Summary: A combination of bisulfite treatment of DNA and high-throughput sequencing (BS-Seq) can capture a snapshot of a cell's epigenomic state by revealing its genome-wide cytosine methylation at single base resolution. Bismark is a flexible tool for the time-efficient analysis of BS-Seq data which performs both read mapping and methylation calling in a single convenient step. Its output discriminates between cytosines in CpG, CHG and CHH context and enables bench scientists to visualize and interpret their methylation data soon after the sequencing run is completed.Availability and implementation: Bismark is released under the GNU GPLv3+ licence. The source code is freely available from www.bioinformatics.bbsrc.ac.uk/projects/bismark/.Contact: felix.krueger@bbsrc.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

Methylation at the 5' position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

SummaryGenome-wide DNA methylation reprogramming occurs in mouse primordial germ cells (PGCs) and preimplantation embryos, but the precise dynamics and biological outcomes are largely unknown. We have carried out whole-genome bisulfite sequencing (BS-Seq) and RNA-Seq across key stages from E6.5 epiblast to E16.5 PGCs. Global loss of methylation takes place during PGC expansion and migration with evidence for passive demethylation, but sequences that carry long-term epigenetic memory (imprints, CpG islands on the X chromosome, germline-specific genes) only become demethylated upon entry of PGCs into the gonads. The transcriptional profile of PGCs is tightly controlled despite global hypomethylation, with transient expression of the pluripotency network, suggesting that reprogramming and pluripotency are inextricably linked. Our results provide a framework for the understanding of the epigenetic ground state of pluripotency in the germline.

Rac, a member of the Rho family of monomeric GTPases, is an integrator of intracellular signaling in a wide range of cellular processes. We have purified a PtdIns(3,4,5)P3-sensitive activator of Rac from neutrophil cytosol. It is an abundant, 185 kDa guanine-nucleotide exchange factor (GEF), which we cloned and named P-Rex1. The recombinant enzyme has Rac-GEF activity that is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gbetagammas both in vitro and in vivo. P-Rex1 antisense oligonucleotides reduced endogenous P-Rex1 expression and C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line. P-Rex1 appears to be a coincidence detector in PtdIns(3,4,5)P3 and Gbetagamma signaling pathways that is particularly adapted to function downstream of heterotrimeric G proteins in neutrophils.

Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity. Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives (queens) and nonreproductives (workers). The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults. We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs (miRNAs), and multiple transcriptomes and methylomes from individual brains in a wasp (Polistes canadensis) and an ant (Dinoponera quadriceps) that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs. Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood. These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity.