H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.This work has been financially supported by: the Plan Nacional de I+D+I 2013–2016/FEDER (PI15/00892 to M.F.F. and A.F.F.; RTC-2015-3393-1 to A.F.F.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, and the Plan Nacional de I+D+I 2008–2011/FEDER (CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); the Fundación Científica de la AECC (to R.G.U.); the Fundación Ramón Areces (to M.F.F); FICYT (to E.G.T., M.G.G. and A.C.); and the Asturias Regional Government (GRUPIN14-052 to M.F.F.). Work in P.M. laboratory is supported by the European Research Council (CoG-2014-646903), the Spanish Ministry of Economy-Competitiveness (SAF-SAF2013-43065), the Obra Social La Caixa-Fundaciò Josep Carreras, and the Generalitat de Catalunya. P.M. is an investigator in the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain.Peer reviewe

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death

DNA methylation and hidroxymethylation changes during aging in human mesenchymal stem cells

Programa de Doctorado “Investigación en Cáncer".[ES] La metilación del ADN genómico en la posición 5 de la citosina (5mC) es el mecanismo epigenético más estudiado y está involucrada en numerosos procesos celulares, como la organización de la cromatina, la impronta genómica, inactivación del cromosoma X y la regulación de la expresión génica, entre otros. Las alteraciones en los patrones de metilación del ADN están relacionadas con el desarrollo de numerosas enfermedades, incluido el cáncer. Además, varios estudios han propuesto que también pueden jugar un papel importante en el envejecimiento y en la predisposición de enfermedades relacionadas con la edad. Por otra parte, el reciente descubrimiento de la 5-hidroximetilcitosina (5hmC), y el hecho de que una parte de la metilación del ADN, que asumía que se trataba en su totalidad de 5mC, se trata en realidad de 5hmC, sugiere que esta marca epigenética deba tenerse también en cuenta en futuros estudios de metilación. El envejecimiento se caracteriza por una pérdida de funciones corporales acompañado por una degeneración general de células y tejidos. En este contexto, el deterioro y la pérdida de respuesta al daño que tienen lugar a lo largo de la vida en células no terminalmente diferenciadas, como las células madre adultas, jugarían un papel crucial en el proceso de declive celular y tisular durante el envejecimiento. Esta tesis doctoral tiene como propósito describir los cambios de metilación e hidroximetilación del ADN que tienen lugar a lo largo de la vida de células madre mesenquimales, su relación entre sí y su asociación con las diferentes marcas post-traduccionales de histonas. Para ello, en primer lugar estudiamos los cambios de metilación del ADN en 34 muestras obtenidas de individuos de edades comprendidas entre 2 y 91 años mediante la plataforma Infinium HumanMethylation450 BeadChip Kit 450 (Illumina Inc). Mediante esta tecnología de microarray, identificamos 18735 sitios CpG hipermetilados y 45407 hipometilados con la edad. Estos cambios de metilación son, en su mayoría, dependientes del tipo celular ya que sólo una pequeña parte de los cambios es común a otros tipos celulares. Mientras que la ganancia de metilación durante el envejecimiento está asociada a las marcas represivas de histonas H3K27me3 y H3k9me3, la hipometilación del ADN está relacionada con la marca de activación H3K4me1 en MSCs. Ambas asociaciones tienen también lugar también en células diferenciadas, lo que sugiere que estas marcas de histonas están asociadas a los cambios de metilación durante el envejecimiento independientemente del tipo celular. Además, el hecho de que la H3k4me1 haya sido recientemente relacionada en estudios previos con enhancers, sugiere que, H3k4me1 e hipometilación del ADN, podrían estar implicadas en los cambios de la regulación de expresión génica que tiene lugar durante el envejecimiento. Además de los cambios de metilación del ADN durante el transcurso de la vida, en esta tesis se describen los patrones de 5hmC en MSCs y los cambios que tienen lugar en individuos de avanzada edad. Para ello, estudiamos los perfiles de 5hmC en 17 muestras obtenidas de individuos de edades comprendidas entre 2 y 89 años, nuevamente, a través de la plataforma Infinium HumanMethylation450 BeadChip Kit 450 (Illumina Inc). Los análisis permitieron identificar 10685 sitios CpG frecuentemente hidroximetilados en células madre mesenquimales que estaban enriquecidos en regiones de baja densidad de CpGs, intrones y regiones intergénicas, y en enhancers asociados a la marca de histonas H3K4me1. Estos resultados están en línea con trabajos anteriores tanto de células madre embrionarias, como de células terminalmente diferenciadas. Un análisis posterior de los datos permitió identificar 1595 sitios CpG diferencialmente hidroximetilados con la edad. De ellos, 749 se hiperhidroximetilaban y 846 hipohidroximetilaban en individuos de avanzada edad. Al comparar los datos de 5hmC con los cambios de 5mC con la edad, se encontró que el aumento de 5hmC asociado a la edad ocurre frecuentemente en regiones que pierden metilación en personas de avanzada edad, en línea con lo descrito en otros trabajos, lo que sugiere que la 5hmC podría jugar un papel en los cambios de metilación del ADN que tienen lugar durante el envejecimiento. Durante el desarrollo de esta tesis doctoral se ha puesto a punto el desarrollo de una técnica que permite el análisis de la 5hmC mediante la pirosecuenciación del ADN en regiones específicas, la cual permitirá, en estudios futuros, el análisis de los patrones de 5hmC de una forma región-específica, lo que ayudará a esclarecer el papel funcional de la 5hmC en el ADN y su implicación en los diferentes procesos biológicos como el desarrollo embrionario, la diferenciación celular, el envejecimiento y el desarrollo de diferentes enfermedades, incluido el cáncer.[EN] DNA methylation is one of the best-known epigenetic modifications in mammals. It is an important epigenetic mark involved in gene expression, X chromosome and transposon inactivation, and genomic imprinting, among others. Alterations of DNA methylation patterns are related to many diseases, including cancer. Moreover, it is well known that genomic DNA methylation is modified during the lifetime of higher organisms, and this variation has been regularly ascribed to the epigenetic mark 5-methylcytosine. However, the recent discovery of the presence of 5-hydroxymethylcytosine (5hmC) in DNA implies that this epigenetic mark must be taken into account in studies of DNA methylation and aging. Aging is defined as the time-dependent functional decline characterized by a progressive loss of physiological integrity, accompanied by loss of functionally of cells and tissues. Throughout life, adult stem cells are subject to the environmental stresses and intracellular damages and could play an important role in the aging process. The work presented in this Thesis concerns the description of DNA methylation and hydroxymethylation changes during aging in MSCs, the relationship between both epigenetic marks, and the possible association between these changes and the chromatin marks. DNA methylation profiling of mesenchymal stem cells obtained from 34 bone marrow donors, aged 2 to 92, identified 18735 hypermethylated and 45407 hypomethylated CpG sites associated with aging. Interestingly, cell type seems to play an important role in the regulation of DNA methylation changes over time since many of the sequences were not common to different tissues. As in differentiated cells, hypermethylated sequences in MSCs were enriched in the the chromatin repressive marks H3K9me3 and H3K27me3. Moreover, hypomethylated CpG sites were highly enriched in the active chromatin mark H3K4me1 in MSCs and differentiated cells, suggesting this is a cell type independent chromatin signature of DNA hypomethylation during aging. Interestingly, H3K4me1 has recently been associated with enhancers, which suggests that H3K4me1-associated DNA hypomethylation could play a role in the changes of gene expression during aging. Besides, we analyzed the genome-wide profile of 5hmc in MSCs obtained from 17 bone marrow donors, aged 2 to 89 years. We identified 10685 frequently hydroxymethylated CpG sites in MSCs that were significantly associated with low density CpG regions, introns, the histone posttranslational modification H3k4me1 and enhancers, in line with previous studies carried out in other cell types. Study of the age-associated changes to 5hmC identified 785 hyper- and 846 hypohydroxymethylated CpG sites in the MSCs obtained from older individuals. Interestingly, DNA hyperhydroxymethylation in the advanced-age group was associated with loss of 5-methylcytosine, which suggests that this epigenetic modification plays a role in changes in genomic DNA methylation during lifetime. During the course of this work we have developed a protocol optimization technique that is applicable to analyze 5hmC through bisulfite pyrosequencing approached. This method allows site-specific DNA hydroxymethylation analysis, and will help to elucidate the functional role of 5hmC in the different biological process, such as cell differentiation, development, aging and disease in future projects.Esta tesis doctoral ha sido realizada gracias al disfrute de una Ayuda Predoctoral para la formación en investigación y docencia del Principado de Asturias del programa Severo Ochoa (FICYT, BP11-138). El trabajo experimental ha sido financiado por los proyectos de la Consejería de Economía y Empleo del Principado de Asturias (GRUPIN14-052) y el Fondo de Investigación Sanitaria (FIS), Ministerio de Sanidad y Consumo (PPI12/01080).Peer reviewe

