RESUMEN
Besides the consequences of retrotransposition, long interspersed element 1 (L1) retrotransposons can affect the host genome through their antisense promoter. In addition to the sense promoter, the evolutionarily recent L1 retrotransposons, which are present in several thousand copies, also possess an anti-sense promoter that can produce L1 chimeric transcripts (LCT) composed of the L1 5' UTR followed by the adjacent genomic sequence. The full extent to which LCT expression occurs in a given tissue and whether disruption of the defense mechanisms that normally control L1 retrotransposons affects their expression and function in cancer cells, remain to be established. By using CLIFinder, a dedicated bioinformatics tool, we found that LCT expression was widespread in normal brain and aggressive glioma samples, and that approximately 17% of recent L1 retrotransposons, from the L1PA1 to L1PA7 subfamilies, were involved in their production. Importantly, the transcriptional activities of the L1 antisense promoters and of their host loci were coupled. Accordingly, we detected LCT-producing L1 retrotransposons mainly in transcriptionally active genes and genomic loci. Moreover, changes in the host genomic locus expression level in glioma were associated with a similar change in LCT expression level, regardless of the L1 promoter methylation status. Our findings support a model in which the host genomic locus transcriptional activity is the main driving force of LCT expression. We hypothesize that this model is more applicable when host gene and LCT are transcribed from the same strand.
Asunto(s)
Glioma , Retroelementos , Encéfalo , Glioma/genética , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Regiones Promotoras Genéticas/genética , Retroelementos/genéticaRESUMEN
In cancer cells, aberrant DNA methylation is commonly associated with transcriptional alterations, including silencing of tumor suppressor genes. However, multiple epigenetic mechanisms, including polycomb repressive marks, contribute to gene deregulation in cancer. To dissect the relative contribution of DNA methylation-dependent and -independent mechanisms to transcriptional alterations at CpG island/promoter-associated genes in cancer, we studied 70 samples of adult glioma, a widespread type of brain tumor, classified according to their isocitrate dehydrogenase (IDH1) mutation status. We found that most transcriptional alterations in tumor samples were DNA methylation-independent. Instead, altered histone H3 trimethylation at lysine 27 (H3K27me3) was the predominant molecular defect at deregulated genes. Our results also suggest that the presence of a bivalent chromatin signature at CpG island promoters in stem cells predisposes not only to hypermethylation, as widely documented, but more generally to all types of transcriptional alterations in transformed cells. In addition, the gene expression strength in healthy brain cells influences the choice between DNA methylation- and H3K27me3-associated silencing in glioma. Highly expressed genes were more likely to be repressed by H3K27me3 than by DNA methylation. Our findings support a model in which altered H3K27me3 dynamics, more specifically defects in the interplay between polycomb protein complexes and the brain-specific transcriptional machinery, is the main cause of transcriptional alteration in glioma cells. Our study provides the first comprehensive description of epigenetic changes in glioma and their relative contribution to transcriptional changes. It may be useful for the design of drugs targeting cancer-related epigenetic defects.
Asunto(s)
Metilación de ADN/genética , Epigénesis Genética/genética , Glioma/genética , Transcripción Genética , Adulto , Línea Celular Tumoral , Cromatina/genética , Islas de CpG/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glioma/patología , Histonas/genética , Humanos , Isocitrato Deshidrogenasa/genética , Histona Demetilasas con Dominio de Jumonji/genética , Masculino , Regiones Promotoras GenéticasRESUMEN
Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify "indirect peaks" of multiple IBPs that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common cofactors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Regulación de la Expresión Génica , Elementos Aisladores/fisiología , ARN Polimerasa II/metabolismo , Animales , Sitios de Unión , Factor de Unión a CCCTC , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas del Ojo/metabolismo , Redes Reguladoras de Genes , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Mapeo de Interacción de Proteínas , Interferencia de ARN , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.
