Crystal Structure of the COMPASS H3K4 Methyltransferase Catalytic Module.

The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.

Genome assembly in the telomere-to-telomere era.

Genome sequences largely determine the biology and encode the history of an organism, and de novo assembly - the process of reconstructing the genome sequence of an organism from sequencing reads - has been a central problem in bioinformatics for four decades. Until recently, genomes were typically assembled into fragments of a few megabases at best, but now technological advances in long-read sequencing enable the near-complete assembly of each chromosome - also known as telomere-to-telomere assembly - for many organisms. Here, we review recent progress on assembly algorithms and protocols, with a focus on how to derive near-telomere-to-telomere assemblies. We also discuss the additional developments that will be required to resolve remaining assembly gaps and to assemble non-diploid genomes.

SnapShot: Chromosome organization.

Chromosomes in higher eukaryotes are folded at different length scales into loop extrusion domains, spatial compartments, and chromosome territories and exhibit interactions with nuclear structures such as the lamina. Microscopic methods can probe this structure by measuring positions of chromosomes in the nuclear space in individual cells, while sequencing-based contact capture approaches can report the frequency of contacts of different regions within these structural layers. To view this SnapShot, open or download the PDF.

The complete sequence of a human Y chromosome.

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure that includes long palindromes, tandem repeats and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4,5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029-base-pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, showing the complete ampliconic structures of gene families TSPY, DAZ and RBMY; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a previous assembly of the CHM13 genome4 and mapped available population variation, clinical variants and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

Uniting tensile ductility with ultrahigh strength via composition undulation.

Metals with nanocrystalline grains have ultrahigh strengths approaching two gigapascals. However, such extreme grain-boundary strengthening results in the loss of almost all tensile ductility, even when the metal has a face-centred-cubic structure-the most ductile of all crystal structures1-3. Here we demonstrate that nanocrystalline nickel-cobalt solid solutions, although still a face-centred-cubic single phase, show tensile strengths of about 2.3 gigapascals with a respectable ductility of about 16 per cent elongation to failure. This unusual combination of tensile strength and ductility is achieved by compositional undulation in a highly concentrated solid solution. The undulation renders the stacking fault energy and the lattice strains spatially varying over length scales in the range of one to ten nanometres, such that the motion of dislocations is thus significantly affected. The motion of dislocations becomes sluggish, promoting their interaction, interlocking and accumulation, despite the severely limited space inside the nanocrystalline grains. As a result, the flow stress is increased, and the dislocation storage is promoted at the same time, which increases the strain hardening and hence the ductility. Meanwhile, the segment detrapping along the dislocation line entails a small activation volume and hence an increased strain-rate sensitivity, which also stabilizes the tensile flow. As such, an undulating landscape resisting dislocation propagation provides a strengthening mechanism that preserves tensile ductility at high flow stresses.

The Human Pangenome Project: a global resource to map genomic diversity.

The human reference genome is the most widely used resource in human genetics and is due for a major update. Its current structure is a linear composite of merged haplotypes from more than 20 people, with a single individual comprising most of the sequence. It contains biases and errors within a framework that does not represent global human genomic variation. A high-quality reference with global representation of common variants, including single-nucleotide variants, structural variants and functional elements, is needed. The Human Pangenome Reference Consortium aims to create a more sophisticated and complete human reference genome with a graph-based, telomere-to-telomere representation of global genomic diversity. Here we leverage innovations in technology, study design and global partnerships with the goal of constructing the highest-possible quality human pangenome reference. Our goal is to improve data representation and streamline analyses to enable routine assembly of complete diploid genomes. With attention to ethical frameworks, the human pangenome reference will contain a more accurate and diverse representation of global genomic variation, improve gene-disease association studies across populations, expand the scope of genomics research to the most repetitive and polymorphic regions of the genome, and serve as the ultimate genetic resource for future biomedical research and precision medicine.

Full resolution HLA and KIR gene annotations for human genome assemblies.

The Human leukocyte antigens (HLA) genes and the Killer cell immunoglobulin like receptors (KIR) genes are critical to immune responses and are associated with many immune-related diseases. Located in highly polymorphic regions, they are hard to study with traditional short-read alignment-based methods. Although modern long-read assemblers can often assemble these genes, using existing tools to annotate HLA and KIR genes in these assemblies remains a nontrivial task. Here, we describe Immuannot, a new computation tool to annotate the gene structures of HLA and KIR genes and to type the allele of each gene. Applying Immuannot to 56 regional and 212 whole-genome assemblies from previous studies, we annotated 9,931 HLA and KIR genes and found that almost half of these genes, 4,068, had novel sequences compared to the current Immuno Polymorphism Database (IPD). These novel gene sequences were represented by 2,664 distinct alleles, some of which contained nonsynonymous variations resulting in 92 novel protein sequences. We demonstrated the complex haplotype structures at the two loci and reported the linkage between HLA/KIR haplotypes and gene alleles. We anticipate that Immuannot will speed up the discovery of new HLA/KIR alleles and enable the association of HLA/KIR haplotype structures with clinical outcomes in the future.

