Explore the vast range of titles available.

HiChIP combines chromosome conformation capture with immunoprecipitation- and tagmentation-based library preparation to uncover the 3D chromatin architecture focused around a protein of interest. Genome conformation is central to gene control but challenging to interrogate. Here we present HiChIP, a protein-centric chromatin conformation method. HiChIP improves the yield of conformation-informative reads by over 10-fold and lowers the input requirement over 100-fold relative to that of ChIA-PET. HiChIP of cohesin reveals multiscale genome architecture with greater signal-to-background ratios than those of in situ Hi-C.

An RNA secondary structure (RSS) map of coding and noncoding RNA from a human family (two parents and their child) is produced; this reveals that approximately 15% of all transcribed single nucleotide variants (SNVs) alter local RNA structure, and these SNVs are depleted in certain locations, suggesting that particular RNA structures are important at those sites. Probing the in vivo RNA structurome Being single-stranded, RNA can adopt a diversity of secondary structures via inter- and intramolecular base-pairing. Three studies published in this issue of Nature provide an in-depth view of the variety, dynamics and functional influence of RNA structures in vivo . Sarah Assmann and colleagues map the in vivo RNA structure of over 10,000 transcripts in the model plant Arabidopsis thaliana . Their struc-seq (structure-seqence) approach incorporates in vivo chemical (DMS) probing and next-generation sequencing to provide single-nucleotide resolution on a genome-wide scale. Distinct patterns of structure are found to be correlated with coding regions, splice sites and polyadenylation sites. Comparison of these results with those obtained by earlier technologies reveals that, although predictions for some classes of genes were fairly accurate, others, such as those involved in stress response, were poorly predicted and may reflect changes that made them more adapted to that condition. Jonathan Weissman and colleagues have also developed a DMS-seq method to globally monitor RNA structure to single-nucleotide precision in yeast and mammalian cells. Comparing their findings with in vitro data, the authors conclude that there is less structure within cells than expected. Even thermostable RNA structures can be denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Howard Chang and colleagues asked a different question: how does RNA secondary structure change on a transcriptome-wide level in related individuals? By calculating the RNA secondary structures of two parents and their child, they find that about 15% of transcribed single-nucleotide variants affect local secondary structure. These 'RiboSNitches' are depleted in certain locations, suggesting that a particular RNA structure at that site is important. This study illustrates that there is much to be learned about how changes in RNA structure, particularly as imparted by genetic variation, can alter gene expression. In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program 1 . However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of ‘riboSNitches’ versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3′ untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5 + basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5 + cells in basal organoids revealed a distinct population of ITGA6 + ITGB4 + mitotic cells, whose offspring further segregated into a TNFRSF12A hi subfraction that comprised about ten per cent of KRT5 + basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia. A long-term culture method for organoids derived from single adult human lung cells is used to identify progenitor cells and study SARS-CoV-2 infection.

The use of a biotinylated adaptor conjugated to an infrared dye allows rapid, sensitive, and quantitative analysis of protein–RNA interactions. The complexity of transcriptome-wide protein–RNA interaction networks is incompletely understood. While emerging studies are greatly expanding the known universe of RNA-binding proteins, methods for the discovery and characterization of protein–RNA interactions remain resource intensive and technically challenging. Here we introduce a UV-C crosslinking and immunoprecipitation platform, irCLIP, which provides an ultraefficient, fast, and nonisotopic method for the detection of protein–RNA interactions using far less material than standard protocols.

High-resolution contact maps of active enhancers and target genes generated by H3K27ac HiChIP in primary human cells provide rational guides to link noncoding disease-associated risk variants to candidate causal genes. Genes are validated by CRISPR activation and interference at connected enhancers and eQTL analysis, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases.

The human endogenous retrovirus HERVK is normally silenced, but here the surprising discovery is made that in early human embryo development it is expressed, producing retroviral-like particles. Retroviral activation in the early embryo The open reading frames encoded by the human endogenous retrovirus HERVK are normally transcriptionally silenced. Joanna Wysocka and colleagues report that HERVK is expressed during early human embryo development from the eight-cell stage to the pre-implantation epiblast, leading to the production of retrovirus-like particles. They further show that the process of human embryonic stem cell derivation silences HERVK expression, and that in pluripotent cells an HERVK accessory protein (Rec) can bind cellular RNAs and appears to induce an antiviral defence response. Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome 1 . The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor 2 , 3 . Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins 4 . However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection 5 , 6 , 7 . Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development.

Mutations associated with Treacher Collins syndrome perturb the subnuclear localization of an RNA helicase involved in ribosome biogenesis through activation of p53 protein, illustrating how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations. RNA-related regulation in craniofacial development Many craniofacial disorders are due to defects in cranial neural crest cells, a cell type that gives rise to the majority of facial structures during embryogenesis. Yet, many of the genetic defects underlying these disorders are heterozygous mutations in general transcription and translation regulators, which are not tissue-specific. Why cranial neural crest cells are more sensitive than others to these mutations during development is not well understood. Joanna Wysocka and colleagues show that mutations associated with Treacher Collins syndrome perturb the subnuclear localization of an RNA helicase involved in ribosome biogenesis, and that this effect occurs specifically in cranial neural crest cells. This protein relocalization process, which involves the activation of p53, impairs ribosome biogenesis and causes craniofacial defects. Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis 1 , 2 . Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis 3 , 4 , the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis 5 , we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1 ), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond–Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

The human long non-coding RNA TINCR binds to STAU1 and controls epidermal differentiation by stabilizing key differentiation mRNAs, by means of a TINCR-binding motif found enriched in epidermal differentiation genes. An lncRNA required for tissue differentiation The human genome codes for thousands of long non-coding RNAs (lncRNAs), but their biological functions are mostly unknown. This study reports the identification and characterization of a 3.7-kilobase lncRNA, named TINCR (for terminal differentiation-induced ncRNA). TINCR controls epidermal differentiation by stabilizing the mRNA of an array of differentiation genes. A 25-nucleotide TINCR-binding motif is enriched in these epidermal differentiation genes. TINCR also combines with the RNA-binding protein staufen1 to form a complex that stabilizes differentiation transcripts via direct binding and other mechanisms yet to be identified. Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized 1 , 2 , 3 , 4 ; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3 . TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR–mRNA interaction occurs through a 25-nucleotide ‘TINCR box’ motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2 , both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR–STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80 . These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

Interactions between proteins and non-proteinaceous biopolymers are essential for life; however, many methods used to characterize these interactions lack precision and display significant biases. Now, a genetically encoded method employing sulfur(vi) fluoride exchange (SuFEx)-based chemical crosslinking has been developed for capturing and analysing protein–RNA and protein–carbohydrate interactions in vivo.

Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Herein, we report a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and precision. The GlycanDIA workflow combines higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and precision in quantification compared to conventional glycomic methods. To facilitate its use, we also develop a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification from DIA data. Our results demonstrate that GlycanDIA can distinguish glycan composition and isomers from N -glycans, O -glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity and precision, we perform experiments to profile N -glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N -glycan profile in cellular and tissue glycoRNA samples, we find that RNA-glycans have different abundant forms as compared to protein-glycans and there are also tissue-specific differences, suggesting their distinct functions in biological processes. Glycans regulate cells via glycosylation, and aberrant glycosylation is linked to disease initiation and progression. Here, the authors present GlycanDIA, a DIA-based workflow enabling sensitive, precise glycan analysis, revealing low-abundant modifications and profiling distinct RNA glycan patterns with biological relevance.