Estudi del mecanisme d'autoregulació del gen Trl i caracterització funcional a nivell global del factor GAGA en cèl·lules S2 de Drosophila melanogaster

Consultable des del TDXTítol obtingut de la portada digitalitzadaEn aquest treball hem aprofundit en l'estudi del factor de transcripció GAGA de Drosophila melanogaster, codificat pel gen Trithorax-like (Trl). GAGA s'uneix al DNA per seqüències de tipus d(GA)n, presents a moltes regions reguladores. Ja inicialment es va comprovar que és un factor essencial, que activa multitud de gens. Tot i així, també s'ha vist que reprimeix l'expressió del gen que la codifica, Trl. En aquest treball hem fet especial èmfasi en l'estudi d'aquest mecanisme d'autoregulació negativa. A més, també hem realitzat un estudi funcional global de GAGA en cèl·lules, per tal d'identificar els processos en que hi podria participar. Per mitjà d'assajos a sistemes cel·lulars heteròlegs, hem conclòs que l'autoregulació negativa de Trl està conservada entre D. melanogaster i D. virilis, però no així a cèl·lules HeLa humanes, on lluny de reprimir, GAGA activa Trl. Partint de la idea de que ha d'existir algun element a la seqüència del promotor de Trl que determini aquesta autoregulació, hem buscat aquest element emprant diverses estratègies i metodologies. Tot i que no hem aconseguit definir exactament les regions imprescindibles per a la repressió, pels nostres resultats proposem que GAGA actuaria desplaçant a un activador més potent del promotor de Trl, resultant en una davallada de l'activitat transcripcional. Alternativament, també podria ser que existís algun factor repressor que actués juntament amb GAGA per a reprimir Trl. En aquest sentit, hem analitzat la contribució d'altres factors de transcripció al mecanisme de repressió. D'aquests, només dCtBP ha mostrat una certa activitat repressora de Trl, encara que probablement per un mecanisme independent de GAGA. D'altra banda, hem analitzat la contribució de les diferents regions de GAGA al mecanisme de repressió, determinant que el domini d'unió al DNA, tot i que imprescindible, no és suficient per a que es doni la repressió, ja que la correcta disposició nuclear i per tant, també l'activitat de GAGA, depenen en part de la regió X. Posteriorment, vàrem passar a validar els resultats en un sistema de mosques transgèniques, concloent que la repressió de Trl per GAGA es dóna igualment, de forma independent de teixit i d'estadi de desenvolupament. A més, emprant RT-PCR quantitativa hem demostrat que, tant a mosca com a cèl·lules, la sobreexpressió de GAGA es tradueix en una davallada dels mRNAs transcrits a partir de la còpia endògena de Trl, indicant que la repressió és molt probablement a nivell transcripcional. Finalment, hem realitzat un experiment de transcriptòmica global en funció de la sobreexpressió i depleció de GAGA via RNAi. Els resultats obtinguts indiquen clarament que GAGA és un activador en tots els casos excepte, potser, en uns pocs promotors en els que està per veure si ho fa directa o indirectament. A més, qualsevol sobreexpressió de GAGA és extraordinàriament letal per a la mosca, fet que justifica la rellevància de l'autoregulació de Trl in vivo. A més, hem vist que la sobreexpressió de la isoforma GAGA519 comporta una major letalitat que la de GAGA581, indicant un possible camí funcional diferent per a les dues isoformes.In this work we have gone into the study of the Drosophila melanogaster's GAGA factor in depth. This factor is codified by the Trithorax-like (Trl) gene and it binds to d(GA)n DNA sequences, which are present in many regulatory regions. It is known that GAGA is an essential transcription factor that activates dozens of genes. In spite of this, it has been shown that GAGA factor can repress its own expression. In this work we have studied this negative self-regulation mechanism in detail. Moreover, with the aim to identify the processes in which GAGA factor can participate, a global functional analysis of GAGA in cells has also been performed. Taking advantage of working in heterologous cellular systems, we have concluded that the negative self-regulation of Trl is conserved between D.melanogaster and D.virilis, but not in HeLa human cells where on the contrary GAGA activates Trl. Assuming that it has to be some element in the Trl promoter sequence that should determine this negative regulation, we have made many efforts to find this element with different strategies and methodologies. Even though we have not been able to precisely define the essential regions for the repression, our results suggest that GAGA could be acting by displacing a stronger activator from the Trl promoter that leads to a decrease in transcriptional activation. On the other hand, it could be possible that a repressor factor could work with GAGA to repress Trl. In this direction, we have analyzed the possible contribution of different transcription factors in this repressing mechanism. Only dCtBP has shown some repressing activity but probably due to an independent mechanism not related to GAGA. Apart from that, we have also analyzed the contribution of the different domains of GAGA to the repressing mechanism, determining that the DNA binding domain is necessary but not sufficient to this repression, because the correct nuclear distribution and also the GAGA activity depend in part on the X region. Later on we validated the results in a transgenic fly system, concluding that Trl repression by GAGA exists and it is independent of the tissue and developmental stage. Furthermore, using quantitative RT-PCR we also demonstrate that both in flies and in cells, GAGA over-expression results in a depletion of the mRNA transcripts from the endogenous Trl copy, indicating that repression is probably taking place at transcriptional level. Finally, we have performed a high throughput transcriptome analysis, by over-expressing and also depleting GAGA factor, by using RNAi. The results obtained clearly indicate that GAGA is an activator except in few promoters in which it is still unclear whether it acts in a direct or indirect manner. Besides, any GAGA over-expression is extremely lethal for the fly, and that justifies the great importance of the self-regulation of Trl in vivo. We have also observed that over-expression of GAGA519 isoform results in more lethality than over-expression of GAGA581, indicating a differential way of function for the two isoforms

