A novel strategy to uncover specific GO terms/phosphorylation pathways in phosphoproteomic data in Arabidopsis thaliana

Background: Proteins are the workforce of the cell and their phosphorylation status tailors specific responses efficiently. One of the main challenges of phosphoproteomic approaches is to deconvolute biological processes that specifically respond to an experimental query from a list of phosphoproteins. Comparison of the frequency distribution of GO (Gene Ontology) terms in a given phosphoproteome set with that observed in the genome reference set (GenRS) is the most widely used tool to infer biological significance. Yet, this comparison assumes that GO term distribution between the phosphoproteome and the genome are identical. However, this hypothesis has not been tested due to the lack of a comprehensive phosphoproteome database. Results: In this study, we test this hypothesis by constructing three phosphoproteome databases in Arabidopsis thaliana: one based in experimental data (ExpRS), another based in in silico phosphorylation protein prediction (PredRS) and a third that is the union of both (UnRS). Our results show that the three phosphoproteome reference sets show default enrichment of several GO terms compared to GenRS, indicating that GO term distribution in the phosphoproteomes does not match that of the genome. Moreover, these differences overshadow the identification of GO terms that are specifically enriched in a particular condition. To overcome this limitation, we present an additional comparison of the sample of interest with UnRS to uncover GO terms specifically enriched in a particular phosphoproteome experiment. Using this strategy, we found that mRNA splicing and cytoplasmic microtubule compounds are important processes specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings. Conclusions: This study provides a novel strategy to uncover GO specific terms in phosphoproteome data of Arabidopsis that could be applied to any other organism. We also highlight the importance of specific phosphorylation pathways that take place during dark-grown Arabidopsis development.Fil: Arico, Denise Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Beati, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Wengier, Diego Leonardo. University of Stanford; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Mazzella, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Estudio comparativo de la organización de la cromatina y su impacto en la expresión génica

Trypanosoma cruzi, Trypanosoma brucei y Leishmania major, usualmente conocidos como TriTryp, son causantes de enfermedades en animales y humanos. Se caracterizan por tener ciclos de vida complejos, alternando entre un hospedador mamífero y un insecto vector. En este trabajo, realizamos un estudio comparativo de la organización de la cromatina y su impacto en la expresión génica, de los estadios presentes en el insecto. Para ello, utilizamos datos obtenidos mediante secuenciación profunda (MNase-seq y RNA-seq). En el caso de T. cruzi, optimizamos el análisis de la cepa híbrida CL Brener, resaltando la importancia de alinear los datos crudos al genoma completo. Además, comparamos los alineamientos generados por Bowtie2 y Hisat2, encontrando que para análisis globales Bowtie2 es mejor, mientras que Hisat2 es más eficiente en asignar lecturas únicas. Dado que se ha reportado que en los TriTryp los nucleosomas se organizan en torno al sitio de trans-splicing (TAS), realizamos una predicción de los TAS más probables para los tres organismos y los usamos como punto de referencia para analizar la organización global de la cromatina. Observamos que L. major y T. cruzi presentan una menor densidad de nucleosomas en el TAS, en contraposición con T. brucei que presenta un leve aumento. Analizamos si habría relación con la composición de bases del ADN; pero fue muy similar entre T. cruzi y T. brucei y observamos mayores diferencias en L. major. Por otra parte, buscamos grupos de genes mediante k-means usando como variables predictoras los valores de densidad de nucleosomas y ARNm respecto al TAS. Los valores de silhouette obtenidos para nucleosomas fueron muy bajos, sugiriendo que la organización de la cromatina en torno al TAS es muy similar en todo el genoma. Del análisis de ARNm, obtuvimos un valor de silhouette alto al aplicar k=2 en T. cruzi o k=3 en T. brucei y L. major, indicando que hay dos o tres grupos de genes con patrones de expresión bien diferentes en los TriTryp.Fil: Zambrano Siri, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Beati, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Smircich, Pablo. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ocampo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXI Congreso de la Sociedad de ProtozoologíaMendozaArgentinaSociedad Argentina de Protozoologí

Role of the TcDOT1a and TcDOT1b isoforms in H3K76 differential methylation and their impact in Trypanosoma cruzi life cycle