Asunto(s)
Dioxigenasas/metabolismo , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Neurogénesis , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Células Cultivadas , Dioxigenasas/genética , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Técnicas de Silenciamiento del Gen , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
Glioblastomas represent approximatively half of all gliomas and are the most deadly and aggressive form. Their therapeutic resistance and tumor relapse rely on a subpopulation of cells that are called Glioma Stem Cells (GSCs). Here, we investigated the role of the long non-coding RNA HOXA-AS2 in GSC biology using descriptive and functional analyses of glioma samples classified according to their isocitrate dehydrogenase (IDH) gene mutation status, and of GSC lines. We found that HOXA-AS2 is overexpressed only in aggressive (IDHwt) glioma and GSC lines. ShRNA-based depletion of HOXA-AS2 in GSCs decreased cell proliferation and altered the expression of several hundreds of genes. Integrative analysis revealed that these expression changes were not associated with changes in DNA methylation or chromatin signatures at the promoter of the majority of genes deregulated following HOXA-AS2 silencing in GSCs, suggesting a post-transcriptional regulation. In addition, transcription factor binding motif enrichment and correlation analyses indicated that HOXA-AS2 affects, directly or indirectly, the expression of key transcription factors implicated in GCS biology, including E2F8, E2F1, STAT1, and ATF3, thus contributing to GCS aggressiveness by promoting their proliferation and modulating the inflammation pathway.
Asunto(s)
Glioma , ARN Largo no Codificante , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Humanos , Inflamación/genética , Células Madre Neoplásicas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the high-salt-recovered sequences-sequencing (HRS-seq) method, a straightforward genome-wide approach whereby we isolated and sequenced genomic regions associated with large high-salt insoluble RNP complexes. By using mouse embryonic stem cells (ESCs), we showed that these regions essentially correspond to the most highly expressed genes, and to cis-regulatory sequences like super-enhancers, that belong to the active A chromosomal compartment. They include both cell-type-specific genes, such as pluripotency genes in ESCs, and housekeeping genes associated with nuclear bodies, such as histone and snRNA genes that are central components of Histone Locus Bodies and Cajal bodies. We conclude that HRSs are associated with the active chromosomal compartment and with large RNP complexes including nuclear bodies. Association of such chromosomal regions with nuclear bodies is in agreement with the recently proposed phase separation model for transcription control and might thus play a central role in organizing the active chromosomal compartment in mammals.
Asunto(s)
Cromosomas/química , Ribonucleoproteínas/química , Animales , Células Cultivadas , Fraccionamiento Químico/métodos , Cromosomas/metabolismo , Células Madre Embrionarias/metabolismo , Ratones , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Ribonucleoproteínas/metabolismo , SalinidadRESUMEN
Summary: L1 Chimeric Transcripts (LCTs) are initiated by repeated LINE-1 element antisense promoters and include the L1 5'UTR sequence in antisense orientation followed by the adjacent genomic region. LCTs have been characterized mainly using bioinformatics approaches to query dbEST. To take advantage of NGS data to unravel the transcriptome composition, we developed Chimeric LIne Finder (CLIFinder), a new bioinformatics tool. Using stranded paired-end RNA-seq data, we demonstrated that CLIFinder can identify genome-wide transcribed chimera sequences corresponding to potential LCTs. Moreover, CLIFinder can be adapted to study transcription from other repeat types. Availability and implementation: The code is available at: https://github.com/GReD-Clermont/CLIFinder; and for Galaxy users, it is directly accessible in the tool shed at: https://toolshed.g2.bx.psu.edu/view/clifinder/clifinder/. Contact: catherine.barriere@uca.fr. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Elementos de Nucleótido Esparcido Largo , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Perfilación de la Expresión Génica , Genoma Humano , Genómica , HumanosRESUMEN
Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation.
Asunto(s)
Metilación de ADN/genética , Impresión Genómica/genética , Células Germinativas/metabolismo , Oocitos/metabolismo , Animales , Blastocisto/metabolismo , Islas de CpG/genética , Femenino , Humanos , Masculino , Ratones , Placenta/metabolismo , Embarazo , Primates/genética , Primates/crecimiento & desarrollo , Espermatozoides/metabolismoRESUMEN
Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire life-span. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover, they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters, regardless of the developmental stage, thereby contributing to protect cell identity.
