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17 pages, 6 figures, 1 table, supplementary material https://doi.org/10.1186/s13072-018-0205-1 ; Abstract Background DNA methylation is one of the main epigenetic mechanisms for the regulation of gene expression in eukaryotes. In the standard model, methylation in gene promoters has received the most attention since it is generally associated with transcriptional silencing. Nevertheless, recent studies in human tissues reveal that methylation of the region downstream of the transcription start site is highly informative of gene expression. Also, in some cell types and specific genes it has been found that methylation of the first intron, a gene feature typically rich in enhancers, is linked with gene expression. However, a genome-wide, tissue-independent, systematic comparative analysis of the relationship between DNA methylation in the first intron and gene expression across vertebrates has not been explored yet. Results The most important findings of this study are: (1) using different tissues from a modern fish, we show a clear genome-wide, tissue-independent quasi-linear inverse relationship between DNA methylation of the first intron and gene expression. (2) This relationship is conserved across vertebrates, since it is also present in the genomes of a model pufferfish, a model frog and different human tissues. Among the gene features, tissues and species interrogated, the first intron's negative correlation with the gene expression was most consistent. (3) We identified more tissue-specific differentially methylated regions (tDMRs) in the first intron than in any other gene feature. These tDMRs have positive or negative correlation with gene expression, indicative of distinct mechanisms of tissue-specific regulation. (4) Lastly, we identified CpGs in transcription factor binding motifs, enriched in the first intron, the methylation of which tended to increase with the distance from the first exon–first intron boundary, with a concomitant decrease in gene expression. Conclusions Our integrative analysis clearly reveals the important and conserved role of the methylation level of the first intron and its inverse association with gene expression regardless of tissue and species. These findings not only contribute to our basic understanding of the epigenetic regulation of gene expression but also identify the first intron as an informative gene feature regarding the relationship between DNA methylation and gene expression where future studies should be focused ; DA was supported by a Ph.D. scholarship from the Spanish Government (BES-2011-044860). AEC is funded by the RED-BIO project of the Spanish National Bioinformatics Institute (INB) under grant number PT13/0001/0044. The INB is funded by the Spanish National Health Institute Carlos III (ISCIII) and the Spanish Ministry of Economy and Competitiveness (MINECO). Research supported by MINECO grant "Epifarm" (ref. AGL2013-41047-R) to FP. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI) ; Peer Reviewed

Discovering the means to control the increasing dissemination of pathogenic vibrios driven by recent climate change is challenged by the limited knowledge of the mechanisms in charge of Vibrio spp. persistence and spread in the time of global warming. To learn about physiological and gene expression patterns associated with the long-term persistence of V. harveyi at elevated temperatures, we studied adaptation of this marine bacterium in seawater microcosms at 30 °C which closely mimicked the upper limit of sea surface temperatures around the globe. We found that nearly 90% of cells lost their culturability and became partly damaged after two weeks, thus suggesting a negative impact of the combined action of elevated temperature and shortage of carbon on V. harveyi survival. Moreover, further gene expression analysis revealed that major adaptive mechanisms were poorly coordinated and apparently could not sustain cell fitness. On the other hand, elevated temperature and starvation promoted expression of many virulence genes, thus potentially reinforcing the pathogenicity of this organism. These findings suggest that the increase in disease outbreaks caused by V. harveyi under rising sea surface temperatures may not reflect higher cell fitness, but rather an increase in virulence enabling V. harveyi to escape from adverse environments to nutrient rich, host-pathogen associations. ; This work was supported by the Spanish Ministry of Economy and Competitiveness grant CGL2015-70929-R, Ikerbasque (Basque Foundation for Science) and pre-doctoral grants PRE-2013-1-901 (I.M.) from the Basque Government and PIF15/101 (E.O.) from the University of the Basque Country. A.E.-C. is funded by ISCIII of the MINECO (Ref: PT17/0009/0019) and cofinanced by FEDER.