Trypanosoma cruzi, the etiologic agent of Chagas Disease, affects a large number of the population in Latin America. It has a complex life cycle alternating between a mammalian host and the vector insect, Triatoma infestans. This cycle consists of three well-defined stages: amastigotes, epimastigotes and trypomastigotes. As the parasite faces different environments, it requires changes in gene expression in order to survive. Hence, gene expression regulation might be a key aspect to understand adaptation. Despite Trypanosomes gene expression is mainly regulated post transcriptionally, there are evidences that chromatin state influence it. In T. cruzi, there are two Dot1 methyltransferases homologues, called Dot1a and Dot1b, involved in the sequential mono, di and tri methylation of lysine 76 of histone H3 (H3K76me). Additionally, recent studies have shown that Dot1a deletion is not viable while Dot1b null mutant has aberrant morphology and decreased growth rate.In this project, we investigated the relevance of TcDot1a and TcDot1b during cell cycle and the metacyclogenesis process. Therefore, to analyze the isoforms subcelular location and their effects on cell cycle progression and differentiation, we have cloned the TcDot1 isoforms in a pRibotex vector with an N-terminal HA-tag and transfected epimastigotes of CL Brener strain. Nevertheless, the overexpression was toxic for the cell. Therefore, we decided to switch to an inducible vector.In order to elucidate the epigenetic implications of H3K76 differential methylation in the different stages of the life cycle of T. cruzi, we propose to map H3K76 mono, di and tri-methylation genome wide by MNase-ChIP-seq technique followed by paired-end sequencing.Overall, our data will be useful to further understand the role of the Dot1 isoforms and the epigenetic mechanism mediated by H3K76 in this unique parasite.Fil: Balestrasse, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Massimino Stepñicka, Milena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Beati, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ocampo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXXII Reunión Anual de la Sociedad Argentina de ProtozoologíaCiudad Autónoma de Buenos AiresArgentinaSociedad Argentina de Protozoologí

Contrasting study among Trypanosomatids reveals conserved chromatin organization around trans splicing-acceptor site

Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, usually known as TriTryps, are the causal agents of animal and human sickness. TriTryps are characterized by having complex life cycles, alternating between a mammal host and an insect vector. One peculiarity of these organisms is that their genes are organized in long transcriptional units that give rise to polycistronic transcripts which maturate into mRNA by a process known as trans-splicing. Even though gene expression regulation occurs mainly post-transcriptionally, it has been recently shown that chromatin plays a role in modulation. In this work, we made a comparative analysis of genome-wide chromatin organization and its potential impact on gene expression for the parasite stage present in the insect vector for the TriTryps using MNase-seq and RNAse-seq data, publicly available or generated by our laboratory. To compare average nucleosome positioning and mRNA patterns, we predicted the most likely trans-splicing acceptor sites and used them as reference points to plot average nucleosome or RNA-seq signals. By representing MNase-seq data, we corroborated the presence of a mild nucleosome depleted region (NDR) around trans-splicing acceptor sites (TASs) in T. cruzi and L. major; but not in T. brucei, as previously reported. However, when analyzing H3 ChIP-seq data, we uphold that TAS protection in T. brucei is due to a non-histone complex instead of a well-position nucleosome, as previously claimed. Moreover, we showed that this nucleosome organization around TASs is not just an average, since the same layout is conserved in most of the genome. Furthermore, the strand-specific analysis revealed that the NDR is not exactly at the TAS but a few base pairs upstream of that point. We corroborated that this trough, is coincident with a footprint of DNA-RNA duplex, as previously observed.Additionally, it was previously shown that average nucleosome density around TAS correlates with average RNA-seq signals. To test how strong is that correlation, we performed gene clustering using k-means with either nucleosome occupancy or mRNA signals relative to TAS as predictor variables. From the MNase-seq clustering, we observed a homogenous distribution of average nucleosome density in the three organisms except for a subset of genes with unusually high nucleosome density in T. cruzi. As opposed, from RNA-seq analysis, we obtained well-defined gene clusters for the three organisms supported by high silhouette values. Particularly, we observed that there is a subset of genes with markedly high mRNA levels compared to the rest, but the correspondence between nucleosome density and mRNA signal is only partial. To have a better understanding of the role and conservation of those subsets of genes with unusual characteristics among TriTryps, we are currently performing GO and Metabolic pathway analysis.Fil: Zambrano Siri, Romina T.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Beati, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Smircich, Pablo. Universidad de la República; UruguayFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Ocampo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaLVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology ResearchMendozaArgentinaSociedad Argentina de Investigaciones en Bioquímica y Biología Molecula

