A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

Background: In human Estrogen Receptor alpha (ER alpha)-positive breast cancers, 59 end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ER alpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation. Methodology/Principal Findings: We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ER alpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 59 end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ER alpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ER alpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ER alpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated. Conclusions/Significance: MBD2 and ER alpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ER alpha and MBD2

Genetic And Epigenetic Determinants In Autoinflammatory Diseases

The concept of autoinflammation has evolved over the past 20 years, beginning with the discovery that mutations in the Mediterranean Fever (MEFV) gene were causative of Familial Mediterranean Fever. Currently, autoinflammatory diseases comprise a wide range of disorders with the common features of recurrent fever attacks, prevalence of hyperreactive innate immune cells, and signs of inflammation that can be systemic or organ specific in the absence of pathogenic infection of autoimmunity. Innate immune cells from the myeloid compartment are the main effectors of uncontrolled inflammation that is caused in great extent by the overproduction of inflammatory cytokines such as IL-1 beta and IL-18. Defects in several signaling pathways that control innate immune defense, particularly the hyperreactivity of one or more inflammasomes, are at the core of pathologic autoinflammatory phenotypes. Although many of the autoinflammatory syndromes are known to be monogenic, some of them are genetically complex and are impacted by environmental factors. Recently, epigenetic dysregulation has surfaced as an additional contributor to pathogenesis. In the present review, we discuss data that are currently available to describe the contribution of epigenetic mechanisms in autoinflammatory diseases

A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2/TFF1 Gene

In human Estrogen Receptor alpha (ERalpha)-positive breast cancers, 5' end dense methylation of the estrogen-regulated pS2/TFF1 gene correlates with its transcriptional inhibition. However, in some ERalpha-rich biopsies, pS2 expression is observed despite the methylation of its TATA-box region. Herein, we investigated the methylation-dependent mechanism of pS2 regulation.We observed interplay between Methyl-CpG Binding Domain protein 2 (MBD2) transcriptional repressor and ERalpha transactivator: (i) the pS2 gene is poised for transcription upon demethylation limited to the enhancer region containing the estrogen responsive element (ERE); (ii) MBD2-binding sites overlapped with the methylation status of the pS2 5' end; (iii) MBD2 depletion elevated pS2 expression and ectopic expression of ERalpha partially overcame the inhibitory effect of MBD2 when the ERE is unmethylated. Furthermore, serial chromatin immunoprecipitation assays indicated that MBD2 and ERalpha could simultaneously occupy the same pS2 DNA molecule; (iv) concomitant ectopic ERalpha expression and MBD2 depletion resulted in synergistic transcriptional stimulation, while the pS2 promoter remains methylated.MBD2 and ERalpha drive opposite effects on pS2 expression, which are associated with specific steady state levels of histone H3 acetylation and methylation marks. Thus, epigenetic silencing of pS2 could be dependent on balance of the relative intracellular concentrations of ERalpha and MBD2

Understanding the relevance of DNA methylation changes in immune differentiation and disease

Altres ajuts: We thank CERCA Programme/Generalitat de Catalunya and Josep Carreras Foundation for institutional support. E.B. was funded by [...] and was co-funded by FEDER funds/European Regional Development Fund (ERDF)-a way to build Europe.Immune cells are one of the most complex and diverse systems in the human organism. Such diversity implies an intricate network of different cell types and interactions that are dependently interconnected. The processes by which different cell types differentiate from progenitors, mature, and finally exert their function requires an orchestrated succession of molecular processes that determine cell phenotype and function. The acquisition of these phenotypes is highly dependent on the establishment of unique epigenetic profiles that confer identity and function on the various types of effector cells. These epigenetic mechanisms integrate microenvironmental cues into the genome to establish specific transcriptional programs. Epigenetic modifications bridge environment and genome regulation and play a role in human diseases by their ability to modulate physiological programs through external stimuli. DNA methylation is one of the most ubiquitous, stable, and widely studied epigenetic modifications. Recent technological advances have facilitated the generation of a vast amount of genome-wide DNA methylation data, providing profound insights into the roles of DNA methylation in health and disease. This review considers the relevance of DNA methylation to immune system cellular development and function, as well as the participation of DNA methylation defects in immune-mediated pathologies, illustrated by selected paradigmatic diseases

The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties

The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD-DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16(INK4a) genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing

Genome-Wide Screen for Differential DNA Methylation Associated with Neural Cell Differentiation in Mouse

Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation

La justicia en el tráfico: Conocimiento y valoración de la población española.

