Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1kd,3a−/−,3b−/−]). Sequencing of 960 RRBS clones from Dnmt[1kd,3a−/−,3b−/−] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1kd,3a−/−,3b−/−] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT. T he existence of non-CpG methylation in mammalian DNA has been a contentious issue. Conventional wisdom has it that mammalian methylation occurs predominantly (or even exclusively) at CpG dinucleotides (1, 2). However, some studies analyzing the nearest neighbors of 5-methylcytosine (5mC) on a genome-wide level have indicated that non-CpG methylation is present in significant quantities (3-6). Indeed, one report (6) has indicated that 55% of all methylation in human spleen DNA could be at dinucleotides other than CpG. It is likely that these wide variations in the reported frequency of non-CpG methylation result from artifacts intrinsic in the conventional nearest neighbor technique, but until the advent of specialized sequencing strategies, there was no way of confirming or refuting these studies.In fact, specialized sequencing strategies have offered little support for the existence of significant quantities of non-CpG methylation. One study reported non-CpG methylation in the promoter region of integrated adenovirus DNA, suggesting that it might be part of a de novo methylator response to invading viral genomes (7). Other work using mammalian cell lines and bisulphite sequencing (which enables the positive identification of all 5mC residues) reported non-CpG methylation within a stably integrated segment of plasmid DNA (8). The host cell lines appeared to have the capacity to maintain methylation at symmetric CpNpG sites, but some de novo CpNpG methylation at other sites within the integrated segment was also noted. Two reports indicating that nonCpG methylation might also be present in endogenous DNA elements, notably in mammalian replication origins (9, 10), have subsequently been disputed (11). The findings may have been attributable to failure of cytosine deamination in the bisulphite sequencing protocol. However, Woodcock et al. have observed nonsymmetric CpA and CpT methylation in an L1 element from human embryonic fibroblasts (12).Despite the relative lack of supporting evidence from sequencing, it could still be argued that non-CpG methylation is present but is confined to selective areas of th...

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Modifications in chromatin, including DNA methylation and histone modification, are known to be important epigenetic determinants of gene transcription. DNA methylation levels fluctuate markedly in early mouse development. In preimplantation development, the mouse embryo undergoes active and passive genomic demethylation (3,15). This is restored at the time of implantation by the combined action of de novo and maintenance DNA methyltransferases (Dnmts). Studies of DNA methyltransferase 1-deficient (Dnmt1 Ϫ/Ϫ ) and Dnmt3a/ Dnmt3b-deficient (Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ]) mouse embryos have demonstrated that restoring DNA methylation is essential for development (13,19). Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryos exhibit an early-lethal phenotype. At day 9.5 postcoitus, the embryos appear to have gastrulated but exhibit marked growth delay, having failed to turn or develop somites. In the presence of Dnmt1 deficiency, development is thought to fail because of cell death. Dnmt1-deficient embryoid bodies (EBs) aberrantly express Xist, down-regulate X-linked genes, and apoptose when induced to differentiate (20). Late-passage hypomethylated Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryonic stem (ES) cells are unable to form teratomas in vivo, but the cause of their differentiation failure has not been studied (4).Early embryonic development is characterized by high levels of Dnmt3a and Dnmt3b expression. These enzymes clearly have roles in initiating remethylation of the genome following preimplantation demethylation, but it is not known whether continued de novo methyltransferase activity is required for development once global remethylation has taken place. This was our reason for studying the differentiation of Dnmt[3a Ϫ/Ϫ , 3b Ϫ/Ϫ ] ES cells in vitro. These mutant ES cells were derived from fully methylated wild-type ES cells and would have been predicted to have retained most of their methylation because of the continued presence of the maintenance methyltransferase Dnmt1. In fact, while early-passage Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells are well methylated, DNA methylation levels fall progressively in culture (4). However, the rate of loss and the precise levels of methylation remaining have not been quantified. We have used a quantitative assay of DNA methylation to examine the effects of progressively decreasing genomic methylation levels on differentiation in vitro. Our studies reveal a clear but unexpected difference between the behaviors of hypomethylated Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells in in vitro assays of differentiation. At very low levels of DNA methylation, Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells demonstrate an inability to initiate differentiation upon leukemia inhibitory factor (LIF) withdrawal, remaining viable and retaining markers characteristic of undifferentiated ES cells. MATERIALS AND METHODSES cell culture. ES cells were maintained on gelatin in a Glasgow modification of Eagle medium (Invitrogen) supplemented with 10% fetal calf serum, 100 M 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 ...

Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.

SummaryHigher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A’s AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.