Aberrant DNA methylation patterns of spermatozoa in men with unexplained infertility

STUDY QUESTION Are there DNA methylation alterations in sperm that could explain the reduced biological fertility of male partners from couples with unexplained infertility? SUMMARY ANSWER DNA methylation patterns, not only at specific loci but also at Alu Yb8 repetitive sequences, are altered in infertile individuals compared with fertile controls. STUDY DESIGN, SIZE, DURATION Case and control prospective study. This study compares 46 sperm samples obtained from 17 normospermic fertile men and 29 normospermic infertile patients. MAIN RESULTS AND THE ROLE OF CHANCE In this study we conduct, for the first time, a genome-wide study to identify alterations of sperm DNA methylation in individuals with unexplained infertility that may account for the differences in their biological fertility compared with fertile individuals. We have identified 2752 CpGs showing aberrant DNA methylation patterns, and more importantly, these differentially methylated CpGs were significantly associated with CpG sites which are specifically methylated in sperm when compared with somatic cells. We also found statistically significant (P < 0.001) associations between DNA hypomethylation and regions corresponding to those which, in somatic cells, are enriched in the repressive histone mark H3K9me3, and between DNA hypermethylation and regions enriched in H3K4me1 and CTCF, suggesting that the relationship between chromatin context and aberrant DNA methylation of sperm in infertile men could be locus-dependent. Finally, we also show that DNA methylation patterns, not only at specific loci but also at several repetitive sequences (LINE-1, Alu Yb8, NBL2, D4Z4), were lower in sperm than in somatic cells. Interestingly, sperm samples at Alu Yb8 repetitive sequences of infertile patients showed significantly lower DNA methylation levels than controls

A Note on Two Rare Bibles

Additional file 6: Table S6. Gene ontology analyisis of hyper5hmC CpG sites during aging. RR: relative risk is a measure of effect size describing the change of proportions between our selected set of genes and a given term; Q value is the result from the adjustment of P values in order to control the false discovery rate (FDR) using the Benjamini-Hochberg method