Search

Histone proteins have traditionally been thought to be restricted to eukaryotes and most archaea, with eukaryotic nucleosomal histones deriving from their archaeal ancestors. In contrast, bacteria lack histones as a rule. However, histone proteins have recently been identified in a few bacterial clades, most notably the phylum Bdellovibrionota, and these histones have been proposed to exhibit a range of divergent features compared to histones in archaea and eukaryotes. However, no functional genomic studies of the properties of Bdellovibrionota chromatin have been carried out. In this work, we map the landscape of chromatin accessibility, active transcription and three-dimensional genome organization in a member of Bdellovibrionota (a Bacteriovorax strain). We find that, similar to what is observed in some archaea and in eukaryotes with compact genomes such as yeast, Bacteriovorax chromatin is characterized by preferential accessibility around promoter regions. Similar to eukaryotes, chromatin accessibility in Bacteriovorax positively correlates with gene expression. Mapping active transcription through single-strand DNA (ssDNA) profiling revealed that unlike in yeast, but similar to the state of mammalian and fly promoters, Bacteriovorax promoters exhibit very strong polymerase pausing. Finally, similar to that of other bacteria without histones, the Bacteriovorax genome exists in a three-dimensional (3D) configuration organized by the parABS system along the axis defined by replication origin and termination regions. These results provide a foundation for understanding the chromatin biology of the unique Bdellovibrionota bacteria and the functional diversity in chromatin organization across the tree of life., (Published by Cold Spring Harbor Laboratory Press.)

