Publications by authors named "Samir Merabet"

37 Publications

Developmental Robustness: The Haltere Case in .

Front Cell Dev Biol 2021 23;9:713282. Epub 2021 Jul 23.

IGFL, ENS Lyon, UMR 5242, Lyon, France.

Developmental processes have to be robust but also flexible enough to respond to genetic and environmental variations. Different mechanisms have been described to explain the apparent antagonistic nature of developmental robustness and plasticity. Here, we present a "self-sufficient" molecular model to explain the development of a particular flight organ that is under the control of the Hox gene () in the fruit fly . Our model is based on a candidate RNAi screen and additional genetic analyses that all converge to an autonomous and cofactor-independent mode of action for Ubx. We postulate that this self-sufficient molecular mechanism is possible due to an unusually high expression level of the Hox protein. We propose that high dosage could constitute a so far poorly investigated molecular strategy for allowing Hox proteins to both innovate and stabilize new forms during evolution.
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http://dx.doi.org/10.3389/fcell.2021.713282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8343187PMC
July 2021

Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein-Protein Interactions in Human Living Cells.

Methods Mol Biol 2021 ;2350:173-190

Institut de Génomique Fonctionnelle de Lyon, UMR5242, Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon, Lyon Cedex 07, France.

Deciphering protein-protein interactions (PPIs) in vivo is crucial to understand protein function. Bimolecular fluorescence complementation (BiFC) makes applicable the analysis of PPIs in many different native contexts, including human live cells. It relies on the property of monomeric fluorescent proteins to be reconstituted from two separate subfragments upon spatial proximity. Candidate partners fused to such complementary subfragments can form a fluorescent protein complex upon interaction, allowing visualization of weak and transient PPIs. It can also be applied for investigation of distinct PPIs at the same time using a multicolor setup. In this chapter, we provide a detailed protocol for analyzing PPIs by doing BiFC in cultured cells. Proof-of-principle experiments rely on the complementation property between the N-terminal fragment of mVenus (designated VN173) and the C-terminal fragment of mCerulean (designated CC155) and the partnership between HOXA7 and PBX1 proteins. This protocol is compatible with any other fluorescent complementation pair fragments and any type of candidate interacting proteins.
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http://dx.doi.org/10.1007/978-1-0716-1593-5_12DOI Listing
September 2021

Hox dosage contributes to flight appendage morphology in Drosophila.

Nat Commun 2021 05 17;12(1):2892. Epub 2021 May 17.

IGFL, CNRS UMR5242, ENS Lyon, Lyon, France.

Flying insects have invaded all the aerial space on Earth and this astonishing radiation could not have been possible without a remarkable morphological diversification of their flight appendages. Here, we show that characteristic spatial expression profiles and levels of the Hox genes Antennapedia (Antp) and Ultrabithorax (Ubx) underlie the formation of two different flight organs in the fruit fly Drosophila melanogaster. We further demonstrate that flight appendage morphology is dependent on specific Hox doses. Interestingly, we find that wing morphology from evolutionary distant four-winged insect species is also associated with a differential expression of Antp and Ubx. We propose that variation in the spatial expression profile and dosage of Hox proteins is a major determinant of flight appendage diversification in Drosophila and possibly in other insect species during evolution.
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http://dx.doi.org/10.1038/s41467-021-23293-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129201PMC
May 2021

LACC1 deficiency links juvenile arthritis with autophagy and metabolism in macrophages.

J Exp Med 2021 03;218(3)

Centre International de Recherche en Infectiologie/International Center for Infectiology Research, Institut National de la Santé et de la Recherche Médicale, U1111, Ecole Normale Supérieure de Lyon, Université Lyon 1, Centre National de la Recherche Scientifique, UMR5308, Lyon, France.

Juvenile idiopathic arthritis is the most common chronic rheumatic disease in children, and its etiology remains poorly understood. Here, we explored four families with early-onset arthritis carrying homozygous loss-of-expression mutations in LACC1. To understand the link between LACC1 and inflammation, we performed a functional study of LACC1 in human immune cells. We showed that LACC1 was primarily expressed in macrophages upon mTOR signaling. We found that LACC1 deficiency had no obvious impact on inflammasome activation, type I interferon response, or NF-κB regulation. Using bimolecular fluorescence complementation and biochemical assays, we showed that autophagy-inducing proteins, RACK1 and AMPK, interacted with LACC1. Autophagy blockade in macrophages was associated with LACC1 cleavage and degradation. Moreover, LACC1 deficiency reduced autophagy flux in primary macrophages. This was associated with a defect in the accumulation of lipid droplets and mitochondrial respiration, suggesting that LACC1-dependent autophagy fuels macrophage bioenergetics metabolism. Altogether, LACC1 deficiency defines a novel form of genetically inherited juvenile arthritis associated with impaired autophagy in macrophages.
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http://dx.doi.org/10.1084/jem.20201006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901146PMC
March 2021

Role of a versatile peptide motif controlling Hox nuclear export and autophagy in the fat body.