New insights for Drosophila GAGA factor in larvae

GAGA factor plays important roles during Drosophila embryogenesis and its maternal contribution is essential for early development. Here, the role of GAGA factor was studied in 3rd instar larvae using depletion and overexpression conditions in wing disc and transcriptome analysis. We found that genes changing expression were different to those previously described using GAGA mutants in embryos. No apparent phenotypes on GAGA depletion could usually be observed at larval stages in imaginal discs but a strong effect on salivary gland polytene chromosomes was observed. In the adult, GAGA depletion produced many defects like abnormal cell proliferation in the wing, impaired dorsal closure and resulted in homeotic transformation of abdominal segment A5. Unexpectedly, no effects on Ultrabithorax expression were observed. Short overexpression of GAGA factor in 3rd instar larvae also resulted in activation of a set of genes not previously described to be under GAGA regulation, and in lethality at pupa. Our results suggest a little contribution of GAGA factor on gene transcription in wing discs and a change of the genes regulated in comparison with embryo. GAGA factor activity thus correlates with the global changes in gene expression that take place at the embryo-to-larva and, later, at the larva-to-pupa transitions.This work was supported by grants of the Ministerio de Educación y Ciencia of the Spanish Government (BFU-2007-64395/BMC), the MICINN (CSD2006-49 and BFU2009-07111) and the Generalitat de Catalunya (SGR2009-1023). M.B. was supported by I3P CPG_06_0034 contract from CSIC, and grant BFU-2007-64395/BMC contract of the Spanish Government. D.P. was supported by an FPU fellowship from the Spanish GovernmentPeer Reviewe

General, negative feedback mechanism for regulation of Trithorax-like gene expression in vivo: new roles for GAGA factor in flies

Expression of every gene is first regulated at the transcriptional level. While some genes show acute and discrete periods of expression others show a rather steady expression level throughout development. An example of the latter is Trithorax-like (Trl) a member of the Trithorax group that encodes GAGA factor in Drosophila. Among other functions, GAGA factor has been described to stimulate transcription of several genes, including some homeotic genes. Here we show that GAGA factor is continuously down-regulating the expression of its own promoter using a negative feedback mechanism in vivo. Like its expression, repression by GAGA factor is ubiquitous, prevents its accumulation, and takes place throughout development. Experimental alteration of GAGA factor dosage results in several unexpected phenotypes, not related to alteration of homeotic gene expression, but rather to functions that take place later during development and affect different morphogenetic processes. The results suggest that GAGA factor is essential during development, even after homeotic gene expression is established, and indicate the existence of an upper limit for GAGA factor dosage that should not be exceeded

Remodeling of the m6A RNA landscape in the conversion of acute lymphoblastic leukemia cells to macrophages