Chronic obstructive pulmonary disease (COPD) is a destructive inflammatory disease and the genes expressed within the lung are crucial to its pathophysiology. We have determined the RNAseq transcriptome of bronchial brush cells from 312 stringently defined ex-smoker patients. Compared to healthy controls there were for males 40 differentially expressed genes (DEGs) and 73 DEGs for females with only 26 genes shared. The gene ontology (GO) term "response to bacterium" was shared, with several different DEGs contributing in males and females. Strongly upregulated genes TCN1 and CYP1B1 were unique to males and females, respectively. For male emphysema (E)-dominant and airway disease (A)-dominant COPD (defined by computed tomography) the term "response to stress" was found for both sub-phenotypes, but this included distinct up-regulated genes for the E-sub-phenotype (neutrophil-related CSF3R, CXCL1, MNDA) and for the A-sub-phenotype (macrophage-related KLF4, F3, CD36). In E-dominant disease, a cluster of mitochondria-encoded (MT) genes forms a signature, able to identify patients with emphysema features in a confirmation cohort. The MT-CO2 gene is upregulated transcriptionally in bronchial epithelial cells with the copy number essentially unchanged. Both MT-CO2 and the neutrophil chemoattractant CXCL1 are induced by reactive oxygen in bronchial epithelial cells. Of the female DEGs unique for E- and A-dominant COPD, 88% were detected in females only. In E-dominant disease we found a pronounced expression of mast cell-associated DEGs TPSB2, TPSAB1 and CPA3. The differential genes discovered in this study point towards involvement of different types of leukocytes in the E- and A-dominant COPD sub-phenotypes in males and females. ; This work was supported by the European Union, FP7 project # 200506. We acknowledge the helpful discussions with Emanuele Raineri, Barcelona, Spain and with Wilfried Karmaus, Division of Epidemiology, Biostatistics, and Environmental Health, School of Public Health, University of Memphis, Memphis, Tennessee, USA. CNAG-CRG lab is a member of the Spanish National Bioinformatics Institute (INB), PRB2-ISCIII and is supported by grant PT13/0001 of the PE I+D+i 2013-2016, funded by ISCIII and FEDER. Work by CB at Leicester and by LZH at Munich was also supported the European Union FP7 project # 270194.

Despite the abundant literature on the adverse effects of Bisphenol A (BPA) as endocrine disruptor, its toxicity mechanisms are still poorly understood. We present here a study of its effects on the zebrafish eleutheroembryo transcriptome at concentrations ranging from 0.1 to 4 mg L−1, this latter representing the lowest observed effect concentration (LOEC) found in our study at three different macroscopical endpoints (survival, hatching and swim bladder inflation). Multivariate data analysis methods identified both monotonic and bi-phasic patterns of dose-dependent responses. Functional analyses of genes affected by BPA exposure suggest an interaction of BPA with different signaling pathways, being the estrogenic and retinoid receptors two likely targets. In addition, we identified an apparently unrelated inhibitory effect on, among others, visual function genes. We interpret our data as the result of a sum of underlying, independent molecular mechanisms occurring simultaneously at the exposed animals, well below the macroscopic LOEC, but related to at least some of the observed morphological alterations, particularly in eye size and yolk sac resorption. Our data supports the idea that the physiological effects of BPA cannot be only explained by its rather weak interaction with the estrogen receptor, and that multivariate analyses are required to analyze the effects of toxicants like BPA, which interact with different cellular targets producing complex phenotypes. Estrogenic- and retinoid-like transcriptomic effects of bisphenol A in zebrafish eleutheroembryos and their relationship with morphological alterations. © 2018 Elsevier Ltd ; This work was supported by the European Research Council under the European Union's Seventh Framework Programme ( FP/2007–2013 )/ERC Grant Agreement n. 320737 . Some part of this study was also supported by a grant from the Spanish Ministry of Economy and Competitiveness ( CTQ2014-56777-R ) and by a grant ( PT17/0009/0019 ) from ISCIII (Carlos III Health Institute), part of the Spanish Ministry of Economy and Competitiveness, and cofinanced by the European Regional Development Fund (ERDF). LNM was supported by a Beatriu de Pinos Postdoctoral Fellow ( 2013BP-B-00088 ) awarded by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia and the Cofund programme of the Marie Curie Actions of the 7th R&D Framework Programme of the European Union. RM was supported by a FPU predoctoral fellow from the Spanish Ministry of Education, Culture and Sport (ref. FPU15/03332 ). We would like to thank Ms. Elia Martinez-Prats and David Angelats for helping with the real time qRT-PCRs measurements. Appendix A ; Peer reviewed