J Cell Sci 2020 09 23;133(18). Epub 2020 Sep 23.

Institut de Génomique Fonctionnelle de Lyon, ENS-Lyon, 32/34 Av. Tony Garnier, 69007 Lyon, France

Hox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deletion forms of the Hox protein Ultrabithorax (Ubx), we identified the presence of an unconventional nuclear export signal (NES) that overlaps with a highly conserved motif originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors that act in development and cancer. We show that this unconventional NES is involved in the interaction with the major exportin protein CRM1 (also known as Embargoed in flies) and We find that this interaction is tightly regulated in the fat body to control the autophagy-repressive activity of Ubx during larval development. The role of the PBC interaction motif as part of an unconventional NES was also uncovered in other and human Hox proteins, highlighting the evolutionary conservation of this novel function. Together, our results reveal the extreme molecular versatility of a unique short peptide motif for controlling the context-dependent activity of Hox proteins both at transcriptional and non-transcriptional levels.
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http://dx.doi.org/10.1242/jcs.241943DOI Listing
September 2020

Cooperation of axial and sex specific information controls Drosophila female genitalia growth by regulating the Decapentaplegic pathway.

Dev Biol 2019 10 25;454(2):145-155. Epub 2019 Jun 25.

Centro de Biología Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Universidad Autónoma de Madrid, Nicolás Cabrera 1, Cantoblanco, 28049, Madrid, Spain. Electronic address:

The specification and morphogenesis of an organ requires the coordinate deployment and integration of regulatory information, including sex specific information when the organ is sex specific. Only a few gene networks controlling size and pattern development have been deciphered, which limits the emergence of principles, general or not, underlying the organ-specifying gene networks. Here we elucidate the genetic and molecular network determining the control of size in the Drosophila abdominal A9 primordium, contributing to the female genitalia. This network requires axial regulatory information provided by the Hox protein Abdominal-BR (Abd-BR), the Hox cofactors Extradenticle (Exd) and Homothorax (Hth), and the sex specific transcription factor Doublesex Female (DsxF). These factors synergize to control size in the female A9 by the coordinate regulation of the Decapentaplegic (Dpp) growth pathway. Molecular dissection of the dpp regulatory region and in vivo protein interaction experiments suggest that Abd-BR, Exd, Hth and DsxF coordinately regulate a short dpp enhancer to repress dpp expression and restrict female A9 size. The same regulators can also suppress dpp expression in the A8, but this requires the absence of the Abd-BM isoform, which specifies A8. These results delineate the network controlling female A9 growth in Drosophila.
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http://dx.doi.org/10.1016/j.ydbio.2019.06.014DOI Listing
October 2019

The human HOXA9 protein uses paralog-specific residues of the homeodomain to interact with TALE-class cofactors.

Sci Rep 2019 04 5;9(1):5664. Epub 2019 Apr 5.

Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon, 46 allée d'Italie 69364, Lyon cedex, 07, France.

HOX proteins interact with PBX and MEIS cofactors, which belong to the TALE-class of homeodomain (HD)-containing transcription factors. Although the formation of HOX-PBX complexes depends on a unique conserved HOX motif called hexapeptide (HX), the additional presence of MEIS induces a remodeling of the interaction, leading to a global dispensability of the HX motif for trimeric complex formation in the large majority of HOX proteins. In addition, it was shown that the anterior HOXB3 and central HOXA7 and HOXC8 proteins could use different alternative TALE interaction motifs, with or without the HX motif, depending on the DNA-binding site and cell context. Here we dissected the molecular interaction properties of the human posterior HOXA9 protein with its TALE cofactors, PBX1 and MEIS1. Analysis was performed on different DNA-binding sites in vitro and by doing Bimolecular Fluorescence Complementation (BiFC) in different cell lines. Notably, we observed that the HOXA9-TALE interaction relies consistently on the redundant activity of the HX motif and two paralog-specific residues of the HOXA9 HD. Together with previous work, our results show that HOX proteins interact with their generic TALE cofactors through various modalities, ranging from unique and context-independent to versatile and context-dependent TALE binding interfaces.
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http://dx.doi.org/10.1038/s41598-019-42096-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450960PMC
April 2019

A systematic survey of HOX and TALE expression profiling in human cancers.

