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BACKGROUND AND AIMS Accurate identification of recent HCV infections is critical for tracing the extent and mechanisms of ongoing transmission. We aimed to validate dried blood spot (DBS) samples for the assessment of Hepatitis C virus (HCV) genetic diversity and to determine epidemiological parameters including incidence, determinants of acute infection, and phylogenetic clustering in people who inject drugs (PWID). APPROACH AND RESULTS HCV nonstructural protein 5B next-generation sequencing was performed from plasma and/or DBS in 220 viremic PWID from the HepCdetect II study. No significant differences were found in consensus sequences or Shannon entropy (SE) intrahost diversity estimate between paired plasma/DBS specimens. SE values were used to identify acute infections with 93.3% sensitivity (95% CI, 0.81-1.06) and 95.0% specificity (95% CI, 0.88-1.02) in a set of well-defined controls. An acute HCV infection (either primary infection or reinfection) was detected in 13.5% of viremic participants and was associated with age ≤30 years (OR, 8.09), injecting less than daily (OR, 4.35), ≤5 years of injected drug use (OR, 3.43), sharing cocaine snorting straws (OR, 2.89), and being unaware of their HCV status (OR, 3.62). Annualized HCV incidence was estimated between 31 and 59/100 person-years. On phylogenetic analysis, 46.8% of viremic cases were part of a transmission pair or cluster; age ≤30 years (OR, 6.16), acute infection (OR, 5.73), and infection with subtype 1a (OR, 4.78) were independently associated with this condition. CONCLUSIONS The results obtained from plasma and DBS characterize PWID with acute infection and those involved in ongoing HCV transmission and allow estimating incidence from cross-sectional data. This information is critical for the design and assessment of targeted harm reduction programs and test-and-treat interventions and to facilitate monitoring of HCV elimination in this key population.

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 Cellex Foundation; and “la Caixa” Banking Foundation (LCF/PR/GN18/51140001). P.S.O. is a fellow of the Severo Ochoa Program (Bp17- 165). V.D. is supported by the Spanish Association Against Cancer (AECC).

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"

The human face is one of the most visible features of our unique identity as individuals. Interestingly, monozygotic twins share almost identical facial traits and the same DNA sequence but could exhibit differences in other biometrical parameters. The expansion of the world wide web and the possibility to exchange pictures of humans across the planet has increased the number of people identified online as virtual twins or doubles that are not family related. Herein, we have characterized in detail a set of “look-alike” humans, defined by facial recognition algorithms, for their multiomics landscape. We report that these individuals share similar genotypes and differ in their DNA methylation and microbiome landscape. These results not only provide insights about the genetics that determine our face but also might have implications for the establishment of other human anthropometric properties and even personality characteristics., This work was funded by the governments of Catalonia (2017SGR1080) and Spain (RTI2018-094049-B-I00, SAF2014-55000, and TIN2017-90124-P) and the Cellex Foundation.

Background & Aims: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB. Methods: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies. Results: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth. Conclusions: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB. Lay summary: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer. (c) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)., This article was possible thanks to the inputs from the Instituto de Salud Carlos III, ISCIII (PI09/00751, PI10/02082, PI13/02340). The project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 668596 (ChiLTERN) and grant agreement No 826121 (iPC). JCR is supported by the Catalan Agency for Management of University and Research Grants (AGAUR, 2019 FI_B01024). LT is supported by an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813). DS is supported by the Gilead Research Scholar in Liver Disease. JML is supported by the European Union's Horizon 2020 research and innovation programme (HEPCAR, 667273-2), Institucio Catalana de Recerca i Estudis Avancats (ICREA), U.S. Department of Defense (CA150272P3), an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813), National Cancer Institute, Tisch Cancer Institute (P30-CA196521), Samuel Waxman Cancer Research Foundation, Spanish National Health Institute (SAF2016-76390) and AGAUR (SGR-1358). CA and MRS were supported by Ramon y Cajal (RYC-2010-07249) and Miguel Servet (CPII14/00021) programs of the Ministry of Science and Innovation of Spain and ISCIII, respectively. CA, MRS and MS received funding from CIBERehd (CB06/04/0033) and AGAUR (2017-SGR-490). IGTP is a member of the CERCA network of institutes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A small fraction of cancer patients with advanced disease survive significantly longer than patients with clinically comparable tumors. Molecular mechanisms for exceptional responses to therapy have been identified by genomic analysis of tumor biopsies from individual patients. Here, we analyzed tumor biopsies from an unbiased cohort of 111 exceptional responder patients using multiple platforms to profile genetic and epigenetic aberrations as well as the tumor microenvironment. Integrative analysis uncovered plausible mechanisms for the therapeutic response in nearly a quarter of the patients. The mechanisms were assigned to four broad categories���DNA damage response, intracellular signaling, immune engagement, and genetic alterations characteristic of favorable prognosis���with many tumors falling into multiple categories. These analyses revealed synthetic lethal relationships that may be exploited therapeutically and rare genetic lesions that favor therapeutic success, while also providing a wealth of testable hypotheses regarding oncogenic mechanisms that may influence the response to cancer therapy. Profiling multi-platform genomics of 110 cancer patients with an exceptional therapeutic response, Wheeler et al. identify putative molecular mechanisms explaining this survival phenotype in ���23% of cases. Therapeutic success is related to rare molecular features of responding tumors, exploiting synthetic lethality and oncogene addiction.