We thank CERCA Programme/Generalitat de Catalunya for institutional support. This work was supported by the Health Department PERIS-project no. SLT/002/16/00374 and AGAUR-projects no. 2017SGR1080 of the Catalan Government (Generalitat de Catalunya); Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and European Regional Development Fund (ERDF) project no. RTI2018-094049-B-I00; the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Grant agreement No. 743168); the Varda and Boaz Dotan Research Center in Hemato-oncology affiliated to the Tel Aviv University; the Cellex Foundation; and “la Caixa” Banking Foundation (LCF/PR/GN18/51140001). ME is an ICREA Research Professor.Peer Reviewed"Article signat per 12 autors/es: Alberto Bueno-Costa, David Piñeyro, Carlos A. García-Prieto, Vanessa Ortiz-Barahona, Laura Martinez-Verbo, Natalie A. Webster, Byron Andrews, Nitzan Kol, Chen Avrahami, Sharon Moshitch-Moshkovitz, Gideon Rechavi & Manel Esteller"Postprint (published version

A-to-I editing on tRNAs: Biochemical, biological and evolutionary implications

AbstractInosine on transfer RNAs (tRNAs) are post-transcriptionally formed by a deamination mechanism of adenosines at positions 34, 37 and 57 of certain tRNAs. Despite its ubiquitous nature, the biological role of inosine in tRNAs remains poorly understood. Recent developments in the study of nucleotide modifications are beginning to indicate that the dynamics of such modifications are used in the control of specific genetic programs. Likewise, the essentiality of inosine-modified tRNAs in genome evolution and animal biology is becoming apparent. Here we review our current understanding on the role of inosine in tRNAs, the enzymes that catalyze the modification and the evolutionary link between such enzymes and other deaminases

B-cell leukemia transdifferentiation to macrophage involves reconfiguration of DNA methylation for long-range regulation

Altres ajuts: We thank CERCA Programme/Generalitat de Catalunya for institutional support. [...] BMJ is a Ramon y Cajal fellow (RYC-2016-19655). This work was supported by the Health Department PERIS-project no. SLT/002/16/00374 and [...]; and CIBERONC CB16/12/00312 and CB16/12/00489; [...]; the Cellex Foundation; and "la Caixa" Bank Foundation (LCF/PR/GN18/51140001). We thank Dr Thomas Graf for providing the transdifferentiation model.Altres ajuts: MEFP/FPU17-0242

Multitrait genome association analysis identifies new susceptibility genes for human anthropometric variation in the GCAT cohort

Background Heritability estimates have revealed an important contribution of SNP variants for most common traits; however, SNP analysis by single-trait genome-wide association studies (GWAS) has failed to uncover their impact. In this study, we applied a multitrait GWAS approach to discover additional factor of the missing heritability of human anthropometric variation. Methods We analysed 205 traits, including diseases identified at baseline in the GCAT cohort (Genomes For Life- Cohort study of the Genomes of Catalonia) (n=4988), a Mediterranean adult population-based cohort study from the south of Europe. We estimated SNP heritability contribution and single-trait GWAS for all traits from 15 million SNP variants. Then, we applied a multitrait-related approach to study genome-wide association to anthropometric measures in a two-stage meta-analysis with the UK Biobank cohort (n=336 107). Results Heritability estimates (eg, skin colour, alcohol consumption, smoking habit, body mass index, educational level or height) revealed an important contribution of SNP variants, ranging from 18% to 77%. Single-trait analysis identified 1785 SNPs with genome-wide significance threshold. From these, several previously reported single-trait hits were confirmed in our sample with LINC01432 (p=1.9×10−9) variants associated with male baldness, LDLR variants with hyperlipidaemia (ICD-9:272) (p=9.4×10−10) and variants in IRF4 (p=2.8×10−57), SLC45A2 (p=2.2×10−130), HERC2 (p=2.8×10−176), OCA2 (p=2.4×10−121) and MC1R (p=7.7×10−22) associated with hair, eye and skin colour, freckling, tanning capacity and sun burning sensitivity and the Fitzpatrick phototype score, all highly correlated cross-phenotypes. Multitrait meta-analysis of anthropometric variation validated 27 loci in a two-stage meta-analysis with a large British ancestry cohort, six of which are newly reported here (p value threshold <5×10−9) at ZRANB2-AS2, PIK3R1, EPHA7, MAD1L1, CACUL1 and MAP3K9. Conclusion Considering multiple-related genetic phenotypes improve associated genome signal detection. These results indicate the potential value of data-driven multivariate phenotyping for genetic studies in large population-based cohorts to contribute to knowledge of complex traits.This work was supported in part by the Spanish Ministerio de Economía y Competitividad (MINECO) project ADE 10/00026, by the Catalan Departament de Salut and by the Departament d’Empresa i Coneixement de la Generalitat de Catalunya, the Agència de Gestió d’Estudis Universitaris i de Recerca (AGA UR) (SGR 1269, SGR 1589 and SGR 647). RdC is the recipient of a Ramon y Cajal grant (RYC-2011-07822). The Project GCAT is coordinated by the Germans Trias i Pujol Research Institute (IGTP), in collaboration with the Catalan Institute of Oncology (ICO), and in partnership with the Blood and Tissue Bank of Catalonia (BST). IGTP is part of the CERCA Programme/Generalitat de Catalunya.Peer ReviewedPostprint (published version

