the absence of DNA-bound Pbx/Meis complexes (red asterisk). This mutation also abolishes (for Hoxb6 and Hoxb7) or drastically affects (for Hoxb8) trimeric complex formation with HX-mutated Hox proteins. (D) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS nucleotide probe, as indicated. On this probe, the HX-mutated form of Hoxb8 is not able to form any dimeric or trimeric complex with Pbx1 or Pbx1/Meis1, respectively. (E) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS probe containing a Meis binding site, as indicated. On this probe, the HX-mutated form of Hoxb8 is able to form trimeric complexes with Pbx1/Meis1. An anti-HA against the HA tag of Hoxb8 was added in the last reaction to confirm the presence of trimeric complexes. Note that Pbx1 is able to bind the two TGAT binding sites of PRS (gray arrowheads). For all gels, colored bars and marks are symbolized as in Figure 3. See also Figure S9.

Mentions: The influence of the topology of the Hox/PBC/Meis binding sites suggest that binding of Meis proteins to DNA is a prerequisite for Meis-mediated uncovering of alternative Hox-PBC interaction modes. To investigate this more directly, we repeated EMSAs with DNA binding deficient Meis proteins. In the case of Drosophila proteins, we used a naturally HD-less isoform of Hth (Figure 5A), which contains only the evolutionary conserved HM domain mediating the direct interaction with Exd [49]. EMSAs were performed on Dllcon, which allowed assessing Hth-mediated uncovering of HX dispensability for Exd recruitment by Lab, Scr, Antp, and AbdB. The role of Hth-HM was not analyzed for Ubx and AbdA since these two proteins do not require Hth for establishing HX-independent interactions with Exd (Figure 3A). We observed that the HD-less isoform of Hth is able to form trimeric complexes in the context of wild type Hox proteins (Figure 5B). The HX mutation, however, abolishes the formation of the trimeric complex in all cases (Figure 5B), highlighting that Hth is not able to promote HX-independent interaction modes when it is not binding to Dllcon sequences.


Hox proteins display a common and ancestral ability to diversify their interaction mode with the PBC class cofactors.

Hudry B, Remacle S, Delfini MC, Rezsohazy R, Graba Y, Merabet S - PLoS Biol. (2012)

Meis class proteins cannot reveal HX-independent interaction modes in the absence of DNA-binding.(A) Scheme of the short HD-less isoform of Hth, which contains only the Exd interaction domain (HM). This form was fused to the HA tag (not schematized). (B) EMSAs with wild type or HX-mutated forms of Drosophila Hox proteins and Exd and the HD-less isoform of Hth on the Dllcon probe, as indicated. For Hox proteins making a dimeric complex with Exd (Lab, Scr and Antp), an anti-HA raised against Hth-HM was also used to validate the presence of the trimeric complex. In all cases, the HX mutation abolishes trimeric complex formation with Exd and Hth-HM. (C) EMSAs with wild type or HX-mutated forms of central mouse Hox proteins and Pbx1 and the HD-mutated form of Meis1a on the ant/cent nucleotide probe, as indicated. The HD mutation abolishes DNA-binding [48], as exemplified by the absence of DNA-bound Pbx/Meis complexes (red asterisk). This mutation also abolishes (for Hoxb6 and Hoxb7) or drastically affects (for Hoxb8) trimeric complex formation with HX-mutated Hox proteins. (D) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS nucleotide probe, as indicated. On this probe, the HX-mutated form of Hoxb8 is not able to form any dimeric or trimeric complex with Pbx1 or Pbx1/Meis1, respectively. (E) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS probe containing a Meis binding site, as indicated. On this probe, the HX-mutated form of Hoxb8 is able to form trimeric complexes with Pbx1/Meis1. An anti-HA against the HA tag of Hoxb8 was added in the last reaction to confirm the presence of trimeric complexes. Note that Pbx1 is able to bind the two TGAT binding sites of PRS (gray arrowheads). For all gels, colored bars and marks are symbolized as in Figure 3. See also Figure S9.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383740&req=5