Int J Dev Biol 2018 ;62(11-12):865-876

Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon, Lyon, France.

HOX and TALE genes encode homeodomain (HD)-containing transcription factors that act in concert in different tissues to coordinate cell fates and morphogenesis throughout embryonic development. These two evolutionary conserved families contain several members that form different types of protein complexes on DNA. Mutations affecting the expression of HOX or TALE genes have been reported in a number of cancers, but whether and how the two gene families could be perturbed together has never been explored systematically. As a consequence, the putative collaborative role between HOX and TALE members for promoting or inhibiting oncogenesis remains to be established in most cancer contexts. Here, we address this issue by considering HOX and TALE expression profiling in normal and cancer adult tissues, using normalized RNA-sequencing expression data deriving from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) research projects. Information was extracted from 28 cancer types originating from 21 different tissues, constituting a unique comparative analysis of HOX and TALE expression profiles between normal and cancer contexts in human. We present the general and specific rules that could be deduced from this large-scale comparative analysis. Overall this work provides a precious annotated support to better understand the role of specific HOX/TALE combinatorial codes in human cancers.
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http://dx.doi.org/10.1387/ijdb.180286fbDOI Listing
May 2019

Generation of a versatile BiFC ORFeome library for analyzing protein-protein interactions in live .

Elife 2018 09 24;7. Epub 2018 Sep 24.

IGFL, CNRS, ENS Lyon, Lyon, France.

Transcription factors achieve specificity by establishing intricate interaction networks that will change depending on the cell context. Capturing these interactions in live condition is however a challenging issue that requires sensitive and non-invasive methods.

We present a set of fly lines, called 'multicolor BiFC library', which covers most of the transcription factors for performing Bimolecular Fluorescence Complementation (BiFC). The multicolor BiFC library can be used to probe two different binary interactions simultaneously and is compatible for large-scale interaction screens. The library can also be coupled with established genetic resources to analyze interactions in the developmentally relevant expression domain of each protein partner. We provide proof of principle experiments of these various applications, using Hox proteins in the live embryo as a case study. Overall this novel collection of ready-to-use fly lines constitutes an unprecedented genetic toolbox for the identification and analysis of protein-protein interactions in vivo.
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http://dx.doi.org/10.7554/eLife.38853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177257PMC
September 2018

Human HOX Proteins Use Diverse and Context-Dependent Motifs to Interact with TALE Class Cofactors.

Cell Rep 2018 03;22(11):3058-3071

Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon, 46, allée d'Italie 69364 Lyon Cedex 07, France. Electronic address:

HOX proteins achieve numerous functions by interacting with the TALE class PBX and MEIS cofactors. In contrast to this established partnership in development and disease, how HOX proteins could interact with PBX and MEIS remains unclear. Here, we present a systematic analysis of HOX/PBX/MEIS interaction properties, scanning all paralog groups with human and mouse HOX proteins in vitro and in live cells. We demonstrate that a previously characterized HOX protein motif known to be critical for HOX-PBX interactions becomes dispensable in the presence of MEIS in all except the two most anterior paralog groups. We further identify paralog-specific TALE-binding sites that are used in a highly context-dependent manner. One of these binding sites is involved in the proliferative activity of HOXA7 in breast cancer cells. Together these findings reveal an extraordinary level of interaction flexibility between HOX proteins and their major class of developmental cofactors.
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http://dx.doi.org/10.1016/j.celrep.2018.02.070DOI Listing
March 2018

Homeodomain proteins in action: similar DNA binding preferences, highly variable connectivity.

Curr Opin Genet Dev 2017 Apr 19;43:1-8. Epub 2016 Oct 19.