Type-2 diabetes (T2D) and glucose metabolic imbalances have been linked to neurodegenerative diseases, including Parkinson's disease (PD). To detect potential effects of different glucose levels on gene expression, by RNA-seq we analyzed the transcriptome of dermal fibroblasts from idiopathic PD (iPD) patients, LRRK2-associated PD (L2PD) patients, and healthy controls (total n = 21 cell lines), which were cultured at two different glucose concentrations (25 and 5 mM glucose). In PD patients we identified differentially expressed genes (DEGs) that were related to biological processes mainly involving the plasmatic cell membrane, the extracellular matrix, and also neuronal functions. Such pathway deregulation was largely similar in iPD or L2PD fibroblasts. Overall, the gene expression changes detected in this study were associated with PD independently of glucose concentration. ; This work was supported by CIBERNED / Instituto de Salud Carlos III (ISCIII) (grant PIE14/00061) (to M.-J.M.), and the Fondo de Investigaciones Sanitarias from ISCIII (grant PIE20/00259) (to M.E.). R.F.-S. was supported by a Jóvenes Investigadores (JIN) grant of the Spanish Ministry of Economy and Competitiveness (MINECO) and the Agencia Estatal de Investigación (AEI) (AEI/FEDER/UE) (grant #SAF2015-73508-JIN), and a Miguel Servet grant from the Instituto de Salud Carlos III (grant #CP19/00048). A.E-C. was funded by ISCIII /MINECO (PT17/0009/0019) and co-funded by FEDER. M.F. was funded through a María de Maeztu grant (#MDM-2017-0729). IDIBAPS receives funding from the CERCA Program from the Generalitat de Catalunya and the FEDER Program from the European Union.

Therapeutic resistance after multimodal therapy is the most relevant cause of glioblastoma (GBM) recurrence. Extensive cellular heterogeneity, mainly driven by the presence of GBM stem-like cells (GSCs), strongly correlates with patients' prognosis and limited response to therapies. Defining the mechanisms that drive stemness and control responsiveness to therapy in a GSC-specific manner is therefore essential. Here we investigated the role of integrin a6 (ITGA6) in controlling stemness and resistance to radiotherapy in proneural and mesenchymal GSCs subtypes. Using cell sorting, gene silencing, RNA-Seq, and in vitro assays, we verified that ITGA6 expression seems crucial for proliferation and stemness of proneural GSCs, while it appears not to be relevant in mesenchymal GSCs under basal conditions. However, when challenged with a fractionated protocol of radiation therapy, comparable to that used in the clinical setting, mesenchymal GSCs were dependent on integrin a6 for survival. Specifically, GSCs with reduced levels of ITGA6 displayed a clear reduction of DNA damage response and perturbation of cell cycle pathways. These data indicate that ITGA6 inhibition is able to overcome the radioresistance of mesenchymal GSCs, while it reduces proliferation and stemness in proneural GSCs. Therefore, integrin a6 controls crucial characteristics across GBM subtypes in GBM heterogeneous biology and thus may represent a promising target to improve patient outcomes. ; This work was supported by Agència Gestió Ajuts Universitaris i Recerca, Generalitat de Catalunya, grant number 2017SGR1014); Red Temática de investigación cooperativa en cáncer, grant number RD12/0036/0029); School of Nursing, grant number PREI-UB 17/005I; 18/010I; 19/009A; the Fondation ARC pour la recherche sur le cancer, the INSERM-CNRS ATIP-Avenir grant, the European Research Council (ERC) under the European Union's Horizon 2020 (grant agreement no. 805225) and the NanoTheRad (Paris-Saclay University). High-throughput sequencing was performed by the ICGex platform of the Institut Curie supported by the grants ANR-10-EQPX-03, ANR-10-INBS-09-08, and INCa-DGOS-4654. Work of M.M. and L.P. is supported by Fondazione AIRC per la Ricerca sul Cancro (IG 18851). E.S. is presently supported by Fondazione Veronesi. A.E-C. is funded by ISCIII/MINECO (PT17/0009/0019) and co-funded by FEDER.

