Procurando sinais de selecção no genoma nuclear da abelha ibérica (Apis mellifera iberiensis Engel)

O estudo da adaptação local é de extrema importância pois permite perceber quais os factores/genes cruciais que permitiram a sobrevivência dos indivíduos num certo habitat. A compreensão dos mecanismos de adaptação local pode ajudar a prever a resposta da espécie a um mundo em acelerada transformação. A Península Ibérica é um laboratório natural para o estudo da adaptação local pois engloba diversos habitats como o Mediterrânico, Deserto, Alpino e Atlântico, permitindo perceber como é que um organismo se adapta aos diferentes ambientes. De forma a representar-se a distribuição natural da abelha ibérica, Apis mellifera iberiensis Engel, ao longo dos diferentes habitats na Península Ibérica, foram amostrados 87 indivíduos, representado 87 colónias e 16 locais, distribuídos por 3 transectos longitudinais (Atlântico, Central e Mediterrânico). Para cada local amostrado foram recolhidas as coordenadas geográficas. Os dados ambientais correspondentes a cada coordenada geográfica foram retirados das bases de dados http://www.worldclim.org e www.cru.uea.ac.uk. O genoma completo dos 87 indivíduos foi sequenciado utilizando a sequenciação “paired-end” e a plataforma Illumina HiSeq 2500. A cobertura mínima obtida do genoma da abelha ibérica foi de 6X. Diversos filtros foram aplicados de forma a eliminar potenciais erros de sequenciação e no final obtiveram-se 1 289 449 polimorfismos de nucleótido simples (SNPs) distribuídos ao longo dos 16 cromossomas da abelha. Para detectar sinais de selecção ao longo do genoma foram utilizadas diversas estatísticas (estatísticas baseadas no FST, iHS, EHH). Adicionalmente, de forma a perceber que variáveis ambientais podem ter moldado a adaptação local, foram utilizados programas como o SAMβADA e o LFMM, pois estes procuram encontrar associações entre os marcadores moleculares e variáveis ambientais.info:eu-repo/semantics/publishedVersio

Developing reduced SNP assays from whole-genome sequence data to estimate C-lineage introgression in the Iberian honeybee (Apis mellifera iberiensis)

The honeybee has been subject to a growing number of threats. In Western Europe one such threat is large-scale introductions of commercial strains (C-lineage), which is leading to introgressive hybridization and even the local extinction of native populations (M-lineage). Here, we developed reduced assays of highly informative SNPs from 176 whole genomes to estimate C-lineage introgression in ;M-lineage subspecies Apis mellifera iberiensis. We started by evaluating the effects of sample size and sampling a geographically restricted area on the number of highly informative SNPs. We demonstrated that a bias in the number of fixed SNPs (FST=1) is introduced when the sample size is small (N≤10) and when sampling only captures a small fraction of a population’s genetic diversity. These results underscore the importance of having a representative sample when developing reliable reduced SNP assays for organisms with complex genetic patterns. We used a training dataset to design four independent SNP assays selected from pairwise FST between the Iberian and C-lineage honeybees. The designed assays, which were validated in holdout and simulated hybrid datasets, proved to be highly accurate and can be readily used for monitoring populations not only in the native range of A. m. iberiensis in Iberia but also in the introduced range in the Balearic islands, Macaronesia, and South America, in a time- and cost-effective manner. While our approach used the Iberian honeybee as model system, it has a high value in a wide range of scenarios for the monitoring and conservation of potentially hybridized domestic and wildlife populations.info:eu-repo/semantics/publishedVersio

Função de genes associados à precipitação envolvidos na adaptação local da abelha ibérica

O aparecimento de novas tecnologias em estudos genómicos permite-nos realizar análises mais aprofundadas e compreender de que modo as forças evolutivas atuam sobre o genoma dos organismos. Um scan genómico realizado previamente na abelha ibérica, revelou genes associados a imunidade, detoxificação e mecanismo da visão, estando alguns deles associados a variáveis ambientais (Chávez-Galarza et al. 2013). Na sequência deste estudo procurou se sinais de seleção em genomas completos de 84 indivíduos de abelha ibérica, integrando informação genética, geográfica e ambiental. Detetou-se a presença 315 genes associados à precipitação. O principal objetivo é caracterizar a função destes genes, e também tentar perceber que tipos de substituições nucleotídicas ocorrem nas sequências codificantes das proteínas. Para isso utilizaram se diversas bases genómicas, como o NCBI, BeeBase e Flybase, as quais mostraram que 51 das variações, presentes em 28 genes, são não-sinónimas, originando aminoácidos diferentes.info:eu-repo/semantics/publishedVersio

Searching for signatures of selection in Iberian honey bee (Apis mellifera iberiensis) using whole genome sequences