La norma existe sea cual sea su manifestación, porque es producto y necesidad de la interacción entre las personas. Pero no tiene sentido si nadie controla que ésta se cumpla. Necesitamos a alguien que administre finalmente las sanciones cuando no se cumpla la norma. Necesitamos algo/alguien que administre la veracidad de que los comportamientos realmente se han producido, de la intención de los mismos, que evalúe la gravedad, que los ponga en relación con lo que las normas dicen, sus excepciones… Necesitamos juzgar antes de sancionar. Es necesario aplicar la justicia. Cada uno de nosotros en algún momento asumimos alguno o todos estos roles a la vez. En efecto, en muchas de nuestras actuaciones diarias, ya sean laborales, familiares…, dictamos normas, controlamos que éstas se cumplan y si se nos permite decirlo, dictamos sentencias para los individuos sobre los que tenemos influencias. En el tema del tráfico, de los accidentes e incidentes, como en toda conducta que se lleva a cabo en un sistema interactivo, hay víctimas y culpables/responsables. En nuestro país existe una tendencia abrumadora en los últimos tiempos, de “intensificar temporalmente los controles”, a intentar establecer nuevas fórmulas sancionadoras e incluso aumentar la calificación de gravedad de la infracción, así como su cuantía. Una de las preguntas que surgen a colación de esto sería ¿se justifica este empeño en detrimento de otras medidas?, ¿se atiende a las variables que, al menos teóricamente aumentarán la eficacia de estas medidas? Hemos constatado que realizando un estudio poblacional facilitamos los posicionamientos de las personas e incrementamos la riqueza en las discusiones a través de todas las variables que hemos podido tratar. Conocer los pensamientos de la población española nos va a permitir, sin lugar a dudas, analizar el problema con mayor rigor, así como proponer soluciones (medidas y contramedidas) más ajustadas a la realidad social sobre la que se va a aplicar. Con ello, pretendemos convertir este libro en manual de consulta para aquellos que intervienen en el marco de la seguridad vial tanto a nivel general como a nivel de los que participan más activamente en el tema más especifico que aquí trabajamos. El libro se ha estructurado en tres grandes partes: En la primera de ellas, para permitir una composición mejor acerca de las circunstancias de las que se han extraído los datos del estudio, se describe la metodología del mismo. En una segunda parte presentamos los más relevantes y significativos resultados del estudio, capítulo que está dividido en diferentes bloques: Normativa, Supervisión Policial, Sanciones, Justicia, Modelos de Respuesta y Medidas. Los cuatro primeros bloques aunque son independientes, puesto que tratan aspectos distintos de un proceso que tiene una cronología relativamente clara, tienen un cierto grado de relación que indudablemente va a quedar reflejado en un análisis que cruza algunas de estas variables con el quinto bloque. Además, este quinto bloque que aborda una serie de conductas, de las cuales algunas son sancionables y otras no lo son, tiene un análisis autónomo que trata de reflejar las relaciones que tiene el continuo con las conductas, las creencias, los conocimientos, las actitudes, etc. En el sexto bloque se analizan, también desde el doble enfoque: de forma autónoma y en relación con los otros, las medidas que se llevan o se pueden llevar a cabo y que tienen relación con la temática que estamos trabajando. En realidad, “medidas” son todos los puntos tratados, pero en este bloque analizamos algunas características diferenciales de las mismas tal y como existen y tal y como podrían ser que, nos parece, tienen una implicación diferencial En una tercera parte, realizamos un recorrido sobre algunos de los resultados que, siendo significativos, puedan ofrecer un panorama general de los conocimientos obtenidos mediante esta investigación. Y lo hacemos desde la óptica, que nunca debemos perder, de las implicaciones que los mismos pueden tener desde un punto de vista aplicado. Recogemos en este libro “análisis complementarios” y conclusiones que nos ayuden a encontrar respuestas en la búsqueda de la máxima eficacia y eficiencia del sistema, considerando sus posibles alternativas

Circulating cell-free DNA methylation mirrors alterations in cerebral patterns in epilepsy

