Genomic structure predicts metabolite dynamics in microbial communities.

The metabolic activities of microbial communities play a defining role in the evolution and persistence of life on Earth, driving redox reactions that give rise to global biogeochemical cycles. Community metabolism emerges from a hierarchy of processes, including gene expression, ecological interactions, and environmental factors. In wild communities, gene content is correlated with environmental context, but predicting metabolite dynamics from genomes remains elusive. Here, we show, for the process of denitrification, that metabolite dynamics of a community are predictable from the genes each member of the community possesses. A simple linear regression reveals a sparse and generalizable mapping from gene content to metabolite dynamics for genomically diverse bacteria. A consumer-resource model correctly predicts community metabolite dynamics from single-strain phenotypes. Our results demonstrate that the conserved impacts of metabolic genes can predict community metabolite dynamics, enabling the prediction of metabolite dynamics from metagenomes, designing denitrifying communities, and discovering how genome evolution impacts metabolism.

Comprehensive structure and functional adaptations of the yeast nuclear pore complex.

Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.

GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

An atlas of protein-protein interactions across mouse tissues.

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

Opportunities and challenges in assessing climate change vulnerability through genomics.

By investigating how past selection has affected allele frequencies across space, genomic tools are providing new insights into adaptive evolutionary processes. Now researchers are considering how this genomic information can be used to predict the future vulnerability of species under climate change. Genomic vulnerability assessments show promise, but challenges remain.

Mapping the genetic landscape of DNA double-strand break repair.

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.

A modular master regulator landscape controls cancer transcriptional identity.

Despite considerable efforts, the mechanisms linking genomic alterations to the transcriptional identity of cancer cells remain elusive. Integrative genomic analysis, using a network-based approach, identified 407 master regulator (MR) proteins responsible for canalizing the genetics of individual samples from 20 cohorts in The Cancer Genome Atlas (TCGA) into 112 transcriptionally distinct tumor subtypes. MR proteins could be further organized into 24 pan-cancer, master regulator block modules (MRBs), each regulating key cancer hallmarks and predictive of patient outcome in multiple cohorts. Of all somatic alterations detected in each individual sample, >50% were predicted to induce aberrant MR activity, yielding insight into mechanisms linking tumor genetics and transcriptional identity and establishing non-oncogene dependencies. Genetic and pharmacological validation assays confirmed the predicted effect of upstream mutations and MR activity on downstream cellular identity and phenotype. Thus, co-analysis of mutational and gene expression profiles identified elusive subtypes and provided testable hypothesis for mechanisms mediating the effect of genetic alterations.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

Recording of elapsed time and temporal information about biological events using Cas9.

DNA has not been utilized to record temporal information, although DNA has been used to record biological information and to compute mathematical problems. Here, we found that indel generation by Cas9 and guide RNA can occur at steady rates, in contrast to typical dynamic biological reactions, and the accumulated indel frequency can be a function of time. By measuring indel frequencies, we developed a method for recording and measuring absolute time periods over hours to weeks in mammalian cells. These time-recordings were conducted in several cell types, with different promoters and delivery vectors for Cas9, and in both cultured cells and cells of living mice. As applications, we recorded the duration of chemical exposure and the lengths of elapsed time since the onset of biological events (e.g., heat exposure and inflammation). We propose that our systems could serve as synthetic "DNA clocks."

Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ.

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

Massively parallel assessment of human variants with base editor screens.

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.

Microbial exposure during early human development primes fetal immune cells.

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth.

Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity.

While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Key Parameters of Tumor Epitope Immunogenicity Revealed Through a Consortium Approach Improve Neoantigen Prediction.

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.

BRICseq Bridges Brain-wide Interregional Connectivity to Neural Activity and Gene Expression in Single Animals.

Comprehensive analysis of neuronal networks requires brain-wide measurement of connectivity, activity, and gene expression. Although high-throughput methods are available for mapping brain-wide activity and transcriptomes, comparable methods for mapping region-to-region connectivity remain slow and expensive because they require averaging across hundreds of brains. Here we describe BRICseq (brain-wide individual animal connectome sequencing), which leverages DNA barcoding and sequencing to map connectivity from single individuals in a few weeks and at low cost. Applying BRICseq to the mouse neocortex, we find that region-to-region connectivity provides a simple bridge relating transcriptome to activity: the spatial expression patterns of a few genes predict region-to-region connectivity, and connectivity predicts activity correlations. We also exploited BRICseq to map the mutant BTBR mouse brain, which lacks a corpus callosum, and recapitulated its known connectopathies. BRICseq allows individual laboratories to compare how age, sex, environment, genetics, and species affect neuronal wiring and to integrate these with functional activity and gene expression.

Data Sanitization to Reduce Private Information Leakage from Functional Genomics.

The generation of functional genomics datasets is surging, because they provide insight into gene regulation and organismal phenotypes (e.g., genes upregulated in cancer). The intent behind functional genomics experiments is not necessarily to study genetic variants, yet they pose privacy concerns due to their use of next-generation sequencing. Moreover, there is a great incentive to broadly share raw reads for better statistical power and general research reproducibility. Thus, we need new modes of sharing beyond traditional controlled-access models. Here, we develop a data-sanitization procedure allowing raw functional genomics reads to be shared while minimizing privacy leakage, enabling principled privacy-utility trade-offs. Our protocol works with traditional Illumina-based assays and newer technologies such as 10x single-cell RNA sequencing. It involves quantifying the privacy leakage in reads by statistically linking study participants to known individuals. We carried out these linkages using data from highly accurate reference genomes and more realistic environmental samples.

Translation and Replication Dynamics of Single RNA Viruses.

RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.

Defining the Teratoma as a Model for Multi-lineage Human Development.

We propose that the teratoma, a recognized standard for validating pluripotency in stem cells, could be a promising platform for studying human developmental processes. Performing single-cell RNA sequencing (RNA-seq) of 179,632 cells across 23 teratomas from 4 cell lines, we found that teratomas reproducibly contain approximately 20 cell types across all 3 germ layers, that inter-teratoma cell type heterogeneity is comparable with organoid systems, and teratoma gut and brain cell types correspond well to similar fetal cell types. Furthermore, cellular barcoding confirmed that injected stem cells robustly engraft and contribute to all lineages. Using pooled CRISPR-Cas9 knockout screens, we showed that teratomas can enable simultaneous assaying of the effects of genetic perturbations across all germ layers. Additionally, we demonstrated that teratomas can be sculpted molecularly via microRNA (miRNA)-regulated suicide gene expression to enrich for specific tissues. Taken together, teratomas are a promising platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.

MorphoSeq: Full Single-Cell Transcriptome Dynamics Up to Gastrulation in a Chordate.

Single-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell's physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.