Asunto(s)
Alelos , Cromatina/metabolismo , Impresión Genómica , Animales , Células Cultivadas , Cromatina/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Células Madre Embrionarias/fisiología , Femenino , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Especificidad de Órganos , Regiones Promotoras GenéticasRESUMEN
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare cause of pulmonary hypertension in newborns. Maternally inherited point mutations in Forkhead Box F1 gene (FOXF1), deletions of the gene, or its long-range enhancers on the maternal allele are responsible for this neonatal lethal disorder. Here, we describe monozygotic twins and one full-term newborn with ACD and gastrointestinal malformations caused by de novo mutations of FOXF1 on the maternal-inherited alleles. Since this parental transmission is consistent with genomic imprinting, the parent-of-origin specific monoallelic expression of genes, we have undertaken a detailed analysis of both allelic expression and DNA methylation. FOXF1 and its neighboring gene FENDRR were both biallelically expressed in a wide range of fetal tissues, including lung and intestine. Furthermore, detailed methylation screening within the 16q24.1 regions failed to identify regions of allelic methylation, suggesting that disrupted imprinting is not responsible for ACDMPV.
Asunto(s)
Factores de Transcripción Forkhead/genética , Impresión Genómica , Síndrome de Circulación Fetal Persistente/genética , Alveolos Pulmonares/anomalías , Hibridación Genómica Comparativa , Metilación de ADN/genética , Femenino , Humanos , Hipertensión Pulmonar , Recién Nacido , Herencia Materna/genética , Mutación , Síndrome de Circulación Fetal Persistente/complicaciones , Síndrome de Circulación Fetal Persistente/patología , Embarazo , Alveolos Pulmonares/patología , Gemelos MonocigóticosRESUMEN
Differential methylation between the two alleles of a gene has been observed in imprinted regions, where the methylation of one allele occurs on a parent-of-origin basis, the inactive X-chromosome in females, and at those loci whose methylation is driven by genetic variants. We have extensively characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies, and hydatidiform moles, using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as to identify 21 novel loci harboring parent-of-origin methylation, 15 of which are restricted to the placenta. We observe that the extent of imprinted differentially methylated regions (DMRs) is extremely similar between tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Further, we profiled all imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically activated oocytes, individual blastomeres, and blastocysts, in order to identify primary DMRs and reveal the extent of reprogramming during preimplantation development. Intriguingly, we find that in contrast to ubiquitous imprints, the majority of placenta-specific imprinted DMRs are unmethylated in sperm and all human embryonic stem cells. Therefore, placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci.
Asunto(s)
Metilación de ADN/genética , Impresión Genómica/genética , Células Germinativas , Alelos , Islas de CpG/genética , Células Madre Embrionarias/citología , Femenino , Expresión Génica/genética , Genoma Humano , Humanos , Placenta/metabolismo , EmbarazoRESUMEN
Malignant gliomas are the most common primary brain tumors. Grade III and IV gliomas harboring wild-type IDH1/2 are the most aggressive. In addition to surgery and radiotherapy, concomitant and adjuvant chemotherapy with temozolomide (TMZ) significantly improves overall survival (OS). The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter is predictive of TMZ response and a prognostic marker of cancer outcome. However, the promoter regions the methylation of which correlates best with survival in aggressive glioma and whether the promoter methylation status predictive value could be refined or improved by other MGMT-associated molecular markers are not precisely known. In a cohort of 87 malignant gliomas treated with radiotherapy and TMZ-based chemotherapy, we retrospectively determined the MGMT promoter methylation status, genotyped single nucleotide polymorphisms (SNPs) in the promoter region and quantified MGMT mRNA expression level. Each of these variables was correlated with each other and with the patients' OS. We found that methylation of the CpG sites within MGMT exon 1 best correlated with OS and MGMT expression levels, and confirmed MGMT methylation as a stronger independent prognostic factor compared to MGMT transcription levels. Our main finding is that the presence of only the A allele at the rs34180180 SNP in the tumor was significantly associated with shorter OS, independently of the MGMT methylation status. In conclusion, in the clinic, rs34180180 SNP genotyping could improve the prognostic value of the MGMT promoter methylation assay in patients with aggressive glioma treated with TMZ.