The binding of multiple transcription factors (TFs) to genomic enhancers drives gene expression in mammalian cells 1 . However, the molecular details that link enhancer sequence to TF binding, promoter state and transcription levels remain unclear. Here we applied single-molecule footprinting 2,3 to measure the simultaneous occupancy of TFs, nucleosomes and other regulatory proteins on engineered enhancer-promoter constructs with variable numbers of TF binding sites for both a synthetic TF and an endogenous TF involved in the type I interferon response. Although TF binding events on nucleosome-free DNA are independent, activation domains recruit cofactors that destabilize nucleosomes, driving observed TF binding cooperativity. Average TF occupancy linearly determines promoter activity, and we decompose TF strength into separable binding and activation terms. Finally, we develop thermodynamic and kinetic models that quantitatively predict both the enhancer binding microstates and gene expression dynamics. This work provides a template for the quantitative dissection of distinct contributors to gene expression, including TF activation domains, concentration, binding affinity, binding site configuration and recruitment of chromatin regulators., Competing Interests: Competing interests: W.J.G. is a consultant and equity holder for 10x Genomics, Guardant Health, Quantapore and Ultima Genomics, co-founder of Protillion Biosciences and is named on patents describing ATAC–seq. L.B. is a co-founder of Stylus Medicine and a member of its scientific advisory board. All other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Folded RNAs contain tertiary contact motifs whose structures and energetics are conserved across different RNAs. The transferable properties of RNA motifs simplify the RNA folding problem, but measuring energetic and conformational properties of many motifs remains a challenge. Here, we use a high-throughput thermodynamic approach to investigate how sequence changes alter the binding properties of naturally occurring motifs, the GAAA tetraloop • tetraloop receptor (TLR) interactions. We measured the binding energies and conformational preferences of TLR sequences that span mutational pathways from the canonical 11ntR to two other natural TLRs, the IC3R and Vc2R. While the IC3R and Vc2R share highly similar energetic and conformational properties, the landscapes that map the sequence changes for their conversion from the 11ntR to changes in these properties differ dramatically. Differences in the energetic landscapes stem from the mutations needed to convert the 11ntR to the IC3R and Vc2R rather than a difference in the intrinsic energetic architectures of these TLRs. The conformational landscapes feature several nonnative TLR variants with conformational preferences that differ from both the initial and final TLRs; these species represent potential branching points along the multidimensional sequence space to sequences with greater fitness in other RNA contexts with alternative conformational preferences. Our high-throughput, quantitative approach reveals the complex nature of sequence-fitness landscapes and leads to models for their molecular origins. Systematic and quantitative molecular approaches provide critical insights into understanding the evolution of natural RNAs as they traverse complex landscapes in response to selective pressures., (© 2024 Shin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Complex structural variations (cxSVs) are often overlooked in genome analyses due to detection challenges. We developed ARC-SV, a probabilistic and machine-learning-based method that enables accurate detection and reconstruction of cxSVs from standard datasets. By applying ARC-SV across 4,262 genomes representing all continental populations, we identified cxSVs as a significant source of natural human genetic variation. Rare cxSVs have a propensity to occur in neural genes and loci that underwent rapid human-specific evolution, including those regulating corticogenesis. By performing single-nucleus multiomics in postmortem brains, we discovered cxSVs associated with differential gene expression and chromatin accessibility across various brain regions and cell types. Additionally, cxSVs detected in brains of psychiatric cases are enriched for linkage with psychiatric GWAS risk alleles detected in the same brains. Furthermore, our analysis revealed significantly decreased brain-region- and cell-type-specific expression of cxSV genes, specifically for psychiatric cases, implicating cxSVs in the molecular etiology of major neuropsychiatric disorders., Competing Interests: Declaration of interests M.P.S. is a co-founder and on the advisory boards of Personalis, Qbio, January AI, SensOmics, Filtricine, Protos, Mirvie, Onza, Marble Therapeutics, Iollo, and NextThought AI. He is also on the advisory boards of Jupiter, Applied Cognition, Neuvivo, Mitrix, and Enovone. W.J.G. is a consultant for 10× Genomics, Guardant Health, Quantapore, and Ultima Genomics, a co-founder of Protillion Biosciences, and is named on ATAC-seq patents. A.K. is a consulting fellow with Illumina; a member of the SABs of OpenTargets (GSK), PatchBio, and SerImmune; and a co-founder of RavelBio. S.B.M. is an advisor for BioMarin, MyOme, and Tenaya Therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. ecDNA renders tumours treatment resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival 1-7 . At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription-replication conflict enables targeted elimination of ecDNA-containing cancers. Stepwise analyses of ecDNA transcription reveal pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription-replication conflicts and replication stress compared with chromosomal loci. Nucleotide incorporation on ecDNA is markedly slower, and replication stress is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. pRPA2-S33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription-dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition causes extensive and preferential tumour cell death in ecDNA-containing tumours. We advance a highly selective, potent and bioavailable oral CHK1 inhibitor, BBI-2779, that preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 amplified on ecDNA, BBI-2779 suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. Transcription-replication conflict emerges as a target for ecDNA-directed therapy, exploiting a synthetic lethality of excess to treat cancer., (© 2024. The Author(s).)

Familial adenomatous polyposis (FAP) is a genetic disease causing hundreds of premalignant polyps in affected persons and is an ideal model to study transitions of early precancer states to colorectal cancer (CRC). We performed deep multiomic profiling of 93 samples, including normal mucosa, benign polyps and dysplastic polyps, from six persons with FAP. Transcriptomic, proteomic, metabolomic and lipidomic analyses revealed a dynamic choreography of thousands of molecular and cellular events that occur during precancerous transitions toward cancer formation. These involve processes such as cell proliferation, immune response, metabolic alterations (including amino acids and lipids), hormones and extracellular matrix proteins. Interestingly, activation of the arachidonic acid pathway was found to occur early in hyperplasia; this pathway is targeted by aspirin and other nonsteroidal anti-inflammatory drugs, a preventative treatment under investigation in persons with FAP. Overall, our results reveal key genomic, cellular and molecular events during the earliest steps in CRC formation and potential mechanisms of pharmaceutical prophylaxis., Competing Interests: Competing interests: M.P.S. is a cofounder and scientific advisor of Personalis, SensOmics, Qbio, January AI, Fodsel, Filtricine, v Protos, RTHM, Iollo, Marble Therapeutics, Crosshair Therapeutics, NextThought and Mirvie. He is a scientific advisor of Jupiter, Neuvivo, Swaza, Mitrix, Yuvan, TranscribeGlass and Applied Cognition. A. Kundaje has affiliations with Biogen (consultant), SerImmune (scientific advisory board (SAB)), RavelBio (scientific cofounder and SAB) and PatchBio (SAB). K.C. is currently an AstraZeneca employee. W.J.G. has affiliations with Guardant Health (consultant and SAB), Protillion Biosciences (scientific cofounder) and 10x and has licenced patents associated with ATAC-seq. E.D.E. is an employee and stockholder of Labcorp, and advisor and stockholder of Taproot Health, Exir Bio and ROMTech. The other authors declare no competing interests., (© 2024. The Author(s).)

