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[Background]: The abundance of energy metabolites is intimately interconnected with the activity of chromatin-modifying enzymes in order to guarantee the finely tuned modulation of gene expression in response to cellular energetic status. Metabolism-induced epigenetic gene regulation is a key molecular axis for the maintenance of cellular homeostasis, and its deregulation is associated with several pathological conditions. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) using the universal methyl donor S-adenosyl methionine (SAM), directly linking one-carbon metabolism with a cell's methylation balance and nicotinamide adenine dinucleotide (NAD+) levels. NNMT expression and activity are regulated in a tissue-specific-manner, and the protein can act either physiologically or pathologically depending on its distribution. While NNMT exerts a beneficial effect by regulating lipid parameters in the liver, its expression in adipose tissue correlates with obesity and insulin resistance. NNMT upregulation has been observed in a variety of cancers, and increased NNMT expression has been associated with tumor progression, metastasis and worse clinical outcomes. Accordingly, NNMT represents an appealing druggable target for metabolic disorders as well as oncological and other diseases in which the protein is improperly activated. ; [Scope of review]: This review examines emerging findings concerning the complex NNMT regulatory network and the role of NNMT in both NAD metabolism and cell methylation balance. We extensively describe recent findings concerning the physiological and pathological regulation of NNMT with a specific focus on the function of NNMT in obesity, insulin resistance and other associated metabolic disorders along with its well-accepted role as a cancer-associated metabolic enzyme. Advances in strategies targeting NNMT pathways are also reported, together with current limitations of NNMT inhibitor drugs in clinical use. ; [Major conclusions]: NNMT is emerging as a key point of intersection between cellular metabolism and epigenetic gene regulation, and growing evidence supports its central role in several pathologies. The use of molecules that target NNMT represents a current pharmaceutical challenge for the treatment of several metabolic-related disease as well as in cancer. ; This research was funded by the Spanish Association Against Cancer (PROYE18061FERN to M.F.F.), the Health Institute Carlos II (ISCIII/FEDER PI18/01527 to A.F.F. and M.F.F.), the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contract CP11/00131 to A.F.F.), the Plan de Ciencia, Tecnología e Innovación from the Asturias Government cofunding (2018–2022/FEDER to M.F.F.), and FINBA-ISPA (A.R.). The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain. ; Peer reviewed

Vicenzo Calvanese et al. ; Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation. ; This work was primarily supported by the European Union (LSHG-CT-2006-018739; ESTOOLS). MFF is funded by the Spanish Ramon & Cajal Programme and the Health Department of the Spanish Government (PI061267). The Cancer Epigenetics group at the CNIO is supported by the Health (FIS01-04) and Education and Science (I+D+I MCYT08-03, FU2004-02073/BMC and Consolider MEC09-05) Departments of the Spanish Government, the European Grant TRANSFOG LSHC-CT-2004-503438, and the Spanish Association Against Cancer (AECC). VC is a recipient of a Fellowship from the FPU Spanish Research Programme. CLL and BSA are supported by the Health Department of the Spanish Government (PI051707). The BACM is supported by the Consejería de Salud de la Junta de Andalucía (0029 and, 0030/2006 to PM) and, the Spanish Ministry of Health to PM (FIS PI070026). CB is supported by the International Jose Carreras Foundation against Leukemia (EDThomas-05) and the ISCIII (FIS 3+3 contract). The Institute of Reconstructive Neurobiology received additional funding from the DFG and the Hertie Foundation. BS, AbH and AnH are supported by the Fundación Progreso y Salud and Instituto de Salud Carlos III-Red Española de Terapia Celular (RD06/0010/0025 ). PWA, NH and HDM are also supported by the MRC. ; Peer reviewed