The research leading to these results received funding from the Obra Social “la Caixa” (to ME), the Cellex Foundation (to ME), the European Union's Horizon 2020 research and innovation programme under grant agreement No 727264 Epipharm (to ME), the 2015 Endowment fund for Research into Respiratory Health (“Fonds de Dotation Recherche en Santé Respiratoire”; to MD), the “Fondation ARC pour la recherche sur le cancer - Aide individuelle” (to MD), the Spanish Association Against Cancer (to VD), the Pla Estratègic de Recerca i Innovació en Salut (to SM), the Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional (PI16/01821 to LMM and PI15/02223, to IG-B), RETTICS grant (RD12/0036/0040 to IG-B), Fundación Merck Salud (to IG-B), Departamento de Salud, Gobierno de Navarra (074-2017, to IGB), the European Commission MSCA IMMUNOMARK-799818 (to IBara), the China Scholarship Council and Karolinska Institutet Fonder (to QX), and the Health and Science Departments of the Generalitat de Catalunya (to ME). We thank IGTP–HUGTP Biobank (PT13/0010/0009) University Hospital Ramon y Cajal-IRYCIS Biobank (PT13/0010/0002), members of the Spanish National Biobanks Network of the Instituto de Salud Carlos III, Andalusia Public Health System Biobank, the Biological Resource Center of the Hospices Civils de Lyon (Tissu-tumorothèque Est), and the Tumor Bank Network of Catalonia for their help with the collection of samples., Duruisseaux, M., Martínez-Cardús, A., Calleja-Cervantes, M.E., Moran, S., Castro de Moura, M. , Davalos, V., Piñeyro, D., Sanchez-Cespedes, M., Girard, N., Brevet, M.,Giroux-Leprieur, E., Dumenil, C. Pradotto, M., Bironzo, P., Capelletto, E., Novello, S., Cortot, A. , Copin, M.-C., Karachaliou, N.,Gonzalez-Cao, M. ,Peralta, S., Montuenga, L.M.,Gil-Bazo, I. ,Baraibar, I.,Lozano, M.D., Varela, M. ,Ruffinelli, J.C.,Palmero, R., Nadal, E.,Moran, T., Perez, L., Ramos, I., Xiao, Q.,Fernandez, A.F., Fraga, M.F.,Gut, M., Gut, I. ,Teixidó, C. , Vilariño, N., Prat, A., Reguart, N., Benito, A.,Garrido, P., Barragan, I., Emile, J.-F., Rosell, R., Brambilla, E., Esteller, M.

BackgroundHeritability 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.MethodsWe 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).ResultsHeritability 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 −9) at ZRANB2-AS2, PIK3R1, EPHA7, MAD1L1, CACUL1 and MAP3K9.ConclusionConsidering 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.