Determining the effect of DNA methylation on chromatin structure and function in higher organisms is challenging due to the extreme complexity of epigenetic regulation. We studied a simpler model system, budding yeast, that lacks DNA methylation machinery making it a perfect model system to study the intrinsic role of DNA methylation in chromatin structure and function. We expressed the murine DNA methyltransferases in Saccharomyces cerevisiae and analyzed the correlation between DNA methylation, nucleosome positioning, gene expression and 3D genome organization. Despite lacking the machinery for positioning and reading methylation marks, induced DNA methylation follows a conserved pattern with low methylation levels at the 5' end of the gene increasing gradually toward the 3' end, with concentration of methylated DNA in linkers and nucleosome free regions, and with actively expressed genes showing low and high levels of methylation at transcription start and terminating sites respectively, mimicking the patterns seen in mammals. We also see that DNA methylation increases chromatin condensation in peri-centromeric regions, decreases overall DNA flexibility, and favors the heterochromatin state. Taken together, these results demonstrate that methylation intrinsically modulates chromatin structure and function even in the absence of cellular machinery evolved to recognize and process the methylation signal. ; This work has been supported by the Spanish Ministry of Science (BIO2012-32868), the Catalan SGR, the Instituto Nacional de Bioinformática, the European Research Council (ERC_SimDNA) and the BioExcel and MuG VRE H2000 projects. M.O. is an ICREA Academia Fellow. This project also received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754510 [to J.P.A. and M.O.]. The work of S.H. was supported by the Spanish Ministry of Science (PGC2018-099640-B-I00). A.E.C. is funded by ISCIII /MINECO (PT17/0009/0019) and co-funded by FEDER.

The etiology and the molecular basis of lung adenocarcinomas (LuADs) in nonsmokers are currently unknown. Furthermore, the scarcity of available primary cultures continues to hamper our biological understanding of non-smoking-related lung adenocarcinomas (NSK-LuADs). We established patient-derived cancer cell (PDC) cultures from metastatic NSK-LuADs, including two pairs of matched EGFR-mutant PDCs before and after resistance to tyrosine kinase inhibitors (TKIs), and then performed whole-exome and RNA sequencing to delineate their genomic architecture. For validation, we analyzed independent cohorts of primary LuADs. In addition to known non-smoker-associated alterations (e.g. RET, ALK, EGFR, and ERBB2), we discovered novel fusions and recurrently mutated genes, including ATF7IP, a regulator of gene expression, that was inactivated in 5% of primary LuAD cases. We also found germline mutations at dominant familiar-cancer genes, highlighting the importance of genetic predisposition in the origin of a subset of NSK-LuADs. Furthermore, there was an over-representation of inactivating alterations at RB1, mostly through complex intragenic rearrangements, in treatment-naive EGFR-mutant LuADs. Three EGFR-mutant and one EGFR-wild-type tumors acquired resistance to EGFR-TKIs and chemotherapy, respectively, and histology on re-biopsies revealed the development of small-cell lung cancer/squamous cell carcinoma (SCLC/LuSCC) transformation. These features were consistent with RB1 inactivation and acquired EGFR-T790M mutation or FGFR3-TACC3 fusion in EGFR-mutant tumors. We found recurrent alterations in LuADs that deserve further exploration. Our work also demonstrates that a subset of NSK-LuADs arises within cancer-predisposition syndromes. The preferential occurrence of RB1 inactivation, via complex rearrangements, found in EGFR-mutant tumors appears to favor SCLC/LuSCC transformation under growth-inhibition pressures. Thus RB1 inactivation may predict the risk of LuAD transformation to a more aggressive type of lung cancer, and may need to be considered as a part of the clinical management of NSK-LuADs patients. ; This work was supported by the Fundacion Cientifica Asociacion Española Contra el Cancer-AECC (grant number GCB14142170MONT) to LMM, MS-C, and EF; the Spanish Ministry of Economy and Competitivity-MINECO (grant number SAF-2017-82186R to MS-C; Rio Hortega-CM17/00180 to MS; PROYBAR17005NADA to EN); the Health Institute Carlos III-ISCIII, Fondo Europeo de Desarrollo Regional-FEDER (grant Number PT13/0001/0044, PT17/0009/0019, PI16 01821); the Government of Navarra (grant number DIANA project); and the Ramon Areces Foundation (no grant number is applicable) to LMM and RP. ; Sí

