RESUMEN
Sex chromosomes underlie the development of male or female sex organs across species. While systemic signals derived from sex organs prominently contribute to sex-linked differences, it is unclear whether the intrinsic presence of sex chromosomes in somatic tissues has a specific function. Here, we use genetic tools to show that cellular sex is crucial for sexual differentiation throughout the body in Drosophila melanogaster. We reveal that every somatic cell converts the intrinsic presence of sex chromosomes into the active production of a sex determinant, a female specific serine- and arginine-rich (SR) splicing factor. This discovery dismisses the mosaic model which posits that only a subset of cells has the potential to sexually differentiate. Using cell-specific sex reversals, we show that this prevalence of cellular sex drives sex differences in organ size and body weight and is essential for fecundity. These findings demonstrate that cellular sex drives differentiation programs at an organismal scale and highlight the importance of cellular sex pathways in sex trait evolution.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Cromosomas Sexuales , Diferenciación Sexual , Animales , Masculino , Femenino , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Diferenciación Sexual/genética , Diferenciación Sexual/fisiología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cromosomas Sexuales/genética , Fertilidad/genética , Caracteres Sexuales , Tamaño de los Órganos , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genética , Peso Corporal , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
The molecular mechanisms connecting cellular metabolism with differentiation remain poorly understood. Here, we find that metabolic signals contribute to stem cell differentiation and germline homeostasis during Drosophila melanogaster spermatogenesis. We discovered that external citrate, originating outside the gonad, fuels the production of Acetyl-coenzyme A by germline ATP-citrate lyase (dACLY). We show that this pathway is essential during the final spermatogenic stages, where a high Acetyl-coenzyme A level promotes NatB-dependent N-terminal protein acetylation. Using genetic and biochemical experiments, we establish that N-terminal acetylation shields key target proteins, essential for spermatid differentiation, from proteasomal degradation by the ubiquitin ligase dUBR1. Our work uncovers crosstalk between metabolism and proteome stability that is mediated via protein post-translational modification. We propose that this system coordinates the metabolic state of the organism with gamete production. More broadly, modulation of proteome turnover by circulating metabolites may be a conserved regulatory mechanism to control cell functions.
Asunto(s)
Drosophila melanogaster , Proteoma , Masculino , Animales , Proteoma/metabolismo , Acetilación , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Procesamiento Proteico-Postraduccional , Acetilcoenzima A/metabolismo , Diferenciación Celular , ReproducciónRESUMEN
While sex chromosomes carry sex-determining genes, they also often differ from autosomes in size and composition, consisting mainly of silenced heterochromatic repetitive DNA. Even though Y chromosomes show structural heteromorphism, the functional significance of such differences remains elusive. Correlative studies suggest that the amount of Y chromosome heterochromatin might be responsible for several male-specific traits, including sex-specific differences in longevity observed across a wide spectrum of species, including humans. However, experimental models to test this hypothesis have been lacking. Here we use the Drosophila melanogaster Y chromosome to investigate the relevance of sex chromosome heterochromatin in somatic organs in vivo. Using CRISPR-Cas9, we generated a library of Y chromosomes with variable levels of heterochromatin. We show that these different Y chromosomes can disrupt gene silencing in trans, on other chromosomes, by sequestering core components of the heterochromatin machinery. This effect is positively correlated to the level of Y heterochromatin. However, we also find that the ability of the Y chromosome to affect genome-wide heterochromatin does not generate physiological sex differences, including sexual dimorphism in longevity. Instead, we discovered that it is the phenotypic sex, female or male, that controls sex-specific differences in lifespan, rather than the presence of a Y chromosome. Altogether, our findings dismiss the 'toxic Y' hypothesis that postulates that the Y chromosome leads to reduced lifespan in XY individuals.