Scalable telomere-to-telomere assembly for diploid and polyploid genomes with double graph.

Despite advances in long-read sequencing technologies, constructing a near telomere-to-telomere assembly is still computationally demanding. Here we present hifiasm (UL), an efficient de novo assembly algorithm combining multiple sequencing technologies to scale up population-wide near telomere-to-telomere assemblies. Applied to 22 human and two plant genomes, our algorithm produces better diploid assemblies at a cost of an order of magnitude lower than existing methods, and it also works with polyploid genomes.

De novo reconstruction of satellite repeat units from sequence data.

Satellite DNA are long tandemly repeating sequences in a genome and may be organized as high-order repeats (HORs). They are enriched in centromeres and are challenging to assemble. Existing algorithms for identifying satellite repeats either require the complete assembly of satellites or only work for simple repeat structures without HORs. Here we describe Satellite Repeat Finder (SRF), a new algorithm for reconstructing satellite repeat units and HORs from accurate reads or assemblies without prior knowledge on repeat structures. Applying SRF to real sequence data, we show that SRF could reconstruct known satellites in human and well-studied model organisms. We also find satellite repeats are pervasive in various other species, accounting for up to 12% of their genome contents but are often underrepresented in assemblies. With the rapid progress in genome sequencing, SRF will help the annotation of new genomes and the study of satellite DNA evolution even if such repeats are not fully assembled.

Efficient and accurate KIR and HLA genotyping with massively parallel sequencing data.

Killer cell immunoglobulin like receptor (KIR) genes and human leukocyte antigen (HLA) genes play important roles in innate and adaptive immunity. They are highly polymorphic and cannot be genotyped with standard variant calling pipelines. Compared with HLA genes, many KIR genes are similar to each other in sequences and may be absent in the chromosomes. Therefore, although many tools have been developed to genotype HLA genes using common sequencing data, none of them work for KIR genes. Even specialized KIR genotypers could not resolve all the KIR genes. Here we describe T1K, a novel computational method for the efficient and accurate inference of KIR or HLA alleles from RNA-seq, whole-genome sequencing, or whole-exome sequencing data. T1K jointly considers alleles across all genotyped genes, so it can reliably identify present genes and distinguish homologous genes, including the challenging KIR2DL5A/KIR2DL5B genes. This model also benefits HLA genotyping, where T1K achieves high accuracy in benchmarks. Moreover, T1K can call novel single-nucleotide variants and process single-cell data. Applying T1K to tumor single-cell RNA-seq data, we found that KIR2DL4 expression was enriched in tumor-specific CD8+ T cells. T1K may open the opportunity for HLA and KIR genotyping across various sequencing applications.

Structural basis of salicylic acid perception by Arabidopsis NPR proteins.

Salicylic acid (SA) is a plant hormone that is critical for resistance to pathogens1-3. The NPR proteins have previously been identified as SA receptors4-10, although how they perceive SA and coordinate hormonal signalling remain unknown. Here we report the mapping of the SA-binding core of Arabidopsis thaliana NPR4 and its ligand-bound crystal structure. The SA-binding core domain of NPR4 refolded with SA adopts an α-helical fold that completely buries SA in its hydrophobic core. The lack of a ligand-entry pathway suggests that SA binding involves a major conformational remodelling of the SA-binding core of NPR4, which we validated using hydrogen-deuterium-exchange mass spectrometry analysis of the full-length protein and through SA-induced disruption of interactions between NPR1 and NPR4. We show that, despite the two proteins sharing nearly identical hormone-binding residues, NPR1 displays minimal SA-binding activity compared to NPR4. We further identify two surface residues of the SA-binding core, the mutation of which can alter the SA-binding ability of NPR4 and its interaction with NPR1. We also demonstrate that expressing a variant of NPR4 that is hypersensitive to SA could enhance SA-mediated basal immunity without compromising effector-triggered immunity, because the ability of this variant to re-associate with NPR1 at high levels of SA remains intact. By revealing the structural mechanisms of SA perception by NPR proteins, our work paves the way for future investigation of the specific roles of these proteins in SA signalling and their potential for engineering plant immunity.

USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

CoLoRd: compressing long reads.

The cost of maintaining exabytes of data produced by sequencing experiments every year has become a major issue in today's genomic research. In spite of the increasing popularity of third-generation sequencing, the existing algorithms for compressing long reads exhibit a minor advantage over the general-purpose gzip. We present CoLoRd, an algorithm able to reduce the size of third-generation sequencing data by an order of magnitude without affecting the accuracy of downstream analyses.

Metagenome assembly of high-fidelity long reads with hifiasm-meta.

De novo assembly of metagenome samples is a common approach to the study of microbial communities. Current metagenome assemblers developed for short sequence reads or noisy long reads were not optimized for accurate long reads. We thus developed hifiasm-meta, a metagenome assembler that exploits the high accuracy of recent data. Evaluated on seven empirical datasets, hifiasm-meta reconstructed tens to hundreds of complete circular bacterial genomes per dataset, consistently outperforming other metagenome assemblers.

Directed evolution of adeno-associated virus for efficient gene delivery to microglia.

As the resident immune cells in the central nervous system (CNS), microglia orchestrate immune responses and dynamically sculpt neural circuits in the CNS. Microglial dysfunction and mutations of microglia-specific genes have been implicated in many diseases of the CNS. Developing effective and safe vehicles for transgene delivery into microglia will facilitate the studies of microglia biology and microglia-associated disease mechanisms. Here, we report the discovery of adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglial transduction via directed evolution of the AAV capsid protein. These AAV-cMG and AAV-MG variants are capable of delivering various genetic payloads into microglia with high efficiency, and enable sufficient transgene expression to support fluorescent labeling, Ca2+ and neurotransmitter imaging and genome editing in microglia in vivo. Furthermore, single-cell RNA sequencing shows that the AAV-MG variants mediate in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the use of various genetically encoded sensors and effectors in the study of microglia-related biology.

Exploring gene content with pangene graphs.

MOTIVATION: The gene content regulates the biology of an organism. It varies between species and between individuals of the same species. Although tools have been developed to identify gene content changes in bacterial genomes, none is applicable to collections of large eukaryotic genomes such as the human pangenome. RESULTS: We developed pangene, a computational tool to identify gene orientation, gene order and gene copy-number changes in a collection of genomes. Pangene aligns a set of input protein sequences to the genomes, resolves redundancies between protein sequences and constructs a gene graph with each genome represented as a walk in the graph. It additionally finds subgraphs, which we call bibubbles, that capture gene content changes. Applied to the human pangenome, pangene identifies known gene-level variations and reveals complex haplotypes that are not well studied before. Pangene also works with high-quality bacterial pangenome and reports similar numbers of core and accessory genes in comparison to existing tools. AVAILABILITY AND IMPLEMENTATION: Source code at https://github.com/lh3/pangene; pre-built pangene graphs can be downloaded from https://zenodo.org/records/8118576 and visualized at https://pangene.bioinweb.org.

Chemosensory protein regulates the behavioural response of Frankliniella intonsa and Frankliniella occidentalis to tomato zonate spot virus-Infected pepper (Capsicum annuum).

Many herbivorous insects rely on plant volatiles to locate their host plants. Vector-borne viral infections induce changes in plant volatiles, which render infected plants more attractive to insect vectors. However, the detailed mechanisms underlying the olfactory responses of insect vectors induced by the volatiles produced by virus-infected plants are poorly understood. Here, we show that volatiles emitted by pepper (Capsicum annuum) plants infected with tomato zonate spot virus (TZSV), particularly the volatile cis-3-hexenal, which is recognized by chemosensory protein 1 of the thrips Frankliniella intonsa (FintCSP1), are more attractive to F. intonsa than the volatiles emitted by non-infected pepper plants. FintCSP1 is highly abundant in the antenna of F. intonsa. Silencing of FintCSP1 significantly decreased electroantennogram responses of F. intonsa antennae to cis-3-hexenal and impaired thrips' responses to TZSV-infected pepper plants and cis-3-hexenal, as assessed using a Y-tube olfactometer. Three-dimensional model predictions indicated that FintCSP1 consists of seven α-helixes and two disulfide bridges. Molecular docking analysis suggested that cis-3-hexenal is positioned deep inside the binding pocket of FintCSP1 and binds to residues of the protein. We combined site-directed mutagenesis and fluorescence binding assays and identified three hydrophilic residues, Lys26, Thr28, and Glu67, of FintCSP1 as being critical for cis-3-hexenal binding. Furthermore, CSP of F. occidentalis (FoccCSP) is also a key olfactory protein involved in modulating the behaviour of F. occidentalis to TZSV-infected pepper. This study revealed the specific binding characteristics of CSPs to cis-3-hexenal and confirmed the general hypothesis that virus infections induce changes in host volatiles, which can be recognized by the olfactory proteins of the insect vector to enhance vector attraction and this may facilitate viral spread and transmission.

