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Accumulating observations suggest that peripheral somatosensory ganglia may regulate nociceptive transmission, yet direct evidence is sparse. Here, in experiments on rats and mice, we show that the peripheral afferent nociceptive information undergoes dynamic filtering within the dorsal root ganglion (DRG) and suggest that this filtering occurs at the axonal bifurcations (t-junctions). Using synchronous in vivo electrophysiological recordings from the peripheral and central processes of sensory neurons (in the spinal nerve and dorsal root), ganglionic transplantation of GABAergic progenitor cells, and optogenetics, we demonstrate existence of tonic and dynamic filtering of action potentials traveling through the DRG. Filtering induced by focal application of GABA or optogenetic GABA release from the DRG-transplanted GABAergic progenitor cells was specific to nociceptive fibers. Light-sheet imaging and computer modeling demonstrated that, compared to other somatosensory fiber types, nociceptors have shorter stem axons, making somatic control over t-junctional filtering more efficient. Optogenetically induced GABA release within DRG from the transplanted GABAergic cells enhanced filtering and alleviated hypersensitivity to noxious stimulation produced by chronic inflammation and neuropathic injury in vivo. These findings support “gating” of pain information by DRGs and suggest new therapeutic approaches for pain relief.

DNA methylation mapped DNA methylation, an important mechanism of epigenetic modification that produces different patterns of gene expression from a single DNA sequence, is vital to normal development and its malfunction can cause cancer and other abnormalities. A map of DNA methylation in embryonic stem cells, and in various cell types derived from them, has now been produced at nucleotide resolution using high-throughput bisulphite sequencing combined with single molecule-based sequencing. The map reveals specific sites in the genome where methylation changes as cells develop, for instance when embryonic stem cells mature into nerve cells. More generally, the methodology will be of value for the epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine. Genome-scale DNA methylation profiles have been generated at nucleotide resolution in mammalian cells using a combination of high-throughput bisulphite sequencing and single molecule-based sequencing. The DNA methylation maps cover the vast majority of CpG islands, as well as several other genomic regions, in murine embryonic stem cells, cells derived from ES cells and various primary cells. DNA methylation is essential for normal development 1 , 2 , 3 and has been implicated in many pathologies including cancer 4 , 5 . Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing 6 and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that ‘weak’ CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro , in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.

We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations. Chromatin profiling Although they contain the same set of genes, different cell types in a multicellular organism maintain very different behaviours. These cell states are thought to be related to chromatin state — that is, modifications to histones and other proteins that package the genome. Single-molecule sequencing technology has now been used to construct chromatin-state maps for mouse embryonic stem cells and two other more developmentally advanced cell types, revealing the genome-wide distribution of important chromatin modifications. The study provides pointers for the use of chromatin profiling on mammalian cell populations, including those of abnormal cells, such as cancer. Single-molecule-based sequencing technology is applied to generate genome-wide maps of chromatin modifications in mammalian cells. Histone marks can discriminate genes that are active, poised for activation, or stably repressed and therefore reflect cell state and developmental potential.

A major challenge is to understand maladaptive changes in ion channels that sets neurons on a course towards epilepsy development. Voltage- and calcium-activated K+ (BK) channels contribute to early spike timing in neurons, and studies indicate that the BK channel plays a pathological role in increasing excitability early after a seizure. Here, we have investigated changes in BK channels and their accessory β4 subunit (KCNMB4) in dentate gyrus (DG) granule neurons of the hippocampus, key neurons that regulate excitability of the hippocampus circuit. Two days after pilocarpine-induced seizures, we found that the predominant effect is a downregulation of the β4 accessory subunit mRNA. Consistent with reduced expression, single channel recording and pharmacology indicate a switch in the subtype of channels expressed; from iberiotoxin-resistant, type II BK channels (BK α/β4) that have higher channel open probability and slow gating, to iberiotoxin-sensitive type I channels (BK α alone) with low open probability and faster gating. The switch to a majority of type I channel expression following seizure activity is correlated with a loss of BK channel function on spike threshold while maintaining the channel's contribution to increased early spike frequency. Using heterozygous β4 knockout mice, we find reduced expression is sufficient to increase seizure sensitivity. We conclude that seizure-induced downregulation of KCNMB4 is an activity dependent mechanism that increases the excitability of DG neurons. These novel findings indicate that BK channel subtypes are not only defined by cell-specific expression, but can also be plastic depending on the recent history of neuronal excitability.

Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (approximately 100-base) sequence reads at very low cost. Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach. Here, we report the development of an algorithm for genome assembly, ALLPATHS-LG, and its application to massively parallel DNA sequence data from the human and mouse genomes, generated on the Illumina platform. The resulting draft genome assemblies have good accuracy, short-range contiguity, long-range connectivity, and coverage of the genome. In particular, the base accuracy is high (≥99.95%) and the scaffold sizes (N50 size = 11.5 Mb for human and 7.2 Mb for mouse) approach those obtained with capillary-based sequencing. The combination of improved sequencing technology and improved computational methods should now make it possible to increase dramatically the de novo sequencing of large genomes. The ALLPATHS-LG program is available at http://www.broadinstitute.org/science/programs/genome-biology/crd.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens 1 . In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1–CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens 2 – 22 . Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes 23 , 24 . We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.  Among naturally occurring antibodies that have adapted to antigen, those with similar heavy chains usually have similar light chains.

The integration of somatosensory information is generally assumed to be a function of the central nervous system (CNS). Here we describe fully functional GABAergic communication within rodent peripheral sensory ganglia and show that it can modulate transmission of pain-related signals from the peripheral sensory nerves to the CNS. We found that sensory neurons express major proteins necessary for GABA synthesis and release and that sensory neurons released GABA in response to depolarization. In vivo focal infusion of GABA or GABA reuptake inhibitor to sensory ganglia dramatically reduced acute peripherally induced nociception and alleviated neuropathic and inflammatory pain. In addition, focal application of GABA receptor antagonists to sensory ganglia triggered or exacerbated peripherally induced nociception. We also demonstrated that chemogenetic or optogenetic depolarization of GABAergic dorsal root ganglion neurons in vivo reduced acute and chronic peripherally induced nociception. Mechanistically, GABA depolarized the majority of sensory neuron somata, yet produced a net inhibitory effect on the nociceptive transmission due to the filtering effect at nociceptive fiber T-junctions. Our findings indicate that peripheral somatosensory ganglia represent a hitherto underappreciated site of somatosensory signal integration and offer a potential target for therapeutic intervention.

Voltage‐gated K + channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2–S3 linker is critical for this effect. Oxidation‐induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia‐induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M‐channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

Gnirke et al . present a bead-based method for capturing sequences of interest in the human genome for massively parallel sequencing. Using long, biotinylated RNA probes to pull down PCR-amplified DNA fragments, they demonstrate sequencing of 2.5 Mbs of exons in 1,900 genes. Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that ∼60% of target bases in the exonic 'catch', and ∼80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine–freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine–freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature. A reference genome sequence for threespine sticklebacks, and re-sequencing of 20 additional world-wide populations, reveals loci used repeatedly during vertebrate evolution; multiple chromosome inversions contribute to marine-freshwater divergence, and regulatory variants predominate over coding variants in this classic example of adaptive evolution in natural environments. The genomics of stickleback speciation Threespine sticklebacks have become a powerful model for studying the molecular basis of adaptive evolution. This paper presents a high-quality reference genome sequence, along with genomes of 20 further individuals from a global set of marine and freshwater populations. Genomic analysis reveals that reuse of globally shared standing genetic variation plays an important part in repeated evolution of distinct stickleback populations, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. The data are consistent with an important role for regulatory changes during parallel evolution of marine and freshwater sticklebacks.