Although three-dimensional (3D) genome architecture is crucial for gene regulation, its role in disease remains elusive. We traced the evolution and malignant transformation of colorectal cancer (CRC) by generating high-resolution chromatin conformation maps of 33 colon samples spanning different stages of early neoplastic growth in persons with familial adenomatous polyposis (FAP). Our analysis revealed a substantial progressive loss of genome-wide cis-regulatory connectivity at early malignancy stages, correlating with nonlinear gene regulation effects. Genes with high promoter-enhancer (P-E) connectivity in unaffected mucosa were not linked to elevated baseline expression but tended to be upregulated in advanced stages. Inhibiting highly connected promoters preferentially represses gene expression in CRC cells compared to normal colonic epithelial cells. Our results suggest a two-phase model whereby neoplastic transformation reduces P-E connectivity from a redundant state to a rate-limiting one for transcriptional levels, highlighting the intricate interplay between 3D genome architecture and gene regulation during early CRC progression., Competing Interests: Competing interests: G.K., A.J., D.L. and Z.S. are employees and shareholders of Ultima Genomics. M.P.S is a cofounder and scientific advisor of Personalis, Qbio, SensOmics, January AI, Mirvie, Protos, NiMo and Onza and is on the advisory board of Genapsys. E.D.E. is an employee and stockholder of Invitae and an advisor and stockholder of Taproot Health and Exir Bio. W.J.G. has affiliations with Guardant Health (consultant and scientific advisory board), Protillion Biosciences (Scientifica cofounder) and 10x and has licensed patents associated with ATAC-seq. All other authors declare no competing interests., (© 2024. The Author(s).)

Three-dimensional nuclear DNA architecture comprises well-studied intra-chromosomal ( cis ) folding and less characterized inter-chromosomal ( trans ) interfaces. Current predictive models of 3D genome folding can effectively infer pairwise cis -chromatin interactions from the primary DNA sequence but generally ignore trans contacts. There is an unmet need for robust models of trans -genome organization that provide insights into their underlying principles and functional relevance. We present TwinC, an interpretable convolutional neural network model that reliably predicts trans contacts measurable through genome-wide chromatin conformation capture (Hi-C). TwinC uses a paired sequence design from replicate Hi-C experiments to learn single base pair relevance in trans interactions across two stretches of DNA. The method achieves high predictive accuracy (AUROC=0.80) on a cross-chromosomal test set from Hi-C experiments in heart tissue. Mechanistically, the neural network learns the importance of compartments, chromatin accessibility, clustered transcription factor binding and G-quadruplexes in forming trans contacts. In summary, TwinC models and interprets trans genome architecture, shedding light on this poorly understood aspect of gene regulation., Competing Interests: Competing interests A. K. is on the scientific advisory board of SerImmune, TensorBio, and AINovo, a consultant with Arcardia Science and Inari, a consultant with Illumina and PatchBio and has a financial stake in DeepGenomics, Immunai, SerImmune and Freenome.