BACKGROUND: The BCN02-trial combined therapeutic vaccination with a viral latency reversing agent (romidepsin, RMD) in HIV-1-infected individuals and included a monitored antiretroviral pause (MAP) as an efficacy read-out identifying individuals with an early or late ( 4weeks) viral-rebound. Integrated -omics analyses were applied prior treatment interruption to identify markers of virus control during MAP. METHODS: PBMC, whole-genome DNA methylation and transcriptomics were assessed in 14 BCN02 participants, including 8 Early and 4 Late viral-rebound individuals. Chromatin state, histone marks and integration analysis (histone-3 acetylation (H3Ac), viral load, proviral levels and HIV-specific T cells responses) were included. REDUC-trial samples (n = 5) were included as a control group for RMD administration alone. FINDINGS: DNA methylation imprints after receiving the complete intervention discriminated Early versus Late viral-rebound individuals before MAP. Also, differential chromatin accessibility and histone marks at DNA methylation level were detected. Importantly, the differential DNA methylation positions (DMPs) between Early and Late rebounders before MAP were strongly associated with viral load, proviral levels as well as the HIV-specific T-cell responses. Most of these DMPs were already present prior to the intervention and accentuated after RMD infusion. INTERPRETATION: This study identifies host DNA methylation profiles and epigenetic cascades that are predictive of subsequent virus control in a kick-and-kill HIV cure strategy. FUNDING: European Union Horizon 2020 Framework Programme for Research and Innovation under Grant Agreement N°681137-EAVI2020 and N°847943-MISTRAL, the Ministerio de Ciencia e Innovación (SAF2017_89726_R), and the National Institutes of Health–National Institute of Allergy and Infectious Diseases Program Grant P01-AI131568.

Background: The BCN02-trial combined therapeutic vaccination with a viral latency reversing agent (romidepsin, RMD) in HIV-1-infected individuals and included a monitored antiretroviral pause (MAP) as an efficacy read-out identifying individuals with an early or late ( 4weeks) viral-rebound. Integrated -omics analyses were applied prior treatment interruption to identify markers of virus control during MAP. Methods: PBMC, whole-genome DNA methylation and transcriptomics were assessed in 14 BCN02 participants, including 8 Early and 4 Late viral-rebound individuals. Chromatin state, histone marks and integration analysis (histone-3 acetylation (H3Ac), viral load, proviral levels and HIV-specific T cells responses) were included. REDUC-trial samples (n = 5) were included as a control group for RMD administration alone. Findings: DNA methylation imprints after receiving the complete intervention discriminated Early versus Late viral-rebound individuals before MAP. Also, differential chromatin accessibility and histone marks at DNA methylation level were detected. Importantly, the differential DNA methylation positions (DMPs) between Early and Late rebounders before MAP were strongly associated with viral load, proviral levels as well as the HIV-specific T-cell responses. Most of these DMPs were already present prior to the intervention and accentuated after RMD infusion. Interpretation: This study identifies host DNA methylation profiles and epigenetic cascades that are predictive of subsequent virus control in a kick-and-kill HIV cure strategy. Funding: European Union Horizon 2020 Framework Programme for Research and Innovation under Grant Agreement N°681137-EAVI2020 and N°847943-MISTRAL, the Ministerio de Ciencia e Innovación (SAF2017_89726_R), and the National Institutes of Health–National Institute of Allergy and Infectious Diseases Program Grant P01-AI131568.