12 páginas, 3 figuras, 3 tablas.-- et al. ; [Introduction]: Identification of gene expression-based breast cancer subtypes is considered a critical means of prognostication. Genetic mutations along with epigenetic alterations contribute to gene-expression changes occurring in breast cancer. So far, these epigenetic contributions to sporadic breast cancer subtypes have not been well characterized, and only a limited understanding exists of the epigenetic mechanisms affected in those particular breast cancer subtypes. The present study was undertaken to dissect the breast cancer methylome and to deliver specific epigenotypes associated with particular breast cancer subtypes. [Methods]: By using a microarray approach, we analyzed DNA methylation in regulatory regions of 806 cancer-related genes in 28 breast cancer paired samples. We subsequently performed substantial technical and biologic validation by pyrosequencing, investigating the top qualifying 19 CpG regions in independent cohorts encompassing 47 basal-like, 44 ERBB2+ overexpressing, 48 luminal A, and 48 luminal B paired breast cancer/adjacent tissues. With the all-subset selection method, we identified the most subtype-predictive methylation profiles in multivariable logistic regression analysis. [Results]: The approach efficiently recognized 15 individual CpG loci differentially methylated in breast cancer tumor subtypes. We further identified novel subtype-specific epigenotypes that clearly demonstrate the differences in the methylation profiles of basal-like and human epidermal growth factor 2 (HER2)-overexpressing tumors. [Conclusions]: Our results provide evidence that well-defined DNA methylation profiles enable breast cancer subtype prediction and support the utilization of this biomarker for prognostication and therapeutic stratification of patients with breast cancer. ; This work was supported by grants from project CGL2008-01131 (Departamento de Sanidad del Gobierno Vasco), S-PE08UN45 and PE09BF02 (Departamento de Ciencia y Tecnologia del Gobierno Vasco), BIO2008-04212, and RD06/0020/1019 (Red Tematica de Investigacion Cooperativa en Cancer, RTICC) from the MICINN. The CIBER de Enfermedades Raras is an initiative of the ISCIII. NGB had a doctoral fellowship from the Basque Government (Departamento de Educacion, Universidades e Investigacion). ; Peer reviewed

The emergence of nanotechnology applied to medicine has revolutionized the treatment of human cancer. As in the case of classic drugs for the treatment of cancer, epigenetic drugs have evolved in terms of their specificity and efficiency, especially because of the possibility of using more effective transport and delivery systems. The use of nanoparticles (NPs) in oncology management offers promising advantages in terms of the efficacy of cancer treatments, but it is still unclear how these NPs may be affecting the epigenome such that safe routine use is ensured. In this work, we summarize the importance of the epigenetic alterations identified in human cancer, which have led to the appearance of biomarkers or epigenetic drugs in precision medicine, and we describe the transport and release systems of the epigenetic drugs that have been developed to date. ; This work has been financially supported by The Plan Nacional de I+D+I FEDER (PI15/00892 and PI18/01527); the Government of the Principality of Asturias PCTI-Plan de Ciencia, Tecnología e Innovación de Asturias 2018-2022/FEDER (IDI/2018/146); AECC (PROYE18061FERN); FGCSIC (0348_CIE_6_E); IUOPA-ISPA-FINBA (The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain); A.F. Fernández is supported by a Miguel Servet II fellowship (contract CPII16/00007). ; Peer reviewed