The Iberian Peninsula comprises a diverse set of habitats. It was an important glacial refugium during the Pleistocene and has served as a bridge for populations migrating between Africa and Europe, resulting in a complex mix of ancestry and diversity. The Iberian honey bee (A. m. iberiensis) is no exception and has been the subject of numerous incongruent population genetic surveys. Recent mtDNA and SNP analyses indicate a steep northeastern-southwestern cline of African ancestry along the peninsula, which has been explained by selection. Advances in DNA sequencing technology and computational tools provide unprecedented opportunities to study demography, search for signatures of selection across the genome and illuminate its role in shaping genomic diversity. We used Illumina technology to sequence the whole genomes of 86 Iberian honeybees, collected across three longitudinal transects in the Iberian Peninsula and spanning semi-arid climates in the southeastern peninsula to oceanic in the North-West. The dataset was first analyzed for FST-outliers, CLR (composite-likelihood ratio) and EHH (Extended Haplotype Homozygosity) methods were further deployed to evaluate polymorphisms implicated in local adaptation and possibly in the response to human- mediated environmental changes, including known and novel variants in genes related to behavior, vision, xenobiotic detoxification and immune response.info:eu-repo/semantics/publishedVersio

Searching for signatures of selection in the Iberian honey bee (Apis mellifera iberiensis) using allele-environment association approaches

In the current context of a global human-mediated environmental crisis, understanding which genes and mechanisms are responsible for adaptation to different climates will enable predictions on how organisms will respond to a rapidly changing world. This is particularly important for the honey bee, a key-stone species for ecosystem functioning and economy, which is facing increasing pressures from the effects of intensified land use, climate change, and the spread of pests and pathogens. The aim of this work is searching for signatures of selection along the genome of 87 individuals using two different allele-environment association approaches.JC-G and DH are supported by Fundação para a Ciência e Tecnologia (FCT) through the scholarships SFRH/BD/68682/2010 and SFRH/BD/84195/2012, respectively. This research was funded by FCT and COMPETE/QREN/EU through the project PTDC/BIABEC/ 099640/2008 and BiodivERsA-FACCE2014-91. Bioinformatic analyses were performed using resources at the Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX)info:eu-repo/semantics/publishedVersio

Protection of islet grafts through transforming growth factor-beta-induced tolerogenic dendritic cells

In type 1 diabetes, the insulin-producing β-cells are destroyed by the immune system. One way of restoring glucose control is to transplant β-cells from a donor. Although this procedure may restore endogenous insulin production, immunosuppressive treatment is needed to prevent the recipient from rejecting the donor-derived islets. We investigated the possibilities of transient expression of the immunosuppressive cytokine transforming growth factor (TGF)-β within islets to achieve long-term graft tolerance. We found that brief expression of TGF-β prevented rejection of syngeneic islets, that there was reduction of dendritic cell (DC) activation in the graft, and that there was reduced reactivation of T cells in the graft-draining lymph nodes. In vitro exposure of bone marrow–derived DCs to TGF-β reduced expression of costimulatory molecules CD80 and CD86, as well as production of proinflammatory cytokines such as interleukin-12 p70 in DCs, but did not alter levels of major histocompatibility complex classes I and II. Furthermore, the capacity of TGF-β–treated bone marrow–derived DCs to activate both CD4+ and CD8+ T cells was reduced. Adding TGF-β–conditioned tolerogenic DCs to the grafted islets led to long-term survival of the graft, demonstrating that TGF-β–induced tolerogenic DCs can provide an effective means to restore immune tolerance in an already established autoimmune disease

The transcriptional coactivator MAML1 regulates p300 autoacetylation and HAT activity

MAML1 is a transcriptional coregulator originally identified as a Notch coactivator. MAML1 is also reported to interact with other coregulator proteins, such as CDK8 and p300, to modulate the activity of Notch. We, and others, previously showed that MAML1 recruits p300 to Notch-regulated genes through direct interactions with the DNA–CSL–Notch complex and p300. MAML1 interacts with the C/H3 domain of p300, and the p300–MAML1 complex specifically acetylates lysines of histone H3 and H4 tails in chromatin in vitro. In this report, we show that MAML1 potentiates p300 autoacetylation and p300 transcriptional activation. MAML1 directly enhances p300 HAT activity, and this coincides with the translocation of MAML1, p300 and acetylated histones to nuclear bodies

GSK3β is a negative regulator of the transcriptional coactivator MAML1

Glycogen synthase kinase 3β (GSK3β) is involved in several cellular signaling systems through regulation of the activity of diverse transcription factors such as Notch, p53 and β-catenin. Mastermind-like 1 (MAML1) was originally identified as a Notch coactivator, but has also been reported to function as a transcriptional coregulator of p53, β-catenin and MEF2C. In this report, we show that active GSK3β directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity, suggesting that GSK3β might target a coactivator in its regulation of gene expression. We have previously shown that MAML1 increases global acetylation of histones, and here we show that the GSK3 inhibitor SB41, further enhances MAML1-dependent histone acetylation in cells. Finally, MAML1 translocates GSK3β to nuclear bodies; this function requires full-length MAML1 protein

Activated Cdc42-associated kinase 1 (ACK1) binds the sterile α motif (SAM) domain of the adaptor SLP-76 and phosphorylates proximal tyrosines

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, "3Y") as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4(+) primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells.This work was supported by Wellcome Trust Grant 092627/Z/10/Z (to C. E. R.