Asunto(s)
Drosophila melanogaster , Caracteres Sexuales , Humanos , Animales , Femenino , Masculino , Drosophila melanogaster/genética , Heterocromatina/genética , Longevidad/genética , Cromosoma Y/genéticaRESUMEN
Homochirality is a fundamental feature of all known forms of life, maintaining biomolecules (amino-acids, proteins, sugars, nucleic acids) in one specific chiral form. While this condition is central to biology, the mechanisms by which the adverse accumulation of non-L-α-amino-acids in proteins lead to pathophysiological consequences remain poorly understood. To address how heterochirality build-up impacts organism's health, we use chiral-selective in vivo assays to detect protein-bound non-L-α-amino acids (focusing on aspartate) and assess their functional significance in Drosophila. We find that altering the in vivo chiral balance creates a 'heterochirality syndrome' with impaired caspase activity, increased tumour formation, and premature death. Our work shows that preservation of homochirality is a key component of protein function that is essential to maintain homeostasis across the cell, tissue and organ level.
Asunto(s)
Aminoácidos , Proteínas , Estereoisomerismo , Aminoácidos/química , Proteínas/químicaRESUMEN
Nutrient-dependent body size plasticity differs between the sexes in most species, including mammals. Previous work in Drosophila showed that body size plasticity was higher in females, yet the mechanisms underlying increased female body size plasticity remain unclear. Here, we discover that a protein-rich diet augments body size in females and not males because of a female-biased increase in activity of the conserved insulin/insulin-like growth factor signaling pathway (IIS). This sex-biased upregulation of IIS activity was triggered by a diet-induced increase in stunted mRNA in females, and required Drosophila insulin-like peptide 2, illuminating new sex-specific roles for these genes. Importantly, we show that sex determination gene transformer promotes the diet-induced increase in stunted mRNA via transcriptional coactivator Spargel to regulate the male-female difference in body size plasticity. Together, these findings provide vital insight into conserved mechanisms underlying the sex difference in nutrient-dependent body size plasticity.
Asunto(s)
Tamaño Corporal , Drosophila melanogaster/fisiología , Insulina/metabolismo , Transducción de Señal , Animales , Femenino , Masculino , Caracteres Sexuales , Procesos de Determinación del Sexo , Regulación hacia ArribaRESUMEN
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 Drosophila 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) in vivo and in vitro We find that this interaction is tightly regulated in the Drosophila 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 Drosophila 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.
Asunto(s)
Proteínas de Drosophila , Drosophila , Transporte Activo de Núcleo Celular , Animales , Autofagia/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Cuerpo Adiposo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos , Factores de Transcripción/metabolismoRESUMEN
Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized.
Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Drosophila melanogaster/metabolismo , Ingestión de Alimentos/fisiología , Mucosa Intestinal/metabolismo , Caracteres Sexuales , Maduración del Esperma/fisiología , Animales , Ácido Cítrico/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Expresión Génica , Quinasas Janus/metabolismo , Masculino , RNA-Seq , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Azúcares/metabolismo , Testículo/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Genitales Femeninos/crecimiento & desarrollo , Animales , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/crecimiento & desarrollo , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox/genética , Genes de Insecto/genética , Proteínas de Homeodominio/metabolismo , Morfogénesis/genética , Proteínas Nucleares/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/metabolismoRESUMEN
Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.
Asunto(s)
Proteínas de Drosophila/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/genética , Procesos de Determinación del Sexo/genética , Diferenciación Sexual/genética , Testículo/embriología , Empalme Alternativo/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Proteínas del Tejido Nervioso/genética , Ovario/embriología , Ovario/metabolismo , Proteínas de Unión al ARN/fisiología , Testículo/metabolismoRESUMEN
Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Células Epiteliales/fisiología , Mucosa Intestinal/fisiología , Proteínas Proto-Oncogénicas c-ret/metabolismo , Animales , Drosophila , Regulación de la Expresión Génica , Humanos , Ratones , Vía de Señalización WntRESUMEN
Sex differences in physiology and disease susceptibility are commonly attributed to developmental and/or hormonal factors, but there is increasing realization that cell-intrinsic mechanisms play important and persistent roles. Here we use the Drosophila melanogaster intestine to investigate the nature and importance of cellular sex in an adult somatic organ in vivo. We find that the adult intestinal epithelium is a cellular mosaic of different sex differentiation pathways, and displays extensive sex differences in expression of genes with roles in growth and metabolism. Cell-specific reversals of the sexual identity of adult intestinal stem cells uncovers the key role this identity has in controlling organ size, reproductive plasticity and response to genetically induced tumours. Unlike previous examples of sexually dimorphic somatic stem cell activity, the sex differences in intestinal stem cell behaviour arise from intrinsic mechanisms that control cell cycle duration and involve a new doublesex- and fruitless-independent branch of the sex differentiation pathway downstream of transformer. Together, our findings indicate that the plasticity of an adult somatic organ is reversibly controlled by its sexual identity, imparted by a new mechanism that may be active in more tissues than previously recognized.