Cancer is an aging-associated disease, but the underlying molecular links between these processes are still largely unknown. Gene promoters that become hypermethylated in aging and cancer share a common chromatin signature in ES cells. In addition, there is also global DNA hypomethylation in both processes. However, the similarity of the regions where this loss of DNA methylation occurs is currently not well characterized, and it is unknown if such regions also share a common chromatin signature in aging and cancer. To address this issue, we analyzed TCGA DNA methylation data from a total of 2,311 samples, including control and cancer cases from patients with breast, kidney, thyroid, skin, brain, and lung tumors and healthy blood, and integrated the results with histone, chromatin state, and transcription factor binding site data from the NIH Roadmap Epigenomics and ENCODE projects. We identified 98,857 CpG sites differentially methylated in aging and 286,746 in cancer. Hyper- and hypomethylated changes in both processes each had a similar genomic distribution across tissues and displayed tissue-independent alterations. The identified hypermethylated regions in aging and cancer shared a similar bivalent chromatin signature. In contrast, hypomethylated DNA sequences occurred in very different chromatin contexts. DNA hypomethylated sequences were enriched at genomic regions marked with the activating histone posttranslational modification H3K4me1 in aging, while in cancer, loss of DNA methylation was primarily associated with the repressive H3K9me3 mark. Our results suggest that the role of DNA methylation as a molecular link between aging and cancer is more complex than previously thought. ; This work was supported by the Plan Nacional de I+D+I [2013‐2016/FEDER PI15/00892 to M.F.F. and A.F.F., 2008‐2011/FEDER CP11/00131 to A.F.F.]; the ISCIII‐Subdirección General de Evaluación y Fomento de la Investigación [Miguel Servet contract CP11/00131 to A.F.F.]; Instituto Universitario de Oncología del Principado de Asturias (IUOPA) to [G.F.B.]; Fundación Ramón Areces to [M.F.F.]; and the Asturias Regional Government [GRUPIN14‐052 to M.F.F.]. R.F.P. is supported by the Retención de Jóvenes Talentos Fellowship from the Obra Social Cajastur‐Liberbank. J.R.T. is supported by the Ministry of Economy and Competitiveness Through a Juan de la Cierva Postdoctoral Fellowship [FJCI‐2015‐26965]. The IUOPA is supported by the Obra Social Cajastur‐Liberbank, Spain. ; Peer Reviewed

This article belongs to the Special Issue Transcriptional and Epigenetic Regulation of Pluripotency and Differentiation. ; Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture. ; Work in the laboratory of M.F.F. was funded by the Spanish Association Against Cancer (PROYE18061FERN to M.F.F.), the Asturias Government (PCTI) co-funding 2018-2022/FEDER (IDI/2018/146 to M.F.F.), Fundación General CSIC (0348_CIE_6_E to M.F.F.) and the Health Institute Carlos III (Plan Nacional de I+D+I) co-funding FEDER (PI18/01527 to M.F.F). R.G.U. is supported by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). We also acknowledge support from the IUOPA-ISPA-FINBA (the IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain). Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF2017-82613-R), ERC (ERC-2014-AdG/669622), Banco Santander (Santander Universities Global Division), la Caixa Foundation, and Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282). ; Peer reviewed

Background Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential. Aim and scope A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures. Methods We compared hADSC cultures at short (P S ) and prolonged (P L ) passages, both in standard and low [O 2 ] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster. Results Comparison of hADSCs cultured at P L or P S surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in P L cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O 2 ], was also significantly higher in P L than in P S passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions. Conclusion A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice. ; This work was supported by grants to AB from the Spanish Ministry of Economy, Industry (SAF2015-70882-R; AEI/FEDER, UE), Comunidad Autónoma de Madrid (S2010/BMD-2420), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0018) and the European Commission (FP7-HEALTH- 2009/CARE-MI). AMS was supported by grants from the MINECO (SAF2010–17167) and Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013), and MFF and RGU by grants from the Plan Nacional de I+D+I 2013-2016/FEDER (PI15/ 00892), the Asturias Regional Government (GRUPIN14-052), the IUOPA (Obra Social Cajastur) and the Fundación Científica de la AECC. SGL held a predoctoral fellowship from the Spanish Programa de Formación del Profesorado Universitario

[Introduction]: Cell-free DNA (cfDNA) methylation is an important molecular biomarker, which provides information about the regulation of gene expression in the tissue of origin. There is an inverse correlation between SOST gene methylation and expression levels. ; [Methods]: We analyzed SOST promoter methylation in cfDNA from serum, and compared it with DNA from blood and bone cells from patients undergoing hip replacement surgery. We also measured cfDNA methylation in 28 osteoporotic patients at baseline and after 6 months of antiosteoporotic therapy (alendronate, teriparatide, or denosumab). ; [Results]: SOST gene promoter methylation levels in serum cfDNA were very similar to those of bone-derived DNA (79% ± 12% and 82% ± 7%, respectively), but lower than methylation levels in blood cell DNA (87% ± 10%). Furthermore, there was a positive correlation between an individual's SOST DNA methylation values in serum and bone. No differences in either serum sclerostin levels or SOST methylation were found after 6-months of therapy with antiosteoporotic drugs. ; [Conclusions]: Our results suggest that serum cfDNA does not originate from blood cells, but rather from bone. However, since we did not confirm changes in this marker after therapy with bone-active drugs, further studies examining the correlation between bone changes of SOST expression and SOST methylation in cfDNA are needed to confirm its potential role as a bone biomarker. ; This study was supported by two grants from Instituto de Salud Carlos III (PI12/615 and PI16/915), through a program potentially co-funded by FEDER Funds from the European Union. ; Peer reviewed

