Cellular response to external signals in S. cerevisiae

En esta tesis, se utilizó como organismo modelo la levadura Saccharomyces cerevisiae para obtener conocimiento fundamental sobre una variedad de mecanismos moleculares utilizados por la célula para responder y adaptarse a los estímulos externos. En respuesta al aumento de la osmolaridad externa, la ruta de señalización MAPK High Osmolarity Glycerol (HOG) se activa para mediar cambios en diversas funciones celulares, incluyendo la reprogramación global de la transcripción y traducción, con el fin último de lograr la adaptación. Tras la detección de alta osmolaridad externa, la señal se transduce a través de dos ramas funcionalmente redundantes pero mecanísticamente distintas, SLN1 y SHO1, para finalmente activar el efector MAPK Hog1. Aunque múltiples interacciones entre sensores, proteínas adaptadoras y los componentes de señalización de la rama SHO1 se han descrito anteriormente, esta tesis caracteriza aún más la complejidad de los perfiles de las interacciones para dilucidar cómo se propaga la señal con eficacia y cómo se logra la fidelidad de la señal. En este sentido, hemos utilizado el nuevo método M-track que detecta tanto las interacciones entre proteínas de corta duración como las estables y hemos realizado varias observaciones interesantes. Una vez que se transduce la señal de respuesta a estrés, Hog1 activado entra en el núcleo de la célula y, entre sus más destacados funciones aguas abajo, modula los cambios de expresión génica global. El complejo de unión a la caperuza CBC (por cap-binding complex), que está formado por las proteínas de unión a RNA Cbc1 y Cbc2, se asocia con la caperuza en 5' del mensajero co-transcripcionalmente y se ha descrito su función en diversos aspectos de la vida de mensajero, incluyendo la transcripción. Utilizando técnicas de biología molecular y genómica, describimos Cbc1 como regulador de la transcripción global, tanto en condiciones de estrés y no estrés, para mediar la expresión génica de manera que se consigan niveles altos de una forma rápida. En respuesta a feromonas del tipo sexual contrario, la ruta de MAPK de feromonas de células de levadura haploides se activa para mediar una serie de cambios fisiológicos en preparación para el apareamiento, que incluyen la reprogramación de la expresión génica, la parada del ciclo celular, la formación de una proyección sexual denomina ¨shmoo¨, y en última instancia la fusión celular de los tipos de células de apareamiento contrario. El factor de traducción eIF5A, esencial y conservado evolutivamente, se ha descrito recientemente que funciona en la traducción de proteínas que contienen tres o más residuos de prolina consecutivos (polyPro), a través de su unión a ribosomas para aliviar el estancamiento del ribosoma durante la formación del enlace péptidico entre prolinas. La activación de eIF5A requiere una modificación post-traduccional única, la hipusinación, donde el residuo de hipusina se deriva de la espermidina, un factor esencial para la fertilidad de los mamíferos y que se requiere para el apareamiento de la levadura. Aquí se investigó eIF5A como regulador de la respuesta a feromonas a través de la traducción de proteínas polyPro con funciones en el apareamiento.In this thesis, we utilised the model organism the budding yeast Saccharomyces cerevisiae to gain fundamental knowledge on a variety of molecular mechanisms employed by the cell to respond and adapt to external stimuli. In response to increased external osmolarity, the yeast high osmolarity glycerol (HOG) MAPK pathway becomes activated to mediate changes to various cellular functions, including changes in glycerol accumulation, cell-cycle arrest, re-establishment of ion homeostasis and global reprogrammation of transcription and translation of the whole genome, in order to achieve adaptation. Upon detection of high external osmolarity, signal is transduced via two functionally redundant but mechanistically distinct branches, SLN1 and SHO1, to finally activate the effector MAPK Hog1. Although multiple interactions between osmosensors, adaptor proteins and signalling components of the SHO1 branch have been previously described, this thesis further characterises the complexity of their interaction profiles to elucidate how signal is effectively propagated and how signal fidelity is achieved. Here, we utilised the novel M-track method, which detects both short-lived and stable protein interactions, and made several interesting observations. Once the osmostress signal is transduced, activated Hog1 enters the cell nucleus and, amongst its most prominent downstream functions, modulates global gene expression changes. Within minutes of shock, global transcription rate rapidly decreases by 50 %, however the transcription machinery is reallocated to specific genes implicated in osmostress cellular protection and their transcription is strongly and rapidly induced. The cap-binding complex (CBC), consisting of mRNA-binding proteins Cbc1 and Cbc2, associates with the 5´ mRNA cap co-transcriptionally and has been described to function in various aspects of the mRNA life, including transcription. Utilising both genomic and molecular biology techniques, we describe Cbc1 as a global transcription regulator, both under stress and non-stress conditions, to mediate high and timely gene expression. In response to pheromones of the opposite mating type, the yeast pheromone MAPK pathway is activated to mediate a series of physiological changes in preparation for mating; including reprogrammation of gene expression, cell-cycle arrest, formation of a sexual projection termed ¨shmoo¨, and ultimately cell fusion of mating partners. The evolutionarily conserved and essential translation factor eIF5A has recently been described to promote translation of proteins containing three or more consecutive proline residues (polyPro) by binding to ribosomes and alleviating ribosome stalling during the formation of the Pro-Pro peptide bond. The activation of eIF5A requires the addition of a unique post-translational modification, a hypusine residue, which is derived from spermidine, an essential factor for mammalian fertility and required for yeast mating. Here we investigated eIF5A as a regulator of the pheromone response through the translation of polyPro proteins with roles in mating

