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Insulin resistance is the primary pathophysiology underlying metabolic syndrome and type 2 diabetes 1 , 2 . Previous metagenomic studies have described the characteristics of gut microbiota and their roles in metabolizing major nutrients in insulin resistance 3 – 9 . In particular, carbohydrate metabolism of commensals has been proposed to contribute up to 10% of the host’s overall energy extraction 10 , thereby playing a role in the pathogenesis of obesity and prediabetes 3 , 4 , 6 . Nevertheless, the underlying mechanism remains unclear. Here we investigate this relationship using a comprehensive multi-omics strategy in humans. We combine unbiased faecal metabolomics with metagenomics, host metabolomics and transcriptomics data to profile the involvement of the microbiome in insulin resistance. These data reveal that faecal carbohydrates, particularly host-accessible monosaccharides, are increased in individuals with insulin resistance and are associated with microbial carbohydrate metabolisms and host inflammatory cytokines. We identify gut bacteria associated with insulin resistance and insulin sensitivity that show a distinct pattern of carbohydrate metabolism, and demonstrate that insulin-sensitivity-associated bacteria ameliorate host phenotypes of insulin resistance in a mouse model. Our study, which provides a comprehensive view of the host–microorganism relationships in insulin resistance, reveals the impact of carbohydrate metabolism by microbiota, suggesting a potential therapeutic target for ameliorating insulin resistance. Faecal carbohydrates, particularly host-accessible monosaccharides, are increased in individuals with insulin resistance and are associated with microbial carbohydrate metabolisms and host inflammatory cytokines.

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA–chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type–specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3′-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5′-ends with an original sample multiplexing strategy in the C1 TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-β of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization. Single-cell transcriptomic profiling allows the exploration of cellular heterogeneity but commonly focuses on the 3′-end of the transcript. Here the authors introduce C1 CAGE, which detects the 5′ transcript end in a multiplexed microfluidic system.

Small non-coding RNAs have been implicated in the regulation of many processes; now a novel class of these RNAs is identified as having a role in the DNA-damage response. Role of small RNA in DNA-damage response Small noncoding RNAs have been implicated in the regulation of many processes. Francia et al . now identify a role for a class of these RNAs in the response to DNA damage. They find that during senescence or after irradiation, two enzymes involved in small-RNA biogenesis are required to activate and maintain the response. The resulting short RNAs arise from the vicinity of a DNA double-strand break, but the target through which they activate the damage response is not yet clear. Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events 1 . Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi) 2 . The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation 3 . So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11–RAD50–NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.

Neurons from fibroblasts Three papers in this issue demonstrate the production of functional induced neuronal (iN) cells from human fibroblasts, a procedure that holds great promise for regenerative medicine. Pang et al . show that a combination of the three transcription factors Ascl1 (also known as Mash1 ), Brn2 (or Pou3f2 ) and Myt1l greatly enhances the neuronal differentiation of human embryonic stem cells. When combined with the basic helix–loop–helix transcription factor NeuroD1, these factors can also convert fetal and postnatal human fibroblasts into iN cells. Caiazzo et al . use a cocktail of three transcription factors to convert prenatal and adult mouse and human fibroblasts into functional dopaminergic neurons. The three are Mash1 , Nurr1 (or Nr4a2 ) and Lmx1a . Conversion is direct with no reversion to a progenitor cell stage, and it occurs in cells from Parkinson's disease patients as well as from healthy donors. Yoo et al . use an alternative approach. They show that microRNAs can have an instructive role in neural fate determination. Expression of miR-9/9* and miR-124 in human fibroblasts induces their conversion into functional neurons, and the process is facilitated by the addition of some neurogenic transcription factors. Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson’s disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons 1 , 2 . In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency 3 , 4 . However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled 5 . Here we identified a minimal set of three transcription factors— Mash1 (also known as Ascl1 ), Nurr1 (also known as Nr4a2 ) and Lmx1a —that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson’s disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

Repetitive elements in differentiated cells are usually silenced. Genome-wide analyses in early mouse development show that repetitive-element expression decreases during development accompanied by the loss of active chromatin marks. LINE-1 and IAP retrotransposons become reactivated after fertilization, and LINE-1 transcription is regulated by short LINE-1 RNAs, which suggests that repetitive elements may be regulated through RNA during the earliest developmental stages. How a more plastic chromatin state is maintained and reversed during development is unknown. Heterochromatin-mediated silencing of repetitive elements occurs in differentiated cells. Here, we used repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. RNA sequencing throughout early mouse embryogenesis revealed that repetitive-element expression is dynamic and stage specific, with most repetitive elements becoming repressed before implantation. We show that LINE-1 and IAP retrotransposons become reactivated from both parental genomes after fertilization. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicates that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. Furthermore, short LINE-1 RNAs regulate LINE-1 transcription in vivo . Our data indicate that reprogramming after mammalian fertilization comprises a robust transcriptional activation of retrotransposons and that repetitive elements are initially regulated through RNA.

RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA–RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs. Here the authors elucidate structure-function relationships of SINEUPs, antisense long non-coding RNAs that through SINE repeat elements positively regulate protein translation of mRNAs they pair with. SINEUP’s functional domains do not share common ancestors.

Variations in transcription start site (TSS) selection reflect diversity of preinitiation complexes and can impact on post-transcriptional RNA fates. Most metazoan polymerase II-transcribed genes carry canonical initiation with pyrimidine/purine (YR) dinucleotide, while translation machinery-associated genes carry polypyrimidine initiator (5’-TOP or TCT). By addressing the developmental regulation of TSS selection in zebrafish we uncovered a class of dual-initiation promoters in thousands of genes, including snoRNA host genes. 5’-TOP/TCT initiation is intertwined with canonical initiation and used divergently in hundreds of dual-initiation promoters during maternal to zygotic transition. Dual-initiation in snoRNA host genes selectively generates host and snoRNA with often different spatio-temporal expression. Dual-initiation promoters are pervasive in human and fruit fly, reflecting evolutionary conservation. We propose that dual-initiation on shared promoters represents a composite promoter architecture, which can function both coordinately and divergently to diversify RNAs. The functional significance of start site choice in promoter architectures is little understood. Here the authors identify in zebrafish development and mammalian cells a class of dual-initiation promoters, in which non-canonical YC dinucleotides reflecting 5’-TOP/TCT initiation are intertwined with canonical YR-initiation.

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.