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response

DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor in children and adolescents. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming and the DNMT1 enzyme has been demonstrated to have an important role in both maintaining the epigenome and controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor proliferation through a mechanism that is independent on DNA methylation. Depletion of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 phase, and DNA damage, as revealed by the induction of gamma H2AX foci. These effects elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 mutated cells, persistent micronuclei and increased DNA instability was observed. Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 as an adjuvant agent in treating EWS

Multitrait genome association analysis identifies new susceptibility genes for human anthropometric variation in the GCAT cohort

BACKGROUND: Heritability estimates have revealed an important contribution of SNP variants for most common traits; however, SNP analysis by single-trait genome-wide association studies (GWAS) has failed to uncover their impact. In this study, we applied a multitrait GWAS approach to discover additional factor of the missing heritability of human anthropometric variation. METHODS: We analysed 205 traits, including diseases identified at baseline in the GCAT cohort (Genomes For Life- Cohort study of the Genomes of Catalonia) (n=4988), a Mediterranean adult population-based cohort study from the south of Europe. We estimated SNP heritability contribution and single-trait GWAS for all traits from 15 million SNP variants. Then, we applied a multitrait-related approach to study genome-wide association to anthropometric measures in a two-stage meta-analysis with the UK Biobank cohort (n=336 107). RESULTS: Heritability estimates (eg, skin colour, alcohol consumption, smoking habit, body mass index, educational level or height) revealed an important contribution of SNP variants, ranging from 18% to 77%. Single-trait analysis identified 1785 SNPs with genome-wide significance threshold. From these, several previously reported single-trait hits were confirmed in our sample with LINC01432 (p=1.9×10-9) variants associated with male baldness, LDLR variants with hyperlipidaemia (ICD-9:272) (p=9.4×10-10) and variants in IRF4 (p=2.8×10-57), SLC45A2 (p=2.2×10-130), HERC2 (p=2.8×10-176), OCA2 (p=2.4×10-121) and MC1R (p=7.7×10-22) associated with hair, eye and skin colour, freckling, tanning capacity and sun burning sensitivity and the Fitzpatrick phototype score, all highly correlated cross-phenotypes. Multitrait meta-analysis of anthropometric variation validated 27 loci in a two-stage meta-analysis with a large British ancestry cohort, six of which are newly reported here (p value threshold <5×10-9) at ZRANB2-AS2, PIK3R1, EPHA7, MAD1L1, CACUL1 and MAP3K9. CONCLUSION: Considering multiple-related genetic phenotypes improve associated genome signal detection. These results indicate the potential value of data-driven multivariate phenotyping for genetic studies in large population-based cohorts to contribute to knowledge of complex traits

Detection of pseudorabies virus antibody in swine oral fluid using a serum whole-virus indirect ELISA

We evaluated the detection of pseudorabies virus (PRV) antibodies in swine oral fluid. Oral fluid and serum samples were obtained from 40 pigs allocated to 4 treatment groups (10 pigs/group): negative control (NC); wild-type PRV inoculation (PRV 3CR Ossabaw; hereafter PRV); PRV vaccination (Ingelvac Aujeszky MLV; Boehringer Ingelheim; hereafter MLV); and PRV vaccination followed by PRV inoculation at 21 d post-vaccination (MLV-PRV). Using a serum PRV whole-virus indirect IgG ELISA (Idexx Laboratories) adapted to the oral fluid matrix, PRV antibody was detected in oral fluid samples from treatment groups PRV, MLV, and MLV-PRV in a pattern similar to serum. Vaccination alone produced a low oral fluid antibody response (groups MLV and MLV-PRV), but a strong anamnestic response was observed following challenge with wild-type virus (group PRV). Analyses of the oral fluid PRV indirect IgG ELISA results showed good binary diagnostic performance (area under ROC curve = 93%) and excellent assay repeatability (intra-class correlation coefficient = 99.3%). The demonstrable presence of PRV antibodies in swine oral fluids suggests the possible use of oral fluids in pseudorabies surveillance