Institut de Génomique Fonctionnelle de Lyon, Centre National de Recherche Scientifique, Ecole Normale Supérieure de Lyon, France. Electronic address:

Homeodomain proteins are evolutionary conserved proteins present in the entire eukaryote kingdom. They execute functions that are essential for life, both in developing and adult organisms. Most homeodomain proteins act as transcription factors and bind DNA to control the activity of other genes. In contrast to their similar DNA binding specificity, homeodomain proteins execute highly diverse and context-dependent functions. Several factors, including genome accessibility, DNA shape, combinatorial binding and the ability to interact with many transcriptional partners, diversify the activity of homeodomain proteins and culminate in the activation of highly dynamic, context-specific transcriptional programs. Clarifying how homeodomain transcription factors work is central to our understanding of development, disease and evolution.
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http://dx.doi.org/10.1016/j.gde.2016.09.008DOI Listing
April 2017

To Be Specific or Not: The Critical Relationship Between Hox And TALE Proteins.

Trends Genet 2016 06 8;32(6):334-347. Epub 2016 Apr 8.

Columbia University Medical Center, New York, NY, USA. Electronic address:

Hox proteins are key regulatory transcription factors that act in different tissues of the embryo to provide specific spatial and temporal coordinates to each cell. These patterning functions often depend on the presence of the TALE-homeodomain class cofactors, which form cooperative DNA-binding complexes with all Hox proteins. How this family of cofactors contributes to the highly diverse and specific functions of Hox proteins in vivo remains an important unsolved question. We review here the most recent advances in understanding the molecular mechanisms underlying Hox-TALE function. In particular, we discuss the role of DNA shape, DNA-binding affinity, and protein-protein interaction flexibility in dictating Hox-TALE specificity. We propose several models to explain how these mechanisms are integrated with each other in the context of the many distinct functions that Hox and TALE factors carry out in vivo.
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http://dx.doi.org/10.1016/j.tig.2016.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4875764PMC
June 2016

The TALE face of Hox proteins in animal evolution.

Front Genet 2015 18;6:267. Epub 2015 Aug 18.

Department of Genetics and Evolution, Faculty of Science, Institute of Genetics and Genomics in Geneva, University of Geneva Geneva, Switzerland.

Hox genes are major regulators of embryonic development. One of their most conserved functions is to coordinate the formation of specific body structures along the anterior-posterior (AP) axis in Bilateria. This architectural role was at the basis of several morphological innovations across bilaterian evolution. In this review, we traced the origin of the Hox patterning system by considering the partnership with PBC and Meis proteins. PBC and Meis belong to the TALE-class of homeodomain-containing transcription factors and act as generic cofactors of Hox proteins for AP axis patterning in Bilateria. Recent data indicate that Hox proteins acquired the ability to interact with their TALE partners in the last common ancestor of Bilateria and Cnidaria. These interactions relied initially on a short peptide motif called hexapeptide (HX), which is present in Hox and non-Hox protein families. Remarkably, Hox proteins can also recruit the TALE cofactors by using specific PBC Interaction Motifs (SPIMs). We describe how a functional Hox/TALE patterning system emerged in eumetazoans through the acquisition of SPIMs. We anticipate that interaction flexibility could be found in other patterning systems, being at the heart of the astonishing morphological diversity observed in the animal kingdom.
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http://dx.doi.org/10.3389/fgene.2015.00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539518PMC
September 2015

Inhibitory activities of short linear motifs underlie Hox interactome specificity in vivo.

Elife 2015 Apr 14;4. Epub 2015 Apr 14.

Institut de génomique fonctionnelle de Lyon, Centre National de Recherche Scientifique, Lyon, France.

Hox proteins are well-established developmental regulators that coordinate cell fate and morphogenesis throughout embryogenesis. In contrast, our knowledge of their specific molecular modes of action is limited to the interaction with few cofactors. Here, we show that Hox proteins are able to interact with a wide range of transcription factors in the live Drosophila embryo. In this context, specificity relies on a versatile usage of conserved short linear motifs (SLiMs), which, surprisingly, often restrains the interaction potential of Hox proteins. This novel buffering activity of SLiMs was observed in different tissues and found in Hox proteins from cnidarian to mouse species. Although these interactions remain to be analysed in the context of endogenous Hox regulatory activities, our observations challenge the traditional role assigned to SLiMs and provide an alternative concept to explain how Hox interactome specificity could be achieved during the embryonic development.
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http://dx.doi.org/10.7554/eLife.06034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392834PMC
April 2015

Toward a new twist in Hox and TALE DNA-binding specificity.