Ultrasonic augers for improved transport of granular materials

This paper explores the superposition of ultrasonic vibration on vertical rotating augers in a variety of granular media. Ultrasonic vibration is known to facilitate direct penetration of granular media, and it is anticipated that any related reduction in the torque requirements of a rotating system might improve augering performance. Experimental results suggest that, compared to the non-ultrasonic scenario, ultrasonically assisted augering significantly promotes the flow of granular media, while moderately reducing the torque required to operate the device. Furthermore, it was discovered that particle size, helix angle, and auger speed all affect the performance of the auger in different ways as the ultrasonic amplitude is adjusted

Evolutionary conserved role of eukaryotic translation factor eIF5A in the regulation of actin-nucleating formins

Elongation factor eIF5A is required for the translation of consecutive prolines, and was shown in yeast to translate polyproline-containing Bni1, an actin-nucleating formin required for polarized growth during mating. Here we show that Drosophila eIF5A can functionally replace yeast eIF5A and is required for actin-rich cable assembly during embryonic dorsal closure (DC). Furthermore, Diaphanous, the formin involved in actin dynamics during DC, is regulated by and mediates eIF5A effects. Finally, eIF5A controls cell migration and regulates Diaphanous levels also in mammalian cells. Our results uncover an evolutionary conserved role of eIF5A regulating cytoskeleton-dependent processes through translation of formins in eukaryotes

SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

Sirtuins 1 and 2 (SIRT1/2) are two NAD-dependent deacetylases with major roles in inflammation. In addition to deacetylating histones and other proteins, SIRT1/2-mediated regulation is coupled with other epigenetic enzymes. Here, we investigate the links between SIRT1/2 activity and DNA methylation in macrophage differentiation due to their relevance in myeloid cells. SIRT1/2 display drastic upregulation during macrophage differentiation and their inhibition impacts the expression of many inflammation-related genes. In this context, SIRT1/2 inhibition abrogates DNA methylation gains, but does not affect demethylation. Inhibition of hypermethylation occurs at many inflammatory loci, which results in more drastic upregulation of their expression upon macrophage polarization following bacterial lipopolysaccharide (LPS) challenge. SIRT1/2-mediated gains of methylation concur with decreases in activating histone marks, and their inhibition revert these histone marks to resemble an open chromatin. Remarkably, specific inhibition of DNA methyltransferases is sufficient to upregulate inflammatory genes that are maintained in a silent state by SIRT1/2. Both SIRT1 and SIRT2 directly interact with DNMT3B, and their binding to proinflammatory genes is lost upon exposure to LPS or through pharmacological inhibition of their activity. In all, we describe a novel role for SIRT1/2 to restrict premature activation of proinflammatory genes

SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

Altres ajuts: CERCA Programme/Generalitat de Catalunya; [...].Sirtuins 1 and 2 (SIRT1/2) are two NAD-dependent deacetylases with major roles in inflammation. In addition to deacetylating histones and other proteins, SIRT1/2-mediated regulation is coupled with other epigenetic enzymes. Here, we investigate the links between SIRT1/2 activity and DNA methylation in macrophage differentiation due to their relevance in myeloid cells. SIRT1/2 display drastic upregulation during macrophage differentiation and their inhibition impacts the expression of many inflammation-related genes. In this context, SIRT1/2 inhibition abrogates DNA methylation gains, but does not affect demethylation. Inhibition of hypermethylation occurs at many inflammatory loci, which results in more drastic upregulation of their expression upon macrophage polarization following bacterial lipopolysaccharide (LPS) challenge. SIRT1/2-mediated gains of methylation concur with decreases in activating histone marks, and their inhibition revert these histone marks to resemble an open chromatin. Remarkably, specific inhibition of DNA methyltransferases is sufficient to upregulate inflammatory genes that are maintained in a silent state by SIRT1/2. Both SIRT1 and SIRT2 directly interact with DNMT3B, and their binding to proinflammatory genes is lost upon exposure to LPS or through pharmacological inhibition of their activity. In all, we describe a novel role for SIRT1/2 to restrict premature activation of proinflammatory genes

Longitudinal analysis of blood DNA methylation identifies mechanisms of response to tumor necrosis factor inhibitor therapy in rheumatoid arthritis

Epigenetics; Rheumatoid arthritis; Treatment responseEpigenètica; Artritis reumatoide; Resposta al tractamentEpigenética; Artritis reumatoide; Respuesta al tratamientoBackground Rheumatoid arthritis (RA) is a chronic, immune-mediated inflammatory disease of the joints that has been associated with variation in the peripheral blood methylome. In this study, we aim to identify epigenetic variation that is associated with the response to tumor necrosis factor inhibitor (TNFi) therapy. Methods Peripheral blood genome-wide DNA methylation profiles were analyzed in a discovery cohort of 62 RA patients at baseline and at week 12 of TNFi therapy. DNA methylation of individual CpG sites and enrichment of biological pathways were evaluated for their association with drug response. Using a novel cell deconvolution approach, altered DNA methylation associated with TNFi response was also tested in the six main immune cell types in blood. Validation of the results was performed in an independent longitudinal cohort of 60 RA patients. Findings Treatment with TNFi was associated with significant longitudinal peripheral blood methylation changes in biological pathways related to RA (FDR<0.05). 139 biological functions were modified by therapy, with methylation levels changing systematically towards a signature similar to that of healthy controls. Differences in the methylation profile of T cell activation and differentiation, GTPase-mediated signaling, and actin filament organization pathways were associated with the clinical response to therapy. Cell type deconvolution analysis identified CpG sites in CD4+T, NK, neutrophils and monocytes that were significantly associated with the response to TNFi. Interpretation Our results show that treatment with TNFi restores homeostatic blood methylation in RA. The clinical response to TNFi is associated to methylation variation in specific biological pathways, and it involves cells from both the innate and adaptive immune systems.This study was funded by the Instituto de Salud Carlos III

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction

Limfòcits b; Metilació de l'ADN; GenomaLinfocitos b; Metilación de ADN; GenomaB-lymphocytes; DNA methylation; GenomeActivation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R to E.B.]; cofunded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe. E.B is supported by Instituto de Salud Carlos III (ISCIII), Ref. AC18/00057, associated with i-PAD project (ERARE European Union program); P.L. and C.P. are supported by the German Cancer Aid project CO-CLL [70113869]; B.G. is funded by the Deutsche Forschungsgemeinschaft [GR1617/14-1/iPAD, SFB1160/2_B5, RESIST–EXC 2155–Project ID 390874280, CIBSS–EXC-2189–Project ID 390939984]; BMBF [GAIN 01GM1910A]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R]

Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids

Objectives Giant cell arteritis (GCA) is a complex systemic vasculitis mediated by the interplay between both genetic and epigenetic factors. Monocytes are crucial players of the inflammation occurring in GCA. Therefore, characterisation of the monocyte methylome and transcriptome in GCA would be helpful to better understand disease pathogenesis. Methods We performed an integrated epigenome-and transcriptome-wide association study in CD14+ monocytes from 82 patients with GCA, cross-sectionally classified into three different clinical statuses (active, in remission with or without glucocorticoid (GC) treatment), and 31 healthy controls. Results We identified a global methylation and gene expression dysregulation in GCA monocytes. Specifically, monocytes from active patients showed a more proinflammatory phenotype compared with healthy controls and patients in remission. In addition to inflammatory pathways known to be involved in active GCA, such as response to IL-6 and IL-1, we identified response to IL-11 as a new pathway potentially implicated in GCA. Furthermore, monocytes from patients in remission with treatment showed downregulation of genes involved in inflammatory processes as well as overexpression of GC receptor-target genes. Finally, we identified changes in DNA methylation correlating with alterations in expression levels of genes with a potential role in GCA pathogenesis, such as ITGA7 and CD63, as well as genes mediating the molecular response to GC, including FKBP5, ETS2, ZBTB16 and ADAMTS2. Conclusion Our results revealed profound alterations in the methylation and transcriptomic profiles of monocytes from GCA patients, uncovering novel genes and pathways involved in GCA pathogenesis and in the molecular response to GC treatment