RESUMO
Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.
Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Viral/genética , Metilação de DNA/genética , Herpesvirus Humano 4/genética , Linfócitos B/virologia , Carcinogênese , Ilhas de CpG/genética , DNA Viral/genética , Genoma Humano , Humanos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Replication origins fire at different times during S-phase. Such timing is determined by the chromosomal context, which includes the activity of nearby genes, telomeric position effects and chromatin structure, such as the acetylation state of the surrounding chromatin. Activation of replication origins involves the conversion of a pre-replicative complex to a replicative complex. A pivotal step during this conversion is the binding of the replication factor Cdc45, which associates with replication origins at approximately their time of activation in a manner partially controlled by histone acetylation. METHODOLOGY/PRINCIPAL FINDINGS: Here we identify histone H3 K36 methylation (H3 K36me) by Set2 as a novel regulator of the time of Cdc45 association with replication origins. Deletion of SET2 abolishes all forms of H3 K36 methylation. This causes a delay in Cdc45 binding to origins and renders the dynamics of this interaction insensitive to the state of histone acetylation of the surrounding chromosomal region. Furthermore, a decrease in H3 K36me3 and a concomitant increase in H3 K36me1 around the time of Cdc45 binding to replication origins suggests opposing functions for these two methylation states. Indeed, we find K36me3 depleted from early firing origins when compared to late origins genomewide, supporting a delaying effect of this histone modification for the association of replication factors with origins. CONCLUSIONS/SIGNIFICANCE: We propose a model in which K36me1 together with histone acetylation advance, while K36me3 and histone deacetylation delay, the time of Cdc45 association with replication origins. The involvement of the transcriptionally induced H3 K36 methylation mark in regulating the timing of Cdc45 binding to replication origins provides a novel means of how gene expression may affect origin dynamics during S-phase.
Assuntos
Proteínas de Ligação a DNA/genética , Histonas/genética , Metilação , Proteínas Nucleares/genética , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Separação Celular , Cromatina/química , Primers do DNA/química , Citometria de Fluxo , Genoma , Metiltransferases/metabolismo , Modelos Genéticos , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/ultraestruturaRESUMO
Beckwith-Wiedemann syndrome (BWS) is a model human imprinting disorder resulting from altered activity of one or more genes in the 11p15.5 imprinted gene cluster. Approximately 20% of BWS cases have uniparental disomy (UPD) of chromosome 11. Such cases appear to result from mitotic recombination occurring in early embryogenesis and offer a rare opportunity to study mitotic recombination in nonneoplastic cells. We analyzed a cohort of 52 children with BWS and UPD using a panel of microsatellite markers for chromosome 11. All cases demonstrated mosaic paternal isodisomy, and IGF2 and H19 were included in the segment of UPD in all cases. However, the extent of segmental disomy was variable, with no evidence of clustering of the proximal UPD breakpoint. In most cases (92% of those informative) UPD did not involve 11q, but 4 patients demonstrated UPD for the whole of chromosome 11. In contrast to meiotic recombination, the mitotic recombination frequency did not decline near the centromere.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Cromossomos Humanos Par 11 , Mitose/genética , Recombinação Genética , Dissomia Uniparental/genética , Criança , Metilação de DNA , Impressão Genômica , HumanosRESUMO
Recently, the application of array-based comparative genomic hybridization (array CGH) has improved rates of detection of chromosomal imbalances in individuals with mental retardation and dysmorphic features. Here, we describe three individuals with learning disability and a heterozygous deletion at chromosome 17q21.3, detected in each case by array CGH. FISH analysis demonstrated that the deletions occurred as de novo events in each individual and were between 500 kb and 650 kb in size. A recently described 900-kb inversion that suppresses recombination between ancestral H1 and H2 haplotypes encompasses the deletion. We show that, in each trio, the parent of origin of the deleted chromosome 17 carries at least one H2 chromosome. This region of 17q21.3 shows complex genomic architecture with well-described low-copy repeats (LCRs). The orientation of LCRs flanking the deleted segment in inversion heterozygotes is likely to facilitate the generation of this microdeletion by means of non-allelic homologous recombination.