Background: DNA methylation profiling of circulating cell-free DNA (cfDNA) has rapidly become a promising strategy for biomarker identification and development. The cell-type-specific nature of DNA methylation patterns and the direct relationship between cfDNA and apoptosis can potentially be used non-invasively to predict local alterations. In addition, direct detection of altered DNA methylation patterns performs well as a biomarker. In a previous study, we demonstrated marked DNA methylation alterations in brain tissue from patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Results: We performed DNA methylation profiling in cfDNA isolated from the serum of MTLE patients and healthy controls using BeadChip arrays followed by systematic bioinformatic analysis including deconvolution analysis and integration with DNase accessibility data sets. Differential cfDNA methylation analysis showed an overrepresentation of gene ontology terms and transcription factors related to central nervous system function and regulation. Deconvolution analysis of the DNA methylation data sets ruled out the possibility that the observed differences were due to changes in the proportional contribution of cortical neurons in cfDNA. Moreover, we found no overrepresentation of neuron- or glia-specific patterns in the described cfDNA methylation patterns. However, the MTLE-HS cfDNA methylation patterns featured a significant overrepresentation of the epileptic DNA methylation alterations previously observed in the hippocampus. Conclusions: Our results support the use of cfDNA methylation profiling as a rational approach to seeking non-invasive and reproducible epilepsy biomarkers.This study has been supported by R+D+i project PID2020-117212RB-I00 funded by MCIN/AEI/10.13039/501100011033. This work has also been par‑ tially supported by a BICE Tecnifar Grant. RM-F is funded by an FCT (Fundação para a Ciência e Tecnologia) fellowship (SFRH/BD/137900/2018). UMIB is funded by FCT Portugal (UIDB/00215/2020 and UIDP/00215/2020) and ITR (LA/P/006/2020).info:eu-repo/semantics/publishedVersio

Los jóvenes en el tráfico: Una visión en primera persona

Los accidentes de tráfico de los conductores jóvenes son un problema de salud y consecuentemente es importante intentar comprender qué está pasando. Los jóvenes constituyen un grupo ampliamente declarado de alto riesgo, donde se conjuga la juventud con la inexperiencia, es y ha de ser, por su inminente o reciente participación en el sistema de tráfico, un grupo objeto de la educación y la formación vial. Los jóvenes, los jóvenes que exhiben conductas de riesgo, los jóvenes que exhiben una conducción arriesgada, han sido objeto de una extensa investigación. Hay muchos factores que contribuyen a la formación de estas actitudes, creencias y comportamientos. Los medios de comunicación, especialmente la televisión, los padres y los "iguales" son centros de influencia que a menudo actúan mediante la potenciación de las actitudes y comportamientos negativos. Es por ello que no podemos responsabilizar en mayor medida a los jóvenes sin que paralelamente nos responsabilicemos más nosotros en lo que se refiere a nuestros comportamientos en general y en los que tenemos con respecto a ellos, al menos en lo que se refiere al tráfico y la seguridad vial. Hemos considerado necesario realizar un análisis de lo que opina la población española, de lo que opinan los jóvenes, toda vez que indagamos en otras cuestiones como sus actitudes, la relación con sus padres, en relación siempre con la seguridad vial. Sin habernos olvidado de abordar aspectos que tienen que ver con las creencias, las actitudes y los conocimientos, nos vamos a centrar en averiguar otros aspectos como son: los jóvenes en su relación con el tráfico y con sus vehículos (ambos hechos objetivos), el historial de accidentes y la tasa de infracciones que suelen cometer (restringidas a unas infracciones específicas), y en su defensa, cómo no, la relación que tienen estas infracciones y accidentes con las de sus padres en lo que hemos venido a titular “padres y jóvenes unidos por el tráfico”, amén de tratar de establecer qué relación tiene todo esto con sus estilos de vida. Y para ello, el libro se ha estructurado en tres grandes apartados: En el primero de ellos, para permitir una composición mejor acerca de las circunstancias de dónde se han extraído los datos del estudio, se describe la metodología del mismo. En un segundo apartado presentamos los más relevantes y significativos resultados del estudio. En un tercer apartado, realizamos un recorrido sobre algunos de los resultados que, siendo significativos, puedan ofrecer un panorama general de los conocimientos obtenidos mediante esta investigación. Y lo hacemos desde la óptica, que nunca debemos perder, de las implicaciones que los mismos pueden tener desde un punto de vista aplicado

DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E

The present work investigates the occurrence and significance of aberrant DNA methylation patterns during early stages of atherosclerosis. To this end, we asked whether the genetically atherosclerosis-prone APOE-null mice show any changes in DNA methylation patterns before the appearance of histologically detectable vascular lesion. We exploited a combination of various techniques: DNA fingerprinting, in vitro methyl-accepting assay, 5-methylcytosine quantitation, histone post-translational modification analysis, Southern blotting, and PCR. Our results show that alterations in DNA methylation profiles, including both hyper- and hypomethylation, were present in aortas and PBMC of 4-week-old mutant mice with no detectable atherosclerotic lesion. Sequencing and expression analysis of 60 leukocytic polymorphisms revealed that epigenetic changes involve transcribed genic sequences, as well as repeated interspersed elements. Furthermore, we showed for the first time that atherogenic lipoproteins promote global DNA hypermethylation in a human monocyte cell line. Taken together, our results unequivocally show that alterations in DNA methylation profiles are early markers of atherosclerosis in a mouse model and may play a causative role in atherogenesis