Asunto(s)
Neoplasias Encefálicas/genética , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Glioma/genética , Polimorfismo de Nucleótido Simple , Proteínas Supresoras de Tumor/genética , Adulto , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Metilación de ADN/genética , Femenino , Genotipo , Glioma/mortalidad , Glioma/patología , Humanos , Estimación de Kaplan-Meier , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Pronóstico , Regiones Promotoras Genéticas/genética , Modelos de Riesgos Proporcionales , Estudios RetrospectivosRESUMEN
The myogenic regulatory factor Myod and insulin-like growth factor 2 (Igf2) have been shown to interact in vitro during myogenic differentiation. In order to understand how they interact in vivo, we produced double-mutant mice lacking both the Myod and Igf2 genes. Surprisingly, these mice display neonatal lethality due to severe diaphragm atrophy. Alteration of diaphragm muscle development occurs as early as 15.5 days post-coitum in the double-mutant embryos and leads to a defect in the terminal differentiation of muscle progenitor cells. A negative-feedback loop was detected between Myod and Igf2 in embryonic muscles. Igf2 belongs to the imprinted H19-Igf2 locus. Molecular analyses show binding of Myod on a mesodermal enhancer (CS9) of the H19 gene. Chromatin conformation capture experiments reveal direct interaction of CS9 with the H19 promoter, leading to increased H19 expression in the presence of Myod. In turn, the non-coding H19 RNA represses Igf2 expression in trans. In addition, Igf2 also negatively regulates Myod expression, possibly by reducing the expression of the Srf transcription factor, a known Myod activator. In conclusion, Igf2 and Myod are tightly co-regulated in skeletal muscles and act in parallel pathways in the diaphragm, where they affect the progression of myogenic differentiation. Igf2 is therefore an essential player in the formation of a functional diaphragm in the absence of Myod.
Asunto(s)
Diafragma/embriología , Epistasis Genética/fisiología , Factor II del Crecimiento Similar a la Insulina/genética , Proteína MioD/genética , ARN Largo no Codificante/genética , Animales , Animales Recién Nacidos , Diafragma/crecimiento & desarrollo , Diafragma/metabolismo , Embrión de Mamíferos , Femenino , Sitios Genéticos , Factor II del Crecimiento Similar a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Desarrollo de Músculos/genética , Proteína MioD/fisiología , Organogénesis/genética , Embarazo , ARN Largo no Codificante/fisiologíaRESUMEN
The junb gene behaves as an immediate early gene in bacterial lipopolysaccharide (LPS)-stimulated dendritic cells (DCs), where its transient transcriptional activation is necessary for the induction of inflammatory cytokines. junb is a short gene and its transcriptional activation by LPS depends on the binding of NF-κB to an enhancer located just downstream of its 3' UTR. Here, we have addressed the mechanisms underlying the transcriptional hyper-reactivity of junb. Using transfection and pharmacological assays to complement chromatin immunoprecipitation analyses addressing the localization of histones, polymerase II, negative elongation factor (NELF)-, DRB sensitivity-inducing factor (DSIF)- and Positive Transcription Factor b complexes, we demonstrate that junb is a RNA Pol II-paused gene where Pol II is loaded in the transcription start site domain but poorly active. Moreover, High salt-Recovered Sequence, chromosome conformation capture (3C)- and gene transfer experiments show that (i) junb is organized in a nuclear chromatin loop bringing into close spatial proximity the upstream promoter region and the downstream enhancer and (ii) this configuration permits immediate Pol II release on the junb body on binding of LPS-activated NF-κB to the enhancer. Thus, our work unveils a novel topological framework underlying fast junb transcriptional response in DCs. Moreover, it also points to a novel layer of complexity in the modes of action of NF-κB.
