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Viegas Russo da Conceição Martinho, Rui Gonçalo
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- Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodellingPublication . Navarro-Costa, Paulo; McCarthy, Alicia; Prudencio, Pedro; Greer, Christina; Guilgur, Leonardo Gastón; Becker, Jorg D.; Secombe, Julie; Rangan, Prashanth; Martinho, Rui GoncaloOocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.
- theLiTE™: a screening platform to identify compounds that reinforce tight junctionsPublication . Gomes, Teresa Lopes; Oliveira-Marques, Virgínia de; Hampson, Richard John; Jacinto, António; de Moraes, Luciana Vieira; Martinho, Rui GoncaloTight junctions (TJ) are formed by transmembrane and intracellular proteins that seal the intercellular space and control selective permeability of epithelia. Integrity of the epithelial barrier is central to tissue homeostasis and barrier dysfunction has been linked to many pathological conditions. TJ support the maintenance of cell polarity through interactions with the Par complex (Cdc42-Par-6-Par-3-aPKC) in which Par-6 is an adaptor and links the proteins of the complex together. Studies have shown that Par-6 overexpression delays the assembly of TJ proteins suggesting that Par-6 negatively regulates TJ assembly. Because restoring barrier integrity is of key therapeutic and prophylactic value, we focus on finding compounds that have epithelial barrier reinforcement properties; we developed a screening platform (theLiTE™) to identify compounds that modulate Par-6 expression in follicular epithelial cells from Par-6-GFP Drosophila melanogaster egg chambers. Hits identified were then tested whether they improve epithelial barrier function, using measurements of transepithelial electrical resistance (TEER) or dye efflux to evaluate paracellular permeability. We tested 2,400 compounds, found in total 10 hits. Here we present data on six of them: the first four hits allowed us to sequentially build confidence in theLiTE™ and two compounds that were shortlisted for further development (myricetin and quercetin). We selected quercetin due to its clinical and scientific validation as a compound that regulates TJ; food supplement formulated on the basis of this discovery is currently undergoing clinical evaluation in gastroesophageal reflux disease (GERD) sufferers.
- Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in DrosophilaPublication . J. Gonçalves, Emanuel; Henriques, Barbara J.; Rodrigues, Joao V.; Prudencio, Pedro; Rocha, Hugo; Vilarinho, Laura; Martinho, Rui Goncalo; Gomes, Claudio M.Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of beta-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4. C8 and 02) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a beta-strand connected by a short loop to an alpha-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as,a potential model organism for MADD. (C) 2012 Elsevier B.V. All rights reserved.
- N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevityPublication . Varland, Sylvia; Silva, Rui Duarte; Kjosås, Ine; Faustino, Alexandra; Bogaert, Annelies; Billmann, Maximilian; Boukhatmi, Hadi; Kellen, Barbara; Costanzo, Michael; Drazic, Adrian; Osberg, Camilla; Chan, Katherine; Zhang, Xiang; Tong, Amy Hin Yan; Andreazza, Simonetta; Lee, Juliette J.; Nedyalkova, Lyudmila; Ušaj, Matej; Whitworth, Alexander J.; Andrews, Brenda J.; Moffat, Jason; Myers, Chad L.; Gevaert, Kris; Boone, Charles; Martinho, Rui Gonçalo; Arnesen, ThomasMost eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. The most common protein modification in eukaryotes is N-terminal acetylation, but its functional impact has remained enigmatic. Here, the authors find that a key role for N-terminal acetylation is shielding proteins from ubiquitin ligase-mediated degradation, mediating motility and longevity.
- Analysis of mammalian native elongating transcript sequencing (mNET-seq) high-throughput dataPublication . Prudencio, Pedro; Rebelo, Kenny; Grosso, Ana Rita; Martinho, Rui Gonçalo; Carmo-Fonseca, MariaMammalian Native Elongating Transcript sequencing (mNET-seq) is a recently developed technique that generates genome-wide profiles of nascent transcripts associated with RNA polymerase II (Pol II) elongation complexes. The ternary transcription complexes formed by Pol II, DNA template and nascent RNA are first isolated, without crosslinking, by immunoprecipitation with antibodies that specifically recognize the different phosphorylation states of the polymerase large subunit C-terminal domain (CTD). The coordinate of the 3' end of the RNA in the complexes is then identified by high-throughput sequencing. The main advantage of mNET-seq is that it provides global, bidirectional maps of Pol II CTD phosphorylation-specific nascent transcripts and coupled RNA processing at single nucleotide resolution. Here we describe the general pipeline to prepare and analyse high-throughput data from mNET-seq experiments.
- Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic developmentPublication . Guilgur, Leonardo Gastón; Prudencio, Pedro; Sobral, Daniel; Liszekova, Denisa; Rosa, Andre; Martinho, Rui GoncaloDrosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, we reasoned that constraints on splicing were likely to exist in the early embryo, being splicing avoidance a possible explanation why most early zygotic genes are intronless. We isolated two mutant alleles for a subunit of the NTC/Prp19 complexes, which specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. We hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. Our findings demonstrate for the first time the existence of a developmental pre-requisite for highly efficient splicing during Drosophila early embryonic development and suggest in highly proliferative tissues a need for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts.
- The emerging role of transcriptional regulation in the oocyte-to-zygote transitionPublication . Navarro-Costa, Paulo; Martinho, Rui GoncaloFertilization marks the beginning of a new life by converting two terminally differentiated gametes into a single totipotent zygote. Central to this transition is a complex biological program commonly referred to as oocyte activation—an umbrella term for a series of profound changes that prepare the fertilized oocyte for totipotency [1, 2]. These include, among others, the completion of meiosis, the formation of the two pronuclei, and the selective translation of maternal RNAs. A remarkable aspect of oocyte activation is that it occurs in the absence of transcription. Not surprisingly, most of our knowledge of this process is centered on the posttranscriptional regulation of gene expression [3]. Yet, a recent body of evidence has brought new focus on the fundamental importance of transcriptional regulation during oogenesis as a primer for the oocyte-to-zygote transition [4].
- Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organismsPublication . Rathore, Om; Faustino, Alexandra; Prudencio, Pedro; Van Damme, Petra; Cox, C. J.; Martinho, Rui GoncaloProtein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.
- Naa50/San-dependent N-terminal acetylation of Scc1 is potentially important for sister chromatid cohesionPublication . Ribeiro, Ana Luisa; Silva, Rui D.; Foyn, Havard; Tiago, Margarida N.; Rathore, Om; Arnesen, Thomas; Martinho, Rui GoncaloThe gene separation anxiety (san) encodes Naa50/San, a N-terminal acetyltransferase required for chromosome segregation during mitosis. Although highly conserved among higher eukaryotes, the mitotic function of this enzyme is still poorly understood. Naa50/San was originally proposed to be required for centromeric sister chromatid cohesion in Drosophila and human cells, yet, more recently, it was also suggested to be a negative regulator of microtubule polymerization through internal acetylation of beta Tubulin. We used genetic and biochemical approaches to clarify the function of Naa50/San during development. Our work suggests that Naa50/San is required during tissue proliferation for the correct interaction between the cohesin subunits Scc1 and Smc3. Our results also suggest a working model where Naa50/San N-terminally acetylates the nascent Scc1 polypeptide, and that this co-translational modification is subsequently required for the establishment and/or maintenance of sister chromatid cohesion.
- Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesionPublication . Silva, Rui; Mirkovic, Mihailo; Guilgur, Leonardo G.; Rathore, Om; Martinho, Rui Goncalo; Oliveira, Raquel A.The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1-3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4-6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9-12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mpsl, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.