To identify cancer-associated gene regulatory changes, we generated single-cell chromatin accessibility landscapes across eight tumor types as part of The Cancer Genome Atlas. Tumor chromatin accessibility is strongly influenced by copy number alterations that can be used to identify subclones, yet underlying cis-regulatory landscapes retain cancer type-specific features. Using organ-matched healthy tissues, we identified the "nearest healthy" cell types in diverse cancers, demonstrating that the chromatin signature of basal-like-subtype breast cancer is most similar to secretory-type luminal epithelial cells. Neural network models trained to learn regulatory programs in cancer revealed enrichment of model-prioritized somatic noncoding mutations near cancer-associated genes, suggesting that dispersed, nonrecurrent, noncoding mutations in cancer are functional. Overall, these data and interpretable gene regulatory models for cancer and healthy tissue provide a framework for understanding cancer-specific gene regulation.

Epigenetic dysregulation is widespread in cancer. However, the specific epigenetic regulators and the processes they control to drive cancer phenotypes are poorly understood. Here, we employed a novel, scalable and high-throughput in vivo method to perform iterative functional screens of over 250 epigenetic regulatory genes within autochthonous oncogenic KRAS-driven lung tumors. We identified multiple novel epigenetic tumor suppressor and tumor dependency genes. We show that a specific HBO1 complex and the MLL1 complex are among the most impactful tumor suppressive epigenetic regulators in lung. The histone modifications generated by the HBO1 complex are frequently absent or reduced in human lung adenocarcinomas. The HBO1 and MLL1 complexes regulate chromatin accessibility of shared genomic regions, lineage fidelity and the expression of canonical tumor suppressor genes. The HBO1 and MLL1 complexes are epistatic during lung tumorigenesis, and their functional correlation is conserved in human cancer cell lines. Together, these results demonstrate the value of quantitative methods to generate a phenotypic roadmap of epigenetic regulatory genes in tumorigenesis in vivo .

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34 + progenitors prior to B/T cell emergence. While these TdT + cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84 lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis., (© 2024. The Author(s).)

Although single-cell and spatial sequencing methods enable simultaneous measurement of more than one biological modality, no technology can capture all modalities within the same cell. For current data integration methods, the feasibility of cross-modal integration relies on the existence of highly correlated, a priori 'linked' features. We describe matching X-modality via fuzzy smoothed embedding (MaxFuse), a cross-modal data integration method that, through iterative coembedding, data smoothing and cell matching, uses all information in each modality to obtain high-quality integration even when features are weakly linked. MaxFuse is modality-agnostic and demonstrates high robustness and accuracy in the weak linkage scenario, achieving 20~70% relative improvement over existing methods under key evaluation metrics on benchmarking datasets. A prototypical example of weak linkage is the integration of spatial proteomic data with single-cell sequencing data. On two example analyses of this type, MaxFuse enabled the spatial consolidation of proteomic, transcriptomic and epigenomic information at single-cell resolution on the same tissue section., (© 2023. The Author(s).)

Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we present a protocol for mapping three-dimensional (3D) genome organization in dinoflagellates and using it for scaffolding their genome assemblies. We describe steps for crosslinking, nuclear lysis, denaturation, restriction digest, ligation, and DNA shearing and purification. We then detail procedures sequencing library generation and computational analysis, including initial Hi-C read mapping and 3D-DNA scaffolding/assembly correction. For complete details on the use and execution of this protocol, please refer to Marinov et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.

Classical evolutionary theories propose tradeoffs among reproduction, damage repair and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. In this study, we used the turquoise killifish (Nothobranchius furzeri) to genetically arrest germline development at discrete stages and examine how different modes of infertility impact life history. We first constructed a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. We show here that germline depletion-but not arresting germline differentiation-enhances damage repair in female killifish. Conversely, germline-depleted males instead showed an extension in lifespan and rejuvenated metabolic functions. Through further transcriptomic analysis, we highlight enrichment of pro-longevity pathways and genes in germline-depleted male killifish and demonstrate functional conservation of how these factors may regulate longevity in germline-depleted Caenorhabditis elegans. Our results, therefore, demonstrate that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative responses to classical evolutionary tradeoffs., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols., (© 2023. The Author(s).)