CD4+ T cells from chronica Chagas disease patients with different degrees of cardiac compromise exhibit distinct expression patterns of inhibitory receptors TIGIT, TIM-3 and LAG-3

In the chronic stage of Chagas disease, patients show a high frequency of activated T cells. Despite this, persistent presence of antigenic stimulus may affect their functionality and induce an exhausted phenotype with a high expression of inhibitory receptors and loss of effector functions. We hypothesize that inhibitory receptors TIGIT, Tim-3 and Lag-3 may be involved in immune modulation of anti-T. cruzi T cell response.In the present work, we assessed the frequency of CD4+ T cells expressing these receptors and their relation to cellular activation, measured as the expression of activation induced markers (AIM) Ox40 and CD25. Samples from chronic Chagas disease patients (CCD) with different degrees of cardiac compromise (asymptomatic, A, and with cardiopathy, C) and non-infected donors (NI) were analyzed under different stimulation conditions: T. cruzi lysate (Tc), unstimulated, or PHA.We observed statistically significant differences in the frequency of activated (Ox40+CD25+) CD4+ T cells between the CCD and NI subjects when stimulated with Tc. Cells from CCD showed an increased frequency of CD4+TIGIT+ T cells upon stimulation with PHA as compared with the unstimulated condition. CD4+Tim-3+ T cells are more abundant in the circulation of C patients and Lag-3+ cells showed increased frequency in the non-activated subset, within the A group.We demonstrated that the AIM method can be used to detect anti-T.cruzi response in CD4+ T cells, upon in vitro stimulation with Tc. Also, TIGIT, Tim-3 and Lag-3 are expressed differently, or they behave in diverging ways upon in vitro stimulation, in CCD.Fil: Alcaraz, Paula Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Girard, Magalí Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Beati, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Chadi, Raul. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; ArgentinaFil: Fernández, Marisa. Gobierno de la Ciudad Autónoma de Buenos Aires. Hospital General de Agudos Doctor Ignacio Pirovano; ArgentinaFil: Hernandez Vasquez, Yolanda Maria. Gobierno de la Ciudad Autónoma de Buenos Aires. Hospital General de Agudos Doctor Ignacio Pirovano; ArgentinaFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Acevedo, Gonzalo Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaLXVII Reunión Científica Anual de Sociedad Argentina de InmunologíaTucumanArgentinaSociedad Argentina de Inmunologi

Expression of Inhibitory Receptors TIGIT, TIM-3, and LAG-3 on CD4+ T Cells from Patients with Different Clinical Forms of Chronic Chagas Disease

T cells are central to the adaptive immune response against Trypanosoma cruzi infection. In chronic Chagas disease (CCD), circulating parasite-specific memory T cells show reduced functionality and increased expression of inhibitory receptors as a result of persistent antigenic stimulation. This phenotype has been linked to progression of cardiac pathology, whereas the presence of polyfunctional T cells shows association with therapeutic success. In this study, we demonstrate that T. cruzi-specific human CD4+ T cells can be identified by their expression of OX40 and CD25 upon in vitro stimulation. We characterized the expression of the inhibitory receptors T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell Ig and mucin-domain containing-3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in CD4+ T cells from CCD patients with and without cardiac alterations. Our results show that, independently of their clinical stage, CCD patients present an increased frequency of CD4+ T cells expressing TIGIT in comparison with non-T. cruzi-infected donors. Exposure to parasite Ags increases the expression of TIM-3 in CD4+ T cells from CCD patients, especially in those with cardiac compromise. Upregulation of LAG-3 was also detected in CCD individuals without cardiac manifestations, predominantly within the subpopulation of cells that did not become activated upon stimulation. Further differences were found between groups in the coexpression of these receptors. Blockade of each individual receptor did not affect activation or the production of IFN-g and IL-10 by CD4+ T cells in response to parasite Ags. Our results suggest a role for TIGIT, TIM-3, and LAG-3 in the modulation of inflammatory phenomena thought to ultimately lead to tissue damage and cardiac pathology.Fil: Ferragut, Fatima Eneida del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alcaraz, Paula Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Beati, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Girard, Magalí Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ossowski, Micaela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Chadi, Raúl. Gobierno de la Ciudad Autonoma de Buenos Aires. Hospital General de Agudos Doctor Ignacio Pirovano .; ArgentinaFil: Fernández, Marisa. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Hernandez Vasquez, Yolanda Maria. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Acevedo, Gonzalo Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. University of California; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin