Variability and adaptability are necessary for overcoming the challenges of multicellular life. To address this need, nature
has evolved a substantial enzymatic toolbox for altering cytosine within the genome. Methylation of the nucleotide cytosine
(C) at the 5-position of the base has profound impacts on gene expression and cellular identity. The reverse of this process,
DNA demethylation, is equally important for cleaning the genomic slate during embryogenesis or achieving rapid reactivation
of previously silenced genes. Although the mechanism of DNA methylation has been rigorously established, active DNA demethylation
in mammals has remained enigmatic, as disparate observations have failed to coalesce into a consistent model. Cytosine deamination,
oxidation, and base excision repair enzymes have been proposed in a dizzying variety of combinations (1). Against this backdrop, two reports in this issue, by Ito et al. (2) on page 1300 and He et al. on page 1303 (3), help bring new clarity to the mechanistic model for DNA demethylation.
"The comet-FISH approach with single-strand probes can quantify various DNA lesions and DNA modifications at physiological levels, allowing the study of damage induction and removal in each DNA strand in defined sequences at the single-molecule level, and in the genome overall in individual cells. These DNA lesions and modifications include different lesions corrected by NER or BER pathways, DNA single-strand breaks and double-strand breaks generated at chromosomal fragile sites (46) or induced by ionizing radiation, 5-formylcytosine and 5-carboxylcytosine involved in active DNA demethylation through BER (47) and ribonucleotides in DNA removed by ribonuclease H (48). By using multispectral fluorescent tags with distinguishable emission signatures (49) to label the strand-specific probes, the comet-FISH approach could enable the analysis of DNA lesions in multiple designated regions in the genome in single cells. "
[Show abstract][Hide abstract] ABSTRACT: Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m(2), a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.
Nucleic Acids Research 06/2013; 41(16). DOI:10.1093/nar/gkt524 · 9.11 Impact Factor
"Recent findings have raised the possibility that regulation by DNA methylation may be quite dynamic rather being static (47) that led to the emergence of enzymes capable of mediating DNA demethylation in mammalian cells (53). The involvement of DNA demethylation under certain conditions such as extrinsic signals, in early stages of development and in highly specialized post-mitotic cells has been reported recently. "
[Show abstract][Hide abstract] ABSTRACT: Recent genome-wide mapping of the mammalian replication origins has suggested the role of transcriptional regulatory elements in origin activation. However, the nature of chromatin modifications associated with such trans-factors or epigenetic marks imprinted on cis-elements during the spatio-temporal regulation of replication initiation remains enigmatic. To unveil the molecular underpinnings, we studied the human lamin B2 origin that spatially overlaps with TIMM 13 promoter. We observed an early G(1)-specific occupancy of c-Myc that facilitated the loading of mini chromosome maintenance protein (MCM) complex during subsequent mid-G(1) phase rather stimulating TIMM 13 gene expression. Investigations on the Myc-induced downstream events suggested a direct interaction between c-Myc and histone methyltransferase mixed-lineage leukemia 1 that imparted histone H3K4me3 mark essential for both recruitment of acetylase complex HBO1 and hyperacetylation of histone H4. Contemporaneously, the nucleosome remodeling promoted the loading of MCM proteins at the origin. These chromatin modifications were under the tight control of active demethylation of E-box as evident from methylation profiling. The active demethylation was mediated by the Ten-eleven translocation (TET)-thymine DNA glycosylase-base excision repair (BER) pathway, which facilitated spatio-temporal occupancy of Myc. Intriguingly, the genome-wide 43% occurrence of E-box among the human origins could support our hypothesis that epigenetic control of E-box could be a molecular switch for the licensing of early replicating origins.
Nucleic Acids Research 07/2012; 40(18):9021-35. DOI:10.1093/nar/gks617 · 9.11 Impact Factor
"Both 5hmC and 5mC can be further oxidized by the Tet dioxygenases to 5-carboxylcytosine (5acC), which in turn is removed by thymine-DNA glycosylase thereby establishing a mechanism for DNA demethylation (He et al. 2011). These findings suggest a complex chemistry leading to different modification states of cytosine in DNA and allowing methyl-removal to unmethylated DNA (Nabel and Kohli 2011). Similar to chromatin modifications, DNA methylation has to be understood as a dynamic and reversible modification in development. "
[Show abstract][Hide abstract] ABSTRACT: The distinct cell types of the body are established from the fertilized egg in development and assembled into functional tissues. Functional characteristics and gene expression patterns are then faithfully maintained in somatic cell lineages over a lifetime. On the molecular level, transcription factors initiate lineage-specific gene expression programmmes and epigenetic regulation contributes to stabilization of expression patterns. Epigenetic mechanisms are essential for maintaining stable cell identities and their disruption can lead to disease or cellular transformation. Here, we discuss the role of epigenetic regulation in the early mouse embryo, which presents a relatively well-understood system. A number of studies have contributed to the understanding of the function of Polycomb group complexes and the DNA methylation system. The role of many other chromatin regulators in development remains largely unexplored. Albeit the current picture remains incomplete, the view emerges that multiple epigenetic mechanisms cooperate for repressing critical developmental regulators. Some chromatin modifications appear to act in parallel and others might repress the same gene at a different stage of cell differentiation. Studies in pluripotent mouse embryonic stem cells show that epigenetic mechanisms function to repress lineage specific gene expression and prevent extraembryonic differentiation. Insights into this epigenetic "memory" of the first lineage decisions help to provide a better understanding of the function of epigenetic regulation in adult stem cell differentiation.
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