Maria Lluı̈sa Espinás

Adherens junctions (AJs) undergo dynamic remodelling during epithelial morphogenesis, requiring precise coordination between adhesive proteins, intracellular adaptors, and cytoskeletal regulators. In addition to cadherins, which mediate core cell-cell adhesion and connect junctions to the actin cytoskeleton, other adhesion molecules from the immunoglobulin superfamily (IgCAM) also contribute to AJ organisation. In Drosophila, Echinoid (Ed), a nectin-like IgCAM, localises along the entire AJ, whereas Sidekick (Sdk), another IgCAM, is predominantly enriched at tricellular adherens junctions (tAJs). Although both proteins interact with overlapping intracellular partners, how they functionally relate to one another has remained unclear. Here, we investigate the spatial, molecular, and functional interplay between Sdk and Ed during embryonic epithelial morphogenesis. Using SRRF-imaging we show that Sdk and Ed frequently colocalise at tAJs but also display adjacent or spatially separated distributions; together with proximity-labelling experiments, these results suggest that Sdk and Ed engage in transient and dynamic associations rather than forming a stable complex. Functional analyses reveal that they influence each other s accumulation, indicating bidirectional regulatory interactions. We find that Sdk modulates Ed levels along the entire AJs and affects Ed enrichment at tAJs. We provide evidence that this regulation involves changes in Ed intracellular trafficking, suggesting that Sdk modulates Ed levels at AJs at least in part by controlling its trafficking. Genetic analyses uncover previously unreported contributions of ed to tracheal cell intercalation and of sdk to dorsal closure, and reveal strong genetic interactions between the two genes, indicating cooperative yet context-dependent functions. Consistent with this, we find that Sdk and Ed converge on shared intracellular adaptor proteins, including Canoe and Polychaetoid, modulating their levels and junctional enrichment. Together, our findings support a model in which dynamic, multi-component protein complexes assemble at bicellular and tricellular AJs, integrating shared and junction-enriched components that engage in multiple, simultaneous, and mutually influencing interactions. This interconnected network would confer the spatiotemporal robustness and flexibility required to support the distinct cellular behaviours underlying tissue-specific remodelling.

Unveiling the molecular mechanisms of receptor activation has led to much understanding of development as well as the identification of important drug targets. We use the Drosophila tracheal system to study the activity of two families of widely used and conserved receptors, the TNFRs and the RTK-FGFRs. Breathless, an FGFR, controls the program of differentiation of the tracheal terminal cells in response to ligand activation. Here we identify a role for Wengen, a TNFR, in repressing the terminal cell program by regulating the MAPK pathway downstream of Breathless. We find that Wengen acts independently of both its canonical ligand and downstream pathway genes. Wengen does not stably localise at the membrane and is instead internalised—a trafficking that seems essential for activity. We show that Breathless and Wengen colocalise in intracellular vesicles and form a complex. Furthermore, Wengen regulates Breathless accumulation, possibly regulating Breathless trafficking and degradation. We propose that, in the tracheal context, Wengen interacts with Breathless to regulate its activity, and suggest that such unconventional mechanism, involving binding by TNFRs to unrelated proteins, may be a general strategy of TNFRs.

Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, we use Drosophila as a model. We previously showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Here we carry out a cellular and molecular analysis of chitin deposition and we show that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. We find that Kkv activity in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Furthermore, we find that Kkv and Exp/Reb display a largely complementary pattern at the apical domain, and that Exp/Reb activity regulates the topological distribution of Kkv at the apical membrane. We propose a model in which Exp/Reb regulates the organisation of Kkv complexes at the apical membrane which, in turn, regulates the function of Kkv in extracellular chitin translocation.

Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation. Haspin is a highly conserved kinase in eukaryotes involved in chromosome organization during mitosis. In this study we demonstrated that haspin is also required to maintain proper chromatin organization during interphase. Our analyses showed that Drosophila haspin is necessary for insulator activity, nuclear architecture, heterochromatin organization and pairing-sensitive gene silencing. We further found that haspin modulates Pds5-cohesin association with chromatin and it is required for robust Polycomb-mediated homeotic gene silencing. Overall our findings reveal that haspin kinase is a key element in chromatin organization and thereby regulates gene expression.

The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis.

Insulators are DNA‐protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190‐binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190‐interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190‐binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA‐binding proteins that form hetero‐oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer‐blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity. ![][1] CP190 is a common component of various Drosophila insulator complexes, but its role at genomic binding sites not shared with other known insulator factors is unclear. This study now identifies two new proteins that interact and cooperate with CP190 in insulator function. EMBO Journal (2014) 33, 637–647 [1]: pending:yes

Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host.

SAP18, a highly evolutionarily conserved protein, has been proposed to be involved in multiple cellular processes, from gene regulation to mRNA processing. To gain further insight into the role of SAP18, we performed genome-wide expression profiling of dsap18 mutant Drosophila melanogaster embryos and we found that dSAP18 is required for the expression of immune and stress related genes. We show that dSAP18 colocalizes with histone H3 phosphorylation, which has been implicated in the regulation of genes in response to signaling stimuli. dsap18 mutant larvae develop melanotic tumors after heat shock and the viability of dsap18 mutant flies is reduced after fungal infection or in high-salt medium. Altogether, our results indicate that dSAP18 is a key player in transcriptional responses to stress.

The homeotic Abdominal-B ( Abd-B ) gene expression depends on a modular cis -regulatory region divided into discrete functional domains ( iab ) that control the expression of the gene in a particular segment of the fly. These domains contain regulatory elements implicated in both initiation and maintenance of homeotic gene expression and elements that separate the different domains. In this paper we have performed an extensive analysis of the iab-6 regulatory region, which regulates Abd-B expression at abdominal segment A6 (PS11), and we have characterized two new polycomb response elements (PREs) within this domain. We report that PREs at Abd-B cis -regulatory domains present a particular chromatin structure which is nuclease accessible all along Drosophila development and both in active and repressed states. We also show that one of these regions contains a dCTCF and CP190 dependent activity in transgenic enhancer-blocking assays, suggesting that it corresponds to the Fab-6 boundary element of the Drosophila bithorax complex.