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      Drosophila melanogaster Hox Transcription Factors Access the RNA Polymerase II Machinery through Direct Homeodomain Binding to a Conserved Motif of Mediator Subunit Med19

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          Abstract

          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.

          Author Summary

          Mutations of Hox developmental genes in the fruit fly Drosophila melanogaster may provoke spectacular changes in form: transformations of one body part into another, or loss of organs. This attribute identifies them as important developmental genes. Insect and vertebrate Hox proteins contain highly related homeodomain motifs used to bind to regulatory DNA and influence expression of developmental target genes. This occurs at the level of transcription of target gene DNA to messenger RNA by RNA polymerase II and its associated protein machinery (>50 proteins). How Hox homeodomain proteins induce fine-tuned transcription remains an open question. We provide an initial response, finding that Hox proteins also use their homeodomains to bind one machinery protein, Mediator complex subunit 19 (Med19) through a Med19 sequence that is highly conserved in animal phyla. Med19 mutants isolated in this work (the first animal mutants) show that Med19 assists Hox protein functions. Further, they indicate that homeodomain binding to the Med19 motif is required for normal expression of a Hox target gene. Our work provides new clues for understanding how the specific transcriptional inputs of the highly conserved Hox class of transcription factors are integrated at the level of the whole transcription machinery.

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          Most cited references 64

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          A gene complex controlling segmentation in Drosophila.

           Edwin Lewis (1978)
          The bithorax gene complex in Drosophila contains a minimum of eight genes that seem to code for substances controlling levels of thoracic and abdominal development. The state of repression of at least four of these genes is controlled by cis-regulatory elements and a separate locus (Polycomb) seems to code for a repressor of the complex. The wild-type and mutant segmentation patterns are consistent with an antero-posterior gradient in repressor concentration along the embryo and a proximo-distal gradient along the chromosome in the affinities for repressor of each gene's cis-regulatory element.
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            Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.

            Networks of protein interactions coordinate cellular functions. We describe a bimolecular fluorescence complementation (BiFC) assay for determination of the locations of protein interactions in living cells. This approach is based on complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are brought together by interactions between proteins fused to each fragment. BiFC analysis was used to investigate interactions among bZIP and Rel family transcription factors. Regions outside the bZIP domains determined the locations of bZIP protein interactions. The subcellular sites of protein interactions were regulated by signaling. Cross-family interactions between bZIP and Rel proteins affected their subcellular localization and modulated transcription activation. These results attest to the general applicability of the BiFC assay for studies of protein interactions.
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              Homeobox genes and axial patterning.

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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                May 2014
                1 May 2014
                : 10
                : 5
                Affiliations
                [1 ]Centre de Biologie du Développement, CBD, UMR5547 CNRS/UPS, Université de Toulouse, Toulouse, France
                [2 ]Institut de Biologie du Développement de Marseille Luminy, IBDML, UMR6216 CNRS, Université de la méditerranée, Marseille, France
                The University of North Carolina at Chapel Hill, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MB BH SM YG HMB DLC. Performed the experiments: MB BH CI YC SBF HMB DLC. Analyzed the data: MB BH CI YC SBF SM YG HMB DLC. Contributed reagents/materials/analysis tools: MB BH YC SBF SM YG HMB DLC. Wrote the paper: MB DLC.

                [¤a]

                Current address: MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London, United Kingdom

                [¤b]

                Current address: IGFL, CNRS/ENS Lyon, UMR5242, Lyon, France

                Article
                PGENETICS-D-13-02073
                10.1371/journal.pgen.1004303
                4006704
                24786462

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 16
                Funding
                This work was supported by recurring support from Centre National de Recherche Scientifique (CNRS) and from the Université Paul Sabatier, a graduate fellowship from the Ligue Nationale contre le Cancer (attributed to CI), and by grants from the Association pour la Recherche sur le Cancer (ARC; http://www.arc-cancer.net/) and the Agence Nationale de Recherche (ANR NT05-3-42540; http://www.agence-nationale-recherche.fr/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                Enzymology
                Enzyme Chemistry
                Cofactors (Biochemistry)
                Proteins
                DNA-binding proteins
                Protein interactions
                Developmental Biology
                Morphogenesis
                Pattern Formation
                Organism Development
                Organogenesis
                Cell Differentiation
                Evolutionary Developmental Biology
                Genetics
                Gene expression
                DNA transcription
                Animal Genetics
                Organisms
                Animals
                Invertebrates
                Arthropoda
                Insects
                Drosophila
                Drosophila Melanogaster
                Research and Analysis Methods
                Model Organisms
                Animal Models

                Genetics

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