The novel GATA1-interacting protein HES6 is an essential transcriptional cofactor for human erythropoiesis.

Normal erythropoiesis requires the precise regulation of gene expression patterns, and transcription cofactors play a vital role in this process. Deregulation of cofactors has emerged as a key mechanism contributing to erythroid disorders. Through gene expression profiling, we found HES6 as an abundant cofactor expressed at gene level during human erythropoiesis. HES6 physically interacted with GATA1 and influenced the interaction of GATA1 with FOG1. Knockdown of HES6 impaired human erythropoiesis by decreasing GATA1 expression. Chromatin immunoprecipitation and RNA sequencing revealed a rich set of HES6- and GATA1-co-regulated genes involved in erythroid-related pathways. We also discovered a positive feedback loop composed of HES6, GATA1 and STAT1 in the regulation of erythropoiesis. Notably, erythropoietin (EPO) stimulation led to up-regulation of these loop components. Increased expression levels of loop components were observed in CD34+ cells of polycythemia vera patients. Interference by either HES6 knockdown or inhibition of STAT1 activity suppressed proliferation of erythroid cells with the JAK2V617F mutation. We further explored the impact of HES6 on polycythemia vera phenotypes in mice. The identification of the HES6-GATA1 regulatory loop and its regulation by EPO provides novel insights into human erythropoiesis regulated by EPO/EPOR and a potential therapeutic target for the management of polycythemia vera.

Electrothermal Transformations within Graphene-Based Aerogels through High-Temperature Flash Joule Heating.

Flash Joule heating of highly porous graphene oxide (GO) aerogel monoliths to ultrahigh temperatures is exploited as a low carbon footprint technology to engineer functional aerogel materials. Aerogel Joule heating to up to 3000 K is demonstrated for the first time, with fast heating kinetics (∼300 K·min-1), enabling rapid and energy-efficient flash heating treatments. The wide applicability of ultrahigh-temperature flash Joule heating is exploited in a range of material fabrication challenges. Ultrahigh-temperature Joule heating is used for rapid graphitic annealing of hydrothermal GO aerogels at fast time scales (30-300 s) and substantially reduced energy costs. Flash aerogel heating to ultrahigh temperatures is exploited for the in situ synthesis of ultrafine nanoparticles (Pt, Cu, and MoO2) embedded within the hybrid aerogel structure. The shockwave heating approach enables high through-volume uniformity of the formed nanoparticles, while nanoparticle size can be readily tuned through controlling Joule-heating durations between 1 and 10 s. As such, the ultrahigh-temperature Joule-heating approach introduced here has important implications for a wide variety of applications for graphene-based aerogels, including 3D thermoelectric materials, extreme temperature sensors, and aerogel catalysts in flow (electro)chemistry.

A gel-coated air-liquid-interface culture system with tunable substrate stiffness matching healthy and diseased lung tissues.

Since its invention in the late 1980s, the air-liquid-interface (ALI) culture system has been the standard in vitro model for studying human airway biology and pulmonary diseases. However, in a conventional ALI system, cells are cultured on a porous plastic membrane that is much stiffer than human airway tissues. Here, we develop a gel-ALI culture system by simply coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We determine the optimum gel thickness that does not impair the transport of nutrients and biomolecules essential to cell growth. We show that the gel-ALI system allows human bronchial epithelial cells (HBECs) to proliferate and differentiate into pseudostratified epithelium. Furthermore, we discover that HBECs migrate significantly faster on hydrogel substrates with stiffness matching that of fibrotic lung tissues, highlighting the importance of mechanical cues in human airway remodeling. The developed gel-ALI system provides a facile approach to studying the effects of mechanical cues in human airway biology and in modeling pulmonary diseases.NEW & NOTEWORTHY In a conventional ALI system, cells are cultured on a plastic membrane that is much stiffer than human airway tissues. We develop a gel-ALI system by coating the plastic membrane with a thin layer of hydrogel with tunable stiffness matching that of healthy and fibrotic airway tissues. We discover that human bronchial epithelial cells migrate significantly faster on hydrogel substrates with pathological stiffness, highlighting the importance of mechanical cues in human airway remodeling.