Introduction Les anti-PD-1 permettent un gain de survie dans les cancers bronchiques non a petites cellules (CBNPC) metastatiques. Cependant, la majorite des patients ne tire pas de benefice de ces traitements. Nous avons evalue l’interet de l’analyse a haut debit du methylome de CBNPC dans la prediction de l’efficacite des anti-PD-1. Methodes Etude multicentrique (15 centres) internationale (France, Espagne, Italie) retrospective. Criteres d’inclusion : CBNPC metastatique traite par anti-PD-1et prelevement tumoral disponible. Une cohorte decouverte (n = 34) a permis d’evaluer la correlation entre caracteristiques epigenetiques des tumeurs evaluees par methylome a haut debit (Illumina 850 K array beadchip) et efficacite des anti-PD-1. Une signature epigenetique (EPIMMUNE) predictive de l’efficacite des anti-PD-1 a ete elaboree et confirmee dans une cohorte de validation (n = 47). Nous avons isole un site specifique de methylation associe au benefice clinique et confirme son interet predictif par une technique de pyrosequencage dans une troisieme cohorte independante (n = 61). La methode de Kaplan–Meier a ete utilisee pour estimer la survie sans progression (SSP) et la survie globale (SG), le test de log-rank pour comparer les groupes de patients. Un modele de COX multivarie a ete construit pour identifier les variables independamment associees avec la SSP et la SG. Resultats La signature EPIMMUNE+ etait associee a une SSP allongee (HR 0,010, IC95 % 3,29 × 10–0,0282 ; p = 0,0067) et une SG allongee (HR 0,080, IC95 % 0,017–0,373 ; p = 0,0012) avec les anti-PD-1 dans la cohorte decouverte ( Fig. 1 ). Ces resultats etaient confirmes en SSP dans la cohorte validation. EPIMMUNE+ n’etait pas associee a l’expression de PD-L1, a l’infiltrat CD8 ou a la charge mutationnelle. Les tumeurs EPIMMUNE–etaient enrichies en macrophages, en polynucleaires neutrophiles, en fibroblastes et en cellules endotheliales senescentes. Le status demethyle du gene FOXP1 etait associe a une SSP allongee (p = 0,0063) et une SG allongee (p = 0,0094) dans la cohorte decouverte et dans une deuxieme cohorte de validation. EPIMMUNE+ et la demethylation de FOXP1 n’avait pas de valeur pronostique dans le TCGA. Conclusion Les caracteristiques epigenetique des cellules tumorales et du microenvironnement conditionnent l’efficacite des anti-PD-1 dans les CBNPC. La demethylation de FOXP1 pourrait etre un biomarqueur predictif de l’efficacite des anti-PD1 utilisable en clinique, complementaire de PD-L1 ou de la charge mutationnelle. Etude soutenue par le FRSR.

GAGA is a highly conserved Drosophila transcription factor encoded by the Trithorax-like (. Trl) gene. While GAGA usually activates transcription, it represses its own promoter. Here we show that GAGA-mediated repression of Trl is conserved between two distant Drosophila species. A detailed promoter study showed that GAGA repressive activity can't be attributed to any discrete element in the Trl promoter. Genome-wide analysis of the transcriptome in S2 cells indicated that repression of Trl is very likely unique, being GAGA factor a transactivator for all the other promoters. Taken together, our results suggest a new mechanism to explain GAGA-mediated repression that involves a dose-dependent change in the architecture of the Trl promoter. © 2013 Elsevier B.V., 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). D. Piñeyro was supported by FPU fellowship from the Spanish Government. M. Blanch was supported by I3P CPG_06_0034 contract from CSIC, and grant BFU-2007-64395/BMC contract of the Spanish Government. M. Badal was supported by grant BFU-2007-64395/BMC contract of the Spanish Government. This work was carried out in the frame of the “Centre de Referència en Biotecnologia” of the Generalitat de Catalunya.

The translation of genes encoded in the mitochondrial genome requires specific machinery that functions in the organelle. Among the many mutations linked to human disease that affect mitochondrial translation, several are localized to nuclear genes coding for mitochondrial aminoacyl-transfer RNA synthetases. The molecular significance of these mutations is poorly understood, but it is expected to be similar to that of the mutations affecting mitochondrial transfer RNAs. To better understand the molecular features of diseases caused by these mutations, and to improve their diagnosis and therapeutics, we have constructed a Drosophila melanogaster model disrupting the mitochondrial seryl-tRNA synthetase by RNA interference. At the molecular level, the knockdown generates a reduction in transfer RNA serylation, which correlates with the severity of the phenotype observed. The silencing compromises viability, longevity, motility and tissue development. At the cellular level, the knockdown alters mitochondrial morphology, biogenesis and function, and induces lactic acidosis and reactive oxygen species accumulation. We report that administration of antioxidant compounds has a palliative effect of some of these phenotypes. In conclusion, the fly model generated in this work reproduces typical characteristics of pathologies caused by mutations in the mitochondrial aminoacylation system, and can be useful to assess therapeutic approaches.

The epigenomic changes that occur during the process of cellular differentiation, such as in the blood cell lineage, are currently not well understood, especially at distant regulatory regions such as enhancer sequences. To study the effects of DNA methylation on cellular (de)differentiation, we have used a human B Cell Acute Lymphoblastic Leukemia model of transdifferentiation (BLaER1), which has an estradiol-inducible CEBPA construct that allows the conversion of leukemic B cells to non-tumorigenic macrophage-like cells. By analyzing the DNA methylation landscape of these cells at different time points of transdifferentiation with an Illumina EPIC methylation array, we have found enhancer-associated CpGs that shifts their methylation levels at the end of the transdifferentiation. By merging these results with the data obtained by Genome-wide Chromosome Conformation Capture Capture (Hi-C) in naive B cells and macrophages, we studied the putative interaction between several gene-promoters and our differentially methylated CpGs. We then proceed further to characterize the impact of the observed interactions on gene expression. We have identified the DNA methylation dependent enhancer interactomes of B-cells and macrophages. These target genes are related with vesicle trafficking, endocytosis and immune response. Our data highlight the role of DNA methylation to determine cell identity in the blood cell lineage. Disclosures No relevant conflicts of interest to declare.

Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.

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 Government

Inosine 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.

Faithful translation of genomes into proteomes depends, mainly, on the activity of transfer RNA (tRNA) as universal adaptor, as insightfully predicted by Crick. The central role of this relatively simple oligonucleotide depends upon a very large number of intermolecular interactions, some of which require that tRNAs maintain a constant general structure, while others depend on specific features that discriminate any given tRNA from the rest. Posttranscriptional modifications that increase the chemical diversity contained in the nucleotides of tRNAs can serve both purposes. Chemical modifications of tRNAs, thus, come in two general flavors: those that help to maintain the general shape of the molecule, and those that improve its interactions with one or more of its many molecular partners. Although the function of most of the chemical modifications known to occur in tRNAs remain unknown, up-to-date knowledge allows us to analyze the majority of them in some model organisms, including Saccharomyces cerevisiae. Here we will review our current understanding on the function of tRNA modifications, generally dividing them into two families: those that are likely to influence the structure of tRNA, and those that may play a role in the codon-anticodon interaction at the decoding center of the ribosome.

Drosophila telomere maintenance depends on the transposition of the specialized retrotransposons HeT-A, TART, and TAHRE. Controlling the activation and silencing of these elements is crucial for a precise telomere function without compromising genomic integrity. Here we describe two chromosomal proteins, JIL-1 and Z4 (also known as Putzig), which are necessary for establishing a fine-tuned regulation of the transcription of the major component of Drosophila telomeres, the HeT-A retrotransposon, thus guaranteeing genome stability. We found that mutant alleles of JIL-1 have decreased HeT-A transcription, putting forward this kinase as the first positive regulator of telomere transcription in Drosophila described to date. We describe how the decrease in HeT-A transcription in JIL-1 alleles correlates with an increase in silencing chromatin marks such as H3K9me3 and HP1a at the HeT-A promoter. Moreover, we have detected that Z4 mutant alleles show moderate telomere instability, suggesting an important role of the JIL-1-Z4 complex in establishing and maintaining an appropriate chromatin environment at Drosophila telomeres. Interestingly, we have detected a biochemical interaction between Z4 and the HeT-A Gag protein, which could explain how the Z4-JIL-1 complex is targeted to the telomeres. Accordingly, we demonstrate that a phenotype of telomere instability similar to that observed for Z4 mutant alleles is found when the gene that encodes the HeT-A Gag protein is knocked down. We propose a model to explain the observed transcriptional and stability changes in relation to other heterochromatin components characteristic of Drosophila telomeres, such as HP1a. © 2012 Silva-Sousa et al., This work was supported by a grant from the Spanish Ministry of Science and Innovation BFU2009-08318/BMC to EC and by a PhD Fellowship from Fundação para a Ciência e Tecnologia, Portugal, SFRH/BD/36291/2007 to RS-S.

Background Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the HeT-A retrotransposon. The sequence of the HeT-A retrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the HeT-A is entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different HeT-A scenarios. Results A detailed study on the variability of HeT-A reveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of HeT-A subfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the HeT-A RNA. Interestingly, our results also suggest that most HeT-A copies are actively expressed regardless of which telomere and where in the telomere they are located. Conclusions Our study demonstrates how the HeT-A sequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock., This work was supported by a grant from the Spanish Ministry of Science and Innovation BFU2009-08318/BMC to E.C.