HoxB genes regulate neuronal delamination in the trunk neural tube by controlling the expression of Lzts1.

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (Lzts1), the expression of which is also activated by HoxB4 and HoxB9. Gain- and loss-of-function experiments showed that Lzts1, which is expressed endogenously in the IZ, controls neuronal delamination. These data collectively indicate that HoxB genes have a generic function in the developing spinal cord, controlling the expression of Lzts1 and neuronal delamination.

The Generic Facet of Hox Protein Function.

Hox transcription factors are essential to promote morphological diversification of the animal body. A substantial number of studies have focused on how Hox proteins reach functional specificity, an issue that arises from the fact that these transcription factors control distinct developmental functions despite sharing similar molecular properties. In this review, we highlight that, besides specific functions, for which these transcription factors are renowned, Hox proteins also often have nonspecific functions. We next discuss some emerging principles of these generic functions and how they relate to specific functions and explore our current grasp of the underlying molecular mechanisms.

The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription.

In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, we uncovered a molecular mechanism that increases productive transcription. We found that the Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. We found that AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription.

HOXB8 Counteracts MAPK/ERK Oncogenic Signaling in a Chicken Embryo Model of Neoplasia.

HOX transcription factors are members of an evolutionarily conserved family of proteins required for the establishment of the anteroposterior body axis during bilaterian development. Although they are often deregulated in cancers, the molecular mechanisms by which they act as oncogenes or tumor suppressor genes are only partially understood. Since the MAPK/ERK signaling pathway is deregulated in most cancers, we aimed at apprehending if and how the Hox proteins interact with ERK oncogenicity. Using an in vivo neoplasia model in the chicken embryo consisting in the overactivation of the ERK1/2 kinases in the trunk neural tube, we analyzed the consequences of the HOXB8 gain of function at the morphological and transcriptional levels. We found that HOXB8 acts as a tumor suppressor, counteracting ERK-induced neoplasia. The HOXB8 tumor suppressor function relies on a large reversion of the oncogenic transcriptome induced by ERK. In addition to showing that the HOXB8 protein controls the transcriptional responsiveness to ERK oncogenic signaling, our study identified new downstream targets of ERK oncogenic activation in an in vivo context that could provide clues for therapeutic strategies.

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

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.

Hox functional diversity: Novel insights from flexible motif folding and plastic protein interaction.

How the formidable diversity of forms emerges from developmental and evolutionary processes is one of the most fascinating questions in biology. The homeodomain-containing Hox proteins were recognized early on as major actors in diversifying animal body plans. The molecular mechanisms underlying how this transcription factor family controls a large array of context- and cell-specific biological functions is, however, still poorly understood. Clues to functional diversity have emerged from studies exploring how Hox protein activity is controlled through interactions with PBC class proteins, also evolutionary conserved HD-containing proteins. Recent structural data and molecular dynamic simulations add further mechanistic insights into Hox protein mode of action, suggesting that flexible folding of protein motifs allows for plastic protein interaction. As we discuss in this review, these findings define a novel type of Hox-PBC interaction, weak and dynamic instead of strong and static, hence providing novel clues to understanding Hox transcriptional specificity and diversity.

Cellular and molecular insights into Hox protein action.

Hox genes encode homeodomain transcription factors that control morphogenesis and have established functions in development and evolution. Hox proteins have remained enigmatic with regard to the molecular mechanisms that endow them with specific and diverse functions, and to the cellular functions that they control. Here, we review recent examples of Hox-controlled cellular functions that highlight their versatile and highly context-dependent activity. This provides the setting to discuss how Hox proteins control morphogenesis and organogenesis. We then summarise the molecular modalities underlying Hox protein function, in particular in light of current models of transcription factor function. Finally, we discuss how functional divergence between Hox proteins might be achieved to give rise to the many facets of their action.

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

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.

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

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.

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

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.

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

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.

Distinct genetic requirements for BX-C-mediated specification of abdominal denticles.