Asunto(s)
Cromatina/química , Células Dendríticas/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , Animales , Línea Celular , Células Dendríticas/química , Células Dendríticas/enzimología , Elementos de Facilitación Genéticos , Sitios Genéticos , Histonas/análisis , Humanos , Lipopolisacáridos/farmacología , Ratones , Conformación de Ácido Nucleico , ARN Polimerasa II/análisis , Factores de Transcripción/biosíntesis , Sitio de Iniciación de la TranscripciónRESUMEN
Paternal duplications of chromosome 6q24, a region that contains the imprinted PLAGL1 and HYMAI transcripts, are associated with transient neonatal diabetes mellitus. A common feature of imprinted genes is that they tend to cluster together, presumably as a result of sharing common cis-acting regulatory elements. To determine the extent of this imprinted cluster in human and mouse, we have undertaken a systematic analysis of allelic expression and DNA methylation of the genes mapping within an â¼1.4-Mb region flanking PLAGL1/Plagl1. We confirm that all nine neighbouring genes are biallelically expressed in both species. In human we identify two novel paternally expressed PLAGL1 coding transcripts that originate from unique promoter regions. Chromatin immunoprecipitation for CTCF and the cohesin subunits RAD21 and SMC3 reveals evolutionarily conserved binding sites within unmethylated regions â¼5 kb downstream of the PLAGL1 differentially methylated region and within the PLAGL1 3' untranslated region (UTR). Higher-order chromatin looping occurs between these regions in both expressing and non-expressing tissues, forming a non-allelic chromatin loop around the PLAGL1/Plagl1 gene. In placenta and brain tissues, we identify an additional interaction between the PLAGL1 P3/P4 promoters and the unmethylated element downstream of the PLAGL1 differentially methylated region that we propose facilitates imprinted expression of these alternative isoforms.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Impresión Genómica , Proteínas Represoras/metabolismo , Alelos , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Cromosomas Humanos Par 6 , Proteínas de Unión al ADN/metabolismo , Femenino , Genes Supresores de Tumor , Genotipo , Humanos , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Placenta/metabolismo , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Factores de Transcripción/genética , CohesinasRESUMEN
It is only partially understood how constitutive allelic methylation at imprinting control regions (ICRs) interacts with other regulation levels to drive timely parental allele-specific expression along large imprinted domains. The Peg13-Kcnk9 domain is an imprinted domain with important brain functions. To gain insights into its regulation during neural commitment, we performed an integrative analysis of its allele-specific epigenetic, transcriptomic, and cis-spatial organization using a mouse stem cell-based corticogenesis model that recapitulates the control of imprinted gene expression during neurodevelopment. We found that, despite an allelic higher-order chromatin structure associated with the paternally CTCF-bound Peg13 ICR, enhancer-Kcnk9 promoter contacts occurred on both alleles, although they were productive only on the maternal allele. This observation challenges the canonical model in which CTCF binding isolates the enhancer and its target gene on either side and suggests a more nuanced role for allelic CTCF binding at some ICRs.
Asunto(s)
Metilación de ADN , Impresión Genómica , Alelos , Metilación de ADN/genética , Impresión Genómica/genética , Regiones Promotoras Genéticas/genética , Animales , RatonesRESUMEN
Genomic imprinting is the parent-of-origin-specific allelic transcriptional silencing observed in mammals, which is governed by DNA methylation established in the gametes and maintained throughout the development. The frequency and extent of epimutations associated with the nine reported imprinting syndromes varies because it is evident that aberrant preimplantation maintenance of imprinted differentially methylated regions (DMRs) may affect multiple loci. Using a custom Illumina GoldenGate array targeting 27 imprinted DMRs, we profiled allelic methylation in 65 imprinting defect patients. We identify multilocus hypomethylation in numerous Beckwith-Wiedemann syndrome, transient neonatal diabetes mellitus (TNDM), and pseudohypoparathyroidism 1B patients, and an individual with Silver-Russell syndrome. Our data reveal a broad range of epimutations exist in certain imprinting syndromes, with the exception of Prader-Willi syndrome and Angelman syndrome patients that are associated with solitary SNRPN-DMR defects. A mutation analysis identified a 1 bp deletion in the ZFP57 gene in a TNDM patient with methylation defects at multiple maternal DMRs. In addition, we observe missense variants in ZFP57, NLRP2, and NLRP7 that are not consistent with maternal effect and aberrant establishment or methylation maintenance, and are likely benign. This work illustrates that further extensive molecular characterization of these rare patients is required to fully understand the mechanism underlying the etiology of imprint establishment and maintenance.