The presence of nanomaterials in our everyday life is ever increasing, and so too are concerns about the possible health consequences of exposure to them. While evidence of their biological activity is growing, there is still scant knowledge of the epigenetic mechanisms that could be at play in these processes. Moreover, the great variability in the chemical and physical structures of these compounds handicaps the study of their possible health risks. Here we have synthesized reduced graphene oxide (rGO) through the thermal exfoliation/reduction of graphite oxide, and characterized the resulting material. We have then made use of Illumina's MethylationEPIC arrays and bisulphite pyrosequencing to analyse the genome-wide and global DNA methylation dynamics associated with the medium-term exposure of human lung epithelial cells to rGO at concentrations of 1 and 10 µg/mL. The results show no genome-wide or global DNA methylation changes associated with either condition. Our observations thus suggest that medium-term rGO exposure does not have significant effects on the DNA methylation patterns of human lung epithelial cells. ; This work has been financially supported by: The Plan Nacional de I+D+I co-funding FEDER (PI15/00892 and PI18/01527); the Government of the Principality of Asturias PCTI-Plan de Ciencia, Tecnología e Innovación de Asturias co-funding 2018-2022/FEDER (IDI/2018/146 and IDI/2018/121); AECC (PROYE18061FERN); FGCSIC (0348_CIE_6_E); IUOPA-ISPA-FINBA (The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain); A.F. Fernández is supported by a Miguel Servet II fellowship (contract CPII16/00007); R.F. Pérez is supported by ISPA-FINBA. ; Peer reviewed

Aging and cancer are two interrelated processes, with aging being a major risk factor for the development of cancer. Parallel epigenetic alterations have been described for both, although differences, especially within the DNA hypomethylation scenario, have also been recently reported. Although many of these observations arise from the use of mouse models, there is a lack of systematic comparisons of human and mouse epigenetic patterns in the context of disease. However, such comparisons are significant as they allow to establish the extent to which some of the observed similarities or differences arise from pre-existing species-specific epigenetic traits. Here, we have used reduced representation bisulfite sequencing to profile the brain methylomes of young and old, tumoral and nontumoral brain samples from human and mouse. We first characterized the baseline epigenomic patterns of the species and subsequently focused on the DNA methylation alterations associated with cancer and aging. Next, we described the functional genomic and epigenomic context associated with the alterations, and finally, we integrated our data to study interspecies DNA methylation levels at orthologous CpG sites. Globally, we found considerable differences between the characteristics of DNA methylation alterations in cancer and aging in both species. Moreover, we describe robust evidence for the conservation of the specific cancer and aging epigenomic signatures in human and mouse. Our observations point toward the preservation of the functional consequences of these alterations at multiple levels of genomic regulation. Finally, our analyses reveal a role for the genomic context in explaining disease- and species-specific epigenetic traits. ; This work was supported by the Spanish Association Against Cancer (PROYE18061FERN to M.F.F.), the Asturias Government (PCTI) cofunding 2018-2022/FEDER (IDI/2018/146 to M.F.F.), Fundación General CSIC (0348_CIE_6_E to M.F.F.), and the Health Institute Carlos III (Plan Nacional de I + D+I) cofunding FEDER (PI15/00892 and PI18/01527 to M.F.F and A.F.F.). They also acknowledge support from the Ramón Areces Foundation (CIVP18A3891 to P.J.F.M.), the AECC (SIRTBIO to P.J.F.M.), the MICINN (SAF2017-85766-R to P.J.F.M.), a Ramón y Cajal fellowship (MICINN, RYC-2017-22335 to P.J.F.M.), and the European Commission ATTRACT project (777222 to A.P.C.). J.R.T. is supported by a Juan de la Cierva fellowship from the Spanish Ministry of Science and Innovation (FJCI-2015-26965). R.F.P. and P.S.O. are supported by the Severo Ochoa program (BP17-114 and BP17-165, respectively). R.G.U. is supported by the Centro de Investigación Biomédica en Red de Enfermedades Raras (Health Institute Carlos III). L.V. was supported by the UAB Predoctoral training programme (PIF predoctoral fellowships). They also acknowledge support from the IMDEA Food Institute and IUOPA-ISPA-FINBA (the IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain). ; Peer reviewed