BACKGROUND: Hox genes encode transcription factors playing important role in segment specific morphogenesis along the anterior posterior axis. Most work in the Hox field aimed at understanding the basis for specialised Hox functions, while little attention was given to Hox common function. In Drosophila, genes of the Bithorax complex [Ultrabithorax (Ubx), abdominalA (abdA), and AbdominalB (AbdB)] all promote abdominal identity. While Ubx and AbdA share extensive sequence conservation, AbdB is highly divergent, questioning how it can perform similar functions as Ubx and AbdA. RESULTS: In this study, we investigate the genetic requirement for the specification of abdominal-type denticles by Ubx, AbdA, and AbdB. The impact of ectopic expression of Hox proteins in embryos mutant for Exd as well as of Wingless or Hedgehog signaling involved in intrasegmental patterning was analyzed. Results indicated that Ubx and AbdA do not require Exd, Wg, and Hh activity for specifying abdominal-type denticles, while AbdB does. CONCLUSIONS: Our results support that distinct regulatory mechanisms underlie Ubx/AbdA- and AbdB-mediated specification of abdominal-type denticles, highlighting distinct strategies for achieving a similar biological output. This suggests that common function performed by distinct paralogue Hox proteins may also rely on newly acquired property, instead of conserved/ancestral properties.

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

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.

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

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.

M1BP is an essential transcriptional activator of oxidative metabolism during Drosophila development.

Oxidative metabolism is the predominant energy source for aerobic muscle contraction in adult animals. How the cellular and molecular components that support aerobic muscle physiology are put in place during development through their transcriptional regulation is not well understood. Using the Drosophila flight muscle model, we show that the formation of mitochondria cristae harbouring the respiratory chain is concomitant with a large-scale transcriptional upregulation of genes linked with oxidative phosphorylation (OXPHOS) during specific stages of flight muscle development. We further demonstrate using high-resolution imaging, transcriptomic and biochemical analyses that Motif-1-binding protein (M1BP) transcriptionally regulates the expression of genes encoding critical components for OXPHOS complex assembly and integrity. In the absence of M1BP function, the quantity of assembled mitochondrial respiratory complexes is reduced and OXPHOS proteins aggregate in the mitochondrial matrix, triggering a strong protein quality control response. This results in isolation of the aggregate from the rest of the matrix by multiple layers of the inner mitochondrial membrane, representing a previously undocumented mitochondrial stress response mechanism. Together, this study provides mechanistic insight into the transcriptional regulation of oxidative metabolism during Drosophila development and identifies M1BP as a critical player in this process.

Control of DNA replication: a new facet of Hox proteins?

Hox proteins are well-known as developmental transcription factors controlling cell and tissue identity, but recent findings suggest that they are also part of the cell replication machinery. Hox-mediated control of transcription and replication may ensure coordinated control of cell growth and differentiation, two processes that need to be tightly and precisely coordinated to allow proper organ formation and patterning. In this review we summarize the available data linking Hox proteins to the replication machinery and discuss the developmental and pathological implications of this new facet of Hox protein function.

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

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.

Sustained experimental activation of FGF8/ERK in the developing chicken spinal cord models early events in ERK-mediated tumorigenesis.

The MAPK/ERK pathway regulates a variety of physiological cellular functions, including cell proliferation and survival. It is abnormally activated in many types of human cancers in response to driver mutations in regulators of this pathway that trigger tumor initiation. The early steps of oncogenic progression downstream of ERK overactivation are poorly understood due to a lack of appropriate models. We show here that ERK1/2 overactivation in the trunk neural tube of the chicken embryo through expression of a constitutively active form of the upstream kinase MEK1 (MEK1ca), rapidly provokes a profound change in the transcriptional signature of developing spinal cord cells. These changes are concordant with a previously established role of the tyrosine kinase receptor ligand FGF8 acting via the ERK1/2 effectors to maintain an undifferentiated state. Furthermore, we show that MEK1ca-transfected spinal cord cells lose neuronal identity, retain caudal markers, and ectopically express potential effector oncogenes, such as AQP1. MEK1ca expression in the developing spinal cord from the chicken embryo is thus a tractable in vivo model to identify the mechanisms fostering neoplasia and malignancy in ERK-induced tumorigenesis of neural origins.

Reiterative use of signalling pathways controls multiple cellular events during Drosophila posterior spiracle organogenesis.

Organogenesis proceeds in multiple steps and events that need to be coordinated in time and space. Yet the genetic and molecular control of such coordination remains poorly understood. In this study we have investigated the contribution of three signalling pathways, Wnt/Wingless (Wg), Hedgehog (Hh), and epidermal growth factor receptor (EGFR), to posterior spiracle morphogenesis, an organ that forms under Abdominal-B (AbdB) control in the eighth abdominal segment. Using targeted signalling inactivation, we show that these pathways are reiteratively used to control multiple cellular events during posterior spiracle organogenesis, including cell survival and maintenance of cell polarity and adhesion required for tissue integrity. We propose that the reiterative use of the Wg, Hh, and EGFR signalling pathways serves to coordinate in time and space the sequential deployment of events that collectively allow proper organogenesis.