Asunto(s)
Alelos , Metilación de ADN , Estudio de Asociación del Genoma Completo , Impresión Genómica , Secuencia de Bases , Síndrome de Beckwith-Wiedemann/diagnóstico , Síndrome de Beckwith-Wiedemann/genética , Sitios de Unión , Islas de CpG , Femenino , Humanos , Masculino , Mutación , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Seudohipoaldosteronismo/diagnóstico , Seudohipoaldosteronismo/genética , Síndrome de Silver-Russell/diagnóstico , Síndrome de Silver-Russell/genética , Transactivadores/genéticaRESUMEN
For the past three decades, assisted reproductive technologies (ART) have revolutionized infertility treatments. The use of ART is thought to be safe. However, early investigations suggested that children born as a result of ART had higher risk of diseases with epigenetic etiologies, including imprinting disorders caused by a lack of maternal methylation at imprinting control elements. In addition, large epidemiology studies have highlighted an increased risk of obstetric complications, including severe intrauterine growth restriction (IUGR) in babies conceived using ART. It is plausible that the increased frequency of IUGR may be due to abnormal imprinting because these transcripts are key for normal fetal growth and development. To address this, we have collected a large cohort of placenta and cord blood samples from ART conceptions and compared the imprinting status with appropriate non-ART population. Using a custom DNA methylation array that simultaneously quantifies 25 imprinted differentially methylated regions, we observed similar epigenetic profiles between groups. A multiplex Sequenom iPLEX allelic expression assay revealed monoallelic expression for 11 imprinted transcripts in our placenta cohort. We also observe appropriate gestational age-dependent methylation dynamics at retrotransposable elements and promoters associated with growth genes in ART placental biopsies. This study confirms that children conceived by ART do not show variability in imprinted regulation and that loss-of-imprinting is not commonly associated with nonsyndromic IUGR or prematurity.
Asunto(s)
Metilación de ADN , Impresión Genómica , Inestabilidad Genómica , Edad Gestacional , Complicaciones del Embarazo/genética , Técnicas Reproductivas Asistidas/efectos adversos , Adulto , Estudios de Cohortes , Femenino , Humanos , Recién Nacido , Infertilidad/epidemiología , Infertilidad/genética , Infertilidad/terapia , Masculino , Placenta/metabolismo , Placenta/patología , Embarazo , Complicaciones del Embarazo/epidemiología , Embarazo Múltiple/genética , Embarazo Múltiple/metabolismo , Técnicas Reproductivas Asistidas/estadística & datos numéricosRESUMEN
Parental genomic imprinting at the Igf2/H19 locus is controlled by a methylation-sensitive CTCF insulator that prevents the access of downstream enhancers to the Igf2 gene on the maternal chromosome. However, on the paternal chromosome, it remains unclear whether long-range interactions with the enhancers are restricted to the Igf2 promoters or whether they encompass the entire gene body. Here, using the quantitative chromosome conformation capture assay, we show that, in the mouse liver, the endodermal enhancers have low contact frequencies with the Igf2 promoters but display, on the paternal chromosome, strong interactions with the intragenic differentially methylated regions 1 and 2. Interestingly, we found that enhancers also interact with a so-far poorly characterized intergenic region of the locus that produces a novel imprinted long non-coding transcript that we named the paternally expressed Igf2/H19 intergenic transcript (PIHit) RNA. PIHit is expressed exclusively from the paternal chromosome, contains a novel discrete differentially methylated region in a highly conserved sequence and, surprisingly, does not require an intact ICR/H19 gene region for its imprinting. Altogether, our data reveal a novel imprinted domain in the Igf2/H19 locus and lead us to propose a model for chromatin folding of this locus on the paternal chromosome.
Asunto(s)
Cromatina/química , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , ARN no Traducido/genética , Animales , Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Metilación de ADN , ADN Intergénico/metabolismo , Elementos de Facilitación Genéticos , Sitios Genéticos , Ratones , Modelos Genéticos , ARN Largo no Codificante , ARN no Traducido/metabolismoRESUMEN
In human, the 39 coding HOX genes and 18 referenced noncoding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression, DNA methylation, and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative 'master' HOX proteins that might contribute to the tumorigenic potential of glioma stem cells.