Asunto(s)
Células Madre Adultas/citología , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Intestinos/citología , Tamaño de los Órganos , Caracteres Sexuales , Animales , Ciclo Celular , Proliferación Celular , Transformación Celular Neoplásica , Compensación de Dosificación (Genética) , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Masculino , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Reproducción , Ribonucleoproteínas/metabolismo , Diferenciación Sexual/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Mapas de Interacción de Proteínas , Secuencias de Aminoácidos , Animales , Unión Competitiva , ADN/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Evolución Molecular , Fluorescencia , Proteínas Intrínsecamente Desordenadas/metabolismo , Ratones , Mutación/genética , Oligopéptidos/metabolismo , Especificidad de Órganos , Unión Proteica , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/metabolismo , Mapeo de Interacción de Proteínas/métodos , Animales , Drosophila/genética , Ensayo de Cambio de Movilidad Electroforética , Expresión Génica , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Drosophila melanogaster/genética , Proteínas de Homeodominio/metabolismo , Complejo Mediador/metabolismo , ARN Polimerasa II/metabolismo , Animales , Sitios de Unión , Unión ProteicaRESUMEN
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.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Evolución Molecular , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Filogenia , Regiones Promotoras Genéticas , Unión Proteica , Anémonas de Mar/embriología , Anémonas de Mar/genética , Anémonas de Mar/metabolismo , Especificidad de la Especie , Factores de Transcripción/genética , Xenopus/embriología , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMEN
Hox genes encode evolutionarily conserved transcription factors, providing positional information used for differential morphogenesis along the anteroposterior axis. Here, we show that Drosophila Hox proteins are potent repressors of the autophagic process. In inhibiting autophagy, Hox proteins display no apparent paralog specificity and do not provide positional information. Instead, they impose temporality on developmental autophagy and act as effectors of environmental signals in starvation-induced autophagy. Further characterization establishes that temporality is controlled by Pontin, a facultative component of the Brahma chromatin remodeling complex, and that Hox proteins impact on autophagy by repressing the expression of core components of the autophagy machinery. Finally, the potential of central and posterior mouse Hox proteins to inhibit autophagy in Drosophila and in vertebrate COS-7 cells indicates that regulation of autophagy is an evolutionary conserved feature of Hox proteins.
Asunto(s)
Autofagia , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Animales , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Ensamble y Desensamble de Cromatina , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ambiente , Proteínas de Homeodominio/genética , Inanición , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
During adaptive angiogenesis, a key process in the etiology and treatment of cancer and obesity, the vasculature changes to meet the metabolic needs of its target tissues. Although the cues governing vascular remodeling are not fully understood, target-derived signals are generally believed to underlie this process. Here, we identify an alternative mechanism by characterizing the previously unrecognized nutrient-dependent plasticity of the Drosophila tracheal system: a network of oxygen-delivering tubules developmentally akin to mammalian blood vessels. We find that this plasticity, particularly prominent in the intestine, drives--rather than responds to--metabolic change. Mechanistically, it is regulated by distinct populations of nutrient- and oxygen-responsive neurons that, through delivery of both local and systemic insulin- and VIP-like neuropeptides, sculpt the growth of specific tracheal subsets. Thus, we describe a novel mechanism by which nutritional cues modulate neuronal activity to give rise to organ-specific, long-lasting changes in vascular architecture.
Asunto(s)
Drosophila melanogaster/fisiología , Neovascularización Fisiológica , Neuropéptidos/metabolismo , Animales , Calcio/metabolismo , Sistema Digestivo/irrigación sanguínea , Humanos , Modelos Animales , Neovascularización Patológica , Neuronas/metabolismo , Oxígeno/metabolismo , Transducción de Señal , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
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.