[Background]: Age-associated changes in genomic DNA methylation have been primarily attributed to 5-methylcytosine (5mC). However, the recent discovery of 5-hydroxymethylcytosine (5hmC) suggests that this epigenetic mark might also play a role in the process. [Methods]: Here, we analyzed the genome-wide profile of 5hmc in mesenchymal stem cells (MSCs) obtained from bone-marrow donors, aged 2-89 years. [Results]: We identified 10,685 frequently hydroxymethylated CpG sites in MSCs that were, as in other cell types, significantly associated with low density CpG regions, introns, the histone posttranslational modification H3k4me1 and enhancers. Study of the age-associated changes to 5hmC identified 785 hyper- and 846 hypo-hydroxymethylated CpG sites in the MSCs obtained from older individuals. [Conclusions]: DNA hyper-hydroxymethylation in the advanced-age group was associated with loss of 5mC, which suggests that, at specific CpG sites, this epigenetic modification might play a role in DNA methylation changes during lifetime. Since bone-marrow MSCs have many clinical applications, and the fact that the epigenomic alterations in this cell type associated with aging identified in this study could have associated functional effects, the age of donors should be taken into account in clinical settings. ; This work has been financially supported by the Plan Nacional de I + D + I 2008–2011/2013–2016/FEDER (PI11/01728 to A.F.F.; PI12/01080 and PI15/00892 to M.F.F.; PI 12/0615 to J.A.R.; PI05/2217 and PI08/0029 to J.G-C); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contract: CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); Fundacion Cientifica de la AECC (to R.G.U.); Fundación Ramón Areces (to M.F.F); and FICYT (to E.G.T., M.G.G and A.C.);the Madrid Regional Government (S-BIO-02042006 and S2010/BMD-2420 to J.G-C) and the Asturias Regional Government (GRUPIN14-052 to M.F.F.). The IUOPA is supported by the Obra Social Cajastur, Spain. ; Peer Reviewed

BACKGROUND: Age-associated changes in genomic DNA methylation have been primarily attributed to 5-methylcytosine (5mC). However, the recent discovery of 5-hydroxymethylcytosine (5hmC) suggests that this epigenetic mark might also play a role in the process. METHODS: Here, we analyzed the genome-wide profile of 5hmc in mesenchymal stem cells (MSCs) obtained from bone-marrow donors, aged 2-89 years. RESULTS: We identified 10,685 frequently hydroxymethylated CpG sites in MSCs that were, as in other cell types, significantly associated with low density CpG regions, introns, the histone posttranslational modification H3k4me1 and enhancers. Study of the age-associated changes to 5hmC identified 785 hyper- and 846 hypo-hydroxymethylated CpG sites in the MSCs obtained from older individuals. CONCLUSIONS: DNA hyper-hydroxymethylation in the advanced-age group was associated with loss of 5mC, which suggests that, at specific CpG sites, this epigenetic modification might play a role in DNA methylation changes during lifetime. Since bone-marrow MSCs have many clinical applications, and the fact that the epigenomic alterations in this cell type associated with aging identified in this study could have associated functional effects, the age of donors should be taken into account in clinical settings. ; This work has been financially supported by the Plan Nacional de I+D+I 2008–2011/2013–2016/FEDER (PI11/01728 to A.F.F.; PI12/01080 and PI15/00892 to M.F.F.; PI 12/0615 to J.A.R.; PI05/2217 and PI08/0029 to J.G‑C); the ISCIII‑Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contract: CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); Fundacion Cientifica de la AECC (to R.G.U.); Fundación Ramón Areces (to M.F.F); and FICYT (to E.G.T., M.G.G and A.C.); the Madrid Regional Government (S‑BIO‑0204‑2006 and S2010/BMD‑2420 to J.G‑C) and the Asturias Regional Government (GRUPIN14‑052 to M.F.F.). The IUOPA is supported by the Obra Social Cajastur, Spain.

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor in adulthood. Epigenetic mechanisms are known to play a key role in GBM although the involvement of histone methyltransferase KMT5B and its mark H4K20me2 has remained largely unexplored. The present study shows that DNA hypermethylation and loss of DNA hydroxymethylation is associated with KMT5B downregulation and genome-wide reduction of H4K20me2 levels in a set of human GBM samples and cell lines as compared with non-tumoral specimens. Ectopic overexpression of KMT5B induced tumor suppressor-like features in vitro and in a mouse tumor xenograft model, as well as changes in the expression of several glioblastoma-related genes. H4K20me2 enrichment was found immediately upstream of the promoter regions of a subset of deregulated genes, thus suggesting a possible role for KMT5B in GBM through the epigenetic modulation of key target cancer genes. ; This research was funded by the Health Institute Carlos III (Plan Nacional de I+D+I) cofounding FEDER (PI15/00892 and PI18/01527 to MF and AF); the Government of the Principality of Asturias PCTI-Plan de Ciencia, Tecnología e Innovación de Asturias co-funding 2018–2022/FEDER (IDI/2018/146 to MF); AECC (PROYE18061FERN to MF); FGCSIC (0348_CIE_6_E to MF); Severo Ochoa Program BP17-165 to PS-O and BP17-114 to RP); the Ministry of Economy and Competitiveness of Spain (VL, Juan de la Cierva fellowship IJCI-2015-23316; JT, Juan de la Cierva fellowship FJCI-2015-26965); FICYT (AC and MG); FINBA-ISPA (VL); and IUOPA (VL and CM). The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain. ; Peer reviewed

Introduction: Cell-free DNA (cfDNA) methylation is an important molecular biomarker, which provides information about the regulation of gene expression in the tissue of origin. There is an inverse correlation between SOST gene methylation and expression levels. Methods: We analyzed SOST promoter methylation in cfDNA from serum, and compared it with DNA from blood and bone cells from patients undergoing hip replacement surgery. We also measured cfDNA methylation in 28 osteoporotic patients at baseline and after 6 months of antiosteoporotic therapy (alendronate, teriparatide, or denosumab). Results: SOST gene promoter methylation levels in serum cfDNA were very similar to those of bone-derived DNA (79% ± 12% and 82% ± 7%, respectively), but lower than methylation levels in blood cell DNA (87% ± 10%). Furthermore, there was a positive correlation between an individual's SOST DNA methylation values in serum and bone. No differences in either serum sclerostin levels or SOST methylation were found after 6-months of therapy with antiosteoporotic drugs. Conclusions: Our results suggest that serum cfDNA does not originate from blood cells, but rather from bone. However, since we did not confirm changes in this marker after therapy with bone-active drugs, further studies examining the correlation between bone changes of SOST expression and SOST methylation in cfDNA are needed to confirm its potential role as a bone biomarker. ; This study was supported by two grants from Instituto de Salud Carlos III (PI12/615 and PI16/915), through a program potentially co-funded by FEDER Funds from the European Union.

Abstract Background Epstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth. Results We investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-κB p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation. Conclusions Our data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts. ; This work was supported by grants PI081346 (FIS) and SAF2011-29635 from the Spanish Ministry of Science and Innovation (MICINN) and grant 2009SGR184 from AGAUR (Catalan Government). AI was supported by fellowship from AGAUR, Government of Catalonia, Spain. MP is supported by Ramon y Cajal Programme. ; Peer Reviewed