pbio-1001351-g005: Meis class proteins cannot reveal HX-independent interaction modes in the absence of DNA-binding.(A) Scheme of the short HD-less isoform of Hth, which contains only the Exd interaction domain (HM). This form was fused to the HA tag (not schematized). (B) EMSAs with wild type or HX-mutated forms of Drosophila Hox proteins and Exd and the HD-less isoform of Hth on the Dllcon probe, as indicated. For Hox proteins making a dimeric complex with Exd (Lab, Scr and Antp), an anti-HA raised against Hth-HM was also used to validate the presence of the trimeric complex. In all cases, the HX mutation abolishes trimeric complex formation with Exd and Hth-HM. (C) EMSAs with wild type or HX-mutated forms of central mouse Hox proteins and Pbx1 and the HD-mutated form of Meis1a on the ant/cent nucleotide probe, as indicated. The HD mutation abolishes DNA-binding [48], as exemplified by the absence of DNA-bound Pbx/Meis complexes (red asterisk). This mutation also abolishes (for Hoxb6 and Hoxb7) or drastically affects (for Hoxb8) trimeric complex formation with HX-mutated Hox proteins. (D) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS nucleotide probe, as indicated. On this probe, the HX-mutated form of Hoxb8 is not able to form any dimeric or trimeric complex with Pbx1 or Pbx1/Meis1, respectively. (E) EMSAs with wild type or HX-mutated forms of Hoxb8 and Pbx1 or Pbx1/Meis1 on the PRS probe containing a Meis binding site, as indicated. On this probe, the HX-mutated form of Hoxb8 is able to form trimeric complexes with Pbx1/Meis1. An anti-HA against the HA tag of Hoxb8 was added in the last reaction to confirm the presence of trimeric complexes. Note that Pbx1 is able to bind the two TGAT binding sites of PRS (gray arrowheads). For all gels, colored bars and marks are symbolized as in Figure 3. See also Figure S9.
Mentions: The influence of the topology of the Hox/PBC/Meis binding sites suggest that binding of Meis proteins to DNA is a prerequisite for Meis-mediated uncovering of alternative Hox-PBC interaction modes. To investigate this more directly, we repeated EMSAs with DNA binding deficient Meis proteins. In the case of Drosophila proteins, we used a naturally HD-less isoform of Hth (Figure 5A), which contains only the evolutionary conserved HM domain mediating the direct interaction with Exd [49]. EMSAs were performed on Dllcon, which allowed assessing Hth-mediated uncovering of HX dispensability for Exd recruitment by Lab, Scr, Antp, and AbdB. The role of Hth-HM was not analyzed for Ubx and AbdA since these two proteins do not require Hth for establishing HX-independent interactions with Exd (Figure 3A). We observed that the HD-less isoform of Hth is able to form trimeric complexes in the context of wild type Hox proteins (Figure 5B). The HX mutation, however, abolishes the formation of the trimeric complex in all cases (Figure 5B), highlighting that Hth is not able to promote HX-independent interaction modes when it is not binding to Dllcon sequences.

Bottom Line: This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale.We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis.Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie du Développement de Marseille Luminy, IBDML, UMR7288, CNRS, AMU, Parc Scientifique de Luminy, Case 907, Marseille, France.

ABSTRACT
Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro analyses have established that the formation of Hox/PBC complexes relies on a short conserved Hox protein motif called the hexapeptide (HX). This paradigm is at the basis of the vast majority of experimental approaches dedicated to the study of Hox protein function. Here we questioned the unique and general use of the HX for PBC recruitment by using the Bimolecular Fluorescence Complementation (BiFC) assay. This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale. We found that the HX is dispensable for PBC recruitment in the majority of investigated Drosophila and mouse Hox proteins. We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis. Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins. Altogether, our results highlight that flexibility in Hox-PBC interactions is an ancestral and evolutionary conserved character, which has strong implications for the understanding of Hox protein functions during normal development and pathologic processes.

Show MeSH
Related in: MedlinePlus