Dev Cell 2015 Feb;32(3):259-61

Department of Developmental Biology, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany. Electronic address:

Hox proteins gain specificity by interacting with TALE-class cofactors. In a recent issue of Cell and in this issue of Developmental Cell, Crocker et al. (2015) and Amin et al. (2015), respectively, demonstrate that non-canonical Hox/TALE binding sequences play a major role in the regionalized regulation of target gene expression in vivo.
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http://dx.doi.org/10.1016/j.devcel.2015.01.030DOI Listing
February 2015

Bimolecular fluorescence complementation (BiFC) in live Drosophila embryos.

Methods Mol Biol 2014 ;1196:307-18

Institut de Génomique Fonctionnelle de Lyon, ENS de Lyon - CNRS UMR5242, 46 Allée d'Italie, 69364, Lyon Cedex 07, France.

Bimolecular fluorescence complementation (BiFC) is a powerful method for studying protein-protein interactions in different cell types and organisms. This method was recently developed in the fruit fly Drosophila melanogaster, allowing analyzing protein interaction properties in a physiologically relevant developing context. Here we present a detailed protocol for performing BiFC with the Venus fluorescent protein in live Drosophila embryos, taking the Hox-PBC partnership as an illustrative test case. This protocol applies to any transcription factor and split fluorescent protein in general.
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http://dx.doi.org/10.1007/978-1-4939-1242-1_19DOI Listing
May 2015

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

PLoS Genet 2014 May 1;10(5):e1004303. Epub 2014 May 1.

Centre de Biologie du Développement, CBD, UMR5547 CNRS/UPS, Université de Toulouse, Toulouse, France.

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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http://dx.doi.org/10.1371/journal.pgen.1004303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4006704PMC
May 2014

Molecular insights into the origin of the Hox-TALE patterning system.

Elife 2014 Mar 18;3:e01939. Epub 2014 Mar 18.

MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London, United Kingdom.

Despite tremendous body form diversity in nature, bilaterian animals share common sets of developmental genes that display conserved expression patterns in the embryo. Among them are the Hox genes, which define different identities along the anterior-posterior axis. Hox proteins exert their function by interaction with TALE transcription factors. Hox and TALE members are also present in some but not all non-bilaterian phyla, raising the question of how Hox-TALE interactions evolved to provide positional information. By using proteins from unicellular and multicellular lineages, we showed that these networks emerged from an ancestral generic motif present in Hox and other related protein families. Interestingly, Hox-TALE networks experienced additional and extensive molecular innovations that were likely crucial for differentiating Hox functions along body plans. Together our results highlight how homeobox gene families evolved during eukaryote evolution to eventually constitute a major patterning system in Eumetazoans. DOI: http://dx.doi.org/10.7554/eLife.01939.001.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957477PMC
http://dx.doi.org/10.7554/eLife.01939DOI Listing
March 2014

Tracking context-specific transcription factors regulating hox activity.

Dev Dyn 2014 Jan 26;243(1):16-23. Epub 2013 Jul 26.

IGFL, UMR5242, CNRS/ENS Lyon, Lyon, France.

Background: Hox proteins are key developmental regulators involved in almost every embryonic tissue for specifying cell fates along longitudinal axes or during organ formation. It is thought that the panoply of Hox activities relies on interactions with tissue-, stage-, and/or cell-specific transcription factors. High-throughput approaches in yeast or cell culture systems have shown that Hox proteins bind to various types of nuclear and cytoplasmic components, illustrating their remarkable potential to influence many different cell regulatory processes. However, these approaches failed to identify a relevant number of context-specific transcriptional partners, suggesting that these interactions are hard to uncover in non-physiological conditions. Here we discuss this problematic.

Results: In this review, we present intrinsic Hox molecular signatures that are probably involved in multiple (yet specific) interactions with transcriptional partners. We also recapitulate the current knowledge on Hox cofactors, highlighting the difficulty to tracking context-specific cofactors through traditional large-scale approaches.

Conclusion: We propose experimental approaches that will allow a better characterisation of interaction networks underlying Hox contextual activities in the next future.
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http://dx.doi.org/10.1002/dvdy.24002DOI Listing
January 2014

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

PLoS Genet 2013 7;9(3):e1003307. Epub 2013 Mar 7.

Centre National de la Recherche Scientifique, Aix Marseille Université, Institut de Biologie du Développement de Marseille Luminy, UMR 7288, Parc Scientifique de Luminy, Marseille, France.

The 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.
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http://dx.doi.org/10.1371/journal.pgen.1003307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591290PMC
June 2013

Antagonism versus cooperativity with TALE cofactors at the base of the functional diversification of Hox protein function.

PLoS Genet 2013 7;9(2):e1003252. Epub 2013 Feb 7.

CABD, CSIC/JA/Universidad Pablo de Olavide, Seville, Spain.

Extradenticle (Exd) and Homothorax (Hth) function as positive transcriptional cofactors of Hox proteins, helping them to bind specifically their direct targets. The posterior Hox protein Abdominal-B (Abd-B) does not require Exd/Hth to bind DNA; and, during embryogenesis, Abd-B represses hth and exd transcription. Here we show that this repression is necessary for Abd-B function, as maintained Exd/Hth expression results in transformations similar to those observed in loss-of-function Abd-B mutants. We characterize the cis regulatory module directly regulated by Abd-B in the empty spiracles gene and show that the Exd/Hth complex interferes with Abd-B binding to this enhancer. Our results suggest that this novel Exd/Hth function does not require the complex to bind DNA and may be mediated by direct Exd/Hth binding to the Abd-B homeodomain. Thus, in some instances, the main positive cofactor complex for anterior Hox proteins can act as a negative factor for the posterior Hox protein Abd-B. This antagonistic interaction uncovers an alternative way in which MEIS and PBC cofactors can modulate Abd-B like posterior Hox genes during development.
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http://dx.doi.org/10.1371/journal.pgen.1003252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567137PMC
June 2013

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

PLoS Biol 2012 26;10(6):e1001351. Epub 2012 Jun 26.

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

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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http://dx.doi.org/10.1371/journal.pbio.1001351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383740PMC
October 2012

Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

PLoS Genet 2011 Oct 27;7(10):e1002302. Epub 2011 Oct 27.

Institut de Biologie du Développement de Marseille Luminy, UMR6216 CNRS, Parc Scientifique de Luminy, Case 907, Marseille, France.

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.
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http://dx.doi.org/10.1371/journal.pgen.1002302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203194PMC
October 2011

On the border of the homeotic function: re-evaluating the controversial role of cofactor-recruiting motifs: the role of cofactor-recruiting motifs in conferring Hox evolutionary flexibility may critically depend on the protein environment.

Bioessays 2011 Jul 5;33(7):499-507. Epub 2011 May 5.

Institut de Biologie du Développement de Marseille Luminy, IBDML, UMR 6216, CNRS, Université de la Méditerranée, Parc Scientifique de Luminy, Case 907, Marseille, France.

In this review we present concepts that challenge a recently emerging paradigm explaining how similar Hox proteins perform different developmental functions across evolution, despite relatively limited sequence variability. This paradigm relates to the transcription factor, Fushi tarazu (Ftz), whose evolutionary plasticity has been shown to rely on the shuffling between two short protein recognition motifs. We discuss the Ftz paradigm and consider alternative interpretations to the evolutionary flexibility of this Hox protein. In particular, we propose that the protein environment might have played a critical role in the functional shuffling of Ftz during arthropod evolution.
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http://dx.doi.org/10.1002/bies.201100019DOI Listing
July 2011

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay.

BMC Biol 2011 Jan 28;9. Epub 2011 Jan 28.

Institut de Biologie du Développement de Marseille Luminy, IBDML, UMR 6216, CNRS, Université de la méditerranée, Parc Scientifique de Luminy, Case 907, 13288, Marseille Cedex 09, France.

Background: Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels.

Results: Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners.

Conclusion: Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.
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http://dx.doi.org/10.1186/1741-7007-9-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041725PMC
January 2011

Selection of distinct Hox-Extradenticle interaction modes fine-tunes Hox protein activity.

Proc Natl Acad Sci U S A 2011 Feb 24;108(6):2276-81. Epub 2011 Jan 24.

Institut de Biologie du Développement de Marseille Luminy, Centre National de la Recherche Scientifique, Université de la Méditerranée, 13288 Marseille Cedex 09, France.

Hox genes encode transcription factors widely used for diversifying animal body plans in development and evolution. To achieve functional specificity, Hox proteins associate with PBC class proteins, Pre-B cell leukemia homeobox (Pbx) in vertebrates, and Extradenticle (Exd) in Drosophila, and were thought to use a unique hexapeptide-dependent generic mode of interaction. Recent findings, however, revealed the existence of an alternative, UbdA-dependent paralog-specific interaction mode providing diversity in Hox-PBC interactions. In this study, we investigated the basis for the selection of one of these two Hox-PBC interaction modes. Using naturally occurring variations and mutations in the Drosophila Ultrabithorax protein, we found that the linker region, a short domain separating the hexapeptide from the homeodomain, promotes an interaction mediated by the UbdA domain in a context-dependent manner. While using a UbdA-dependent interaction for the repression of the limb-promoting gene Distalless, interaction with Exd during segment-identity specification still relies on the hexapeptide motif. We further show that distinctly assembled Hox-PBC complexes display subtle but distinct repressive activities. These findings identify Hox-PBC interaction as a template for subtle regulation of Hox protein activity that may have played a major role in the diversification of Hox protein function in development and evolution.
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http://dx.doi.org/10.1073/pnas.1006964108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038764PMC
February 2011

Regulation of Hox activity: insights from protein motifs.

Adv Exp Med Biol 2010 ;689:3-16

Institute of Developmental Biology of Marseille Luminy, University of the Mediterranean, Marseille, France.

Deciphering the molecular bases of animal body plan construction is a central question in developmental and evolutionary biology. Genome analyses of a number of metazoans indicate that widely conserved regulatory molecules underlie the amazing diversity of animal body plans, suggesting that these molecules are reiteratively used for multiple purposes. Hox proteins constitute a good example of such molecules and provide the framework to address the mechanisms underlying transcriptional specificity and diversity in development and evolution. Here we examine the current knowledge of the molecular bases of Hox-mediated transcriptional control, focusing on how this control is encoded within protein sequences and structures. The survey suggests that the homeodomain is part of an extended multifunctional unit coordinating DNA binding and activity regulation and highlights the need for further advances in our understanding of Hox protein activity.
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http://dx.doi.org/10.1007/978-1-4419-6673-5_1DOI Listing
September 2010

Classification of sequence signatures: a guide to Hox protein function.

Bioessays 2009 May;31(5):500-11

Institut de Biologie du Développement de Marseille Luminy, IBDML, UMR 6216, CNRS, Université de la Méditerranée, Parc Scientifique de Luminy, Case 907, Marseille Cedex 09, France.

Hox proteins are part of the conserved superfamily of homeodomain-containing transcription factors and play fundamental roles in shaping animal body plans in development and evolution. However, molecular mechanisms underlying their diverse and specific biological functions remain largely enigmatic. Here, we have analyzed Hox sequences from the main evolutionary branches of the Bilateria group. We have found that four classes of Hox protein signatures exist, which together provide sufficient support to explain how different Hox proteins differ in their control and function. The homeodomain and its surrounding sequences accumulate nearly all signatures, constituting an extended module where most of the information distinguishing Hox proteins is concentrated. Only a small fraction of these signatures has been investigated at the functional level, but these show that approaches relying on Hox protein alterations still have a large potential for deciphering molecular mechanisms of Hox differential control.
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http://dx.doi.org/10.1002/bies.200800229DOI Listing
May 2009

A unique Extradenticle recruitment mode in the Drosophila Hox protein Ultrabithorax.

Proc Natl Acad Sci U S A 2007 Oct 17;104(43):16946-51. Epub 2007 Oct 17.

Institut de Biologie du Développement de Marseille Luminy, Centre National de la Recherche Scientifique, Université de la Méditerranée, Parc Scientifique de Luminy, 13288 Marseille Cedex 09, France.

Hox transcription factors are essential for shaping body morphology in development and evolution. The control of Hox protein activity in part arises from interaction with the PBC class of partners, pre-B cell transcription factor (Pbx) proteins in vertebrates and Extradenticle (Exd) in Drosophila. Characterized interactions occur through a single mode, involving a short hexapeptide motif in the Hox protein. This apparent uniqueness in Hox-PBC interaction provides little mechanistic insight in how the same cofactors endow Hox proteins with specific and diverse activities. Here, we identify in the Drosophila Ultrabithorax (Ubx) protein a short motif responsible for an alternative mode of Exd recruitment. Together with previous reports, this finding highlights that the Hox protein Ubx has multiple ways to interact with the Exd cofactor and suggests that flexibility in Hox-PBC contacts contributes to specify and diversify Hox protein function.
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http://dx.doi.org/10.1073/pnas.0705832104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2040397PMC
October 2007
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