Background: In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties are originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools., Results: In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays., Conclusions: Our results provide the first window into the 5-hmU and chromatin accessibility landscapes in dinoflagellates., (© 2024. The Author(s).)

Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome., (© 2024. The Author(s).)

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes, not all archaea possess histones and it is not clear if histones are a main packaging component for all that do. Despite increased interest in archaeal chromatin in recent years, its properties have been little studied using genomic tools., Results: Here, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeote Haloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that while H. volcanii promoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection of H. volcanii and find that Haloferax chromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures., Conclusions: Our results provide the first comprehensive maps of chromatin accessibility and active transcription in Haloferax across conditions and thus a foundation for future functional studies of archaeal chromatin., (© 2023. The Author(s).)

Non-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. Yet, the genomic targets and functions of most ncRNAs are unknown. Here we use chromatin-associated RNA sequencing (ChAR-seq) to map the global network of ncRNA interactions with chromatin in human embryonic stem cells and the dynamic changes in interactions during differentiation into definitive endoderm. We uncover general principles governing the organization of the RNA-chromatin interactome, demonstrating that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their locus and provide a model predicting the interactome. We uncover RNAs that interact with many loci across the genome and unveil thousands of unannotated RNAs that dynamically interact with chromatin. By relating the dynamics of the interactome to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by a single ncRNA., (© 2023. Springer Nature Limited.)

Short tandem repeats (STRs) are enriched in eukaryotic cis -regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)-DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis -regulatory mechanism to target TFs to genomic sites.

Genome-wide association studies have identified many loci associated with hair and skin disease, but identification of causal variants requires deciphering of gene-regulatory networks in relevant cell types. We generated matched single-cell chromatin profiles and transcriptomes from scalp tissue from healthy controls and patients with alopecia areata, identifying diverse cell types of the hair follicle niche. By interrogating these datasets at multiple levels of cellular resolution, we infer 50-100% more enhancer-gene links than previous approaches and show that aggregate enhancer accessibility for highly regulated genes predicts expression. We use these gene-regulatory maps to prioritize cell types, genes and causal variants implicated in the pathobiology of androgenetic alopecia (AGA), eczema and other complex traits. AGA genome-wide association studies signals are enriched in dermal papilla regulatory regions, supporting the role of these cells as drivers of AGA pathogenesis. Finally, we train machine learning models to nominate single-nucleotide polymorphisms that affect gene expression through disruption of transcription factor binding, predicting candidate functional single-nucleotide polymorphism for AGA and eczema., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health 1 . The intesting has a length of over nine metres, along which there are differences in structure and function 2 . The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease., (© 2023. The Author(s).)

RNAs are central to fundamental biological processes in all known organisms. The set of possible intramolecular interactions of RNA nucleotides defines the range of alternative structural conformations of a specific RNA that can coexist, and these structures enable functional catalytic properties of RNAs and/or their productive intermolecular interactions with other RNAs or proteins. However, the immense combinatorial space of potential RNA sequences has precluded predictive mapping between RNA sequence and molecular structure and function. Recent advances in high-throughput approaches in vitro have enabled quantitative thermodynamic and kinetic measurements of RNA-RNA and RNA-protein interactions, across hundreds of thousands of sequence variations. In this Review, we explore these techniques, how they can be used to understand RNA function and how they might form the foundations of an accurate model to predict the structure and function of an RNA directly from its nucleotide sequence. The experimental techniques and modelling frameworks discussed here are also highly relevant for the sampling of sequence-structure-function space of DNAs and proteins., (© 2023. Springer Nature Limited.)

Single-cell technologies have revealed the extensive heterogeneity and complexity of the immune system. Systems biology approaches in immunology have taken advantage of the high-parameter, high-throughput data and analyzed immune cell types in a 'bottom-up' data-driven method. This approach has discovered previously unrecognized cell types and functions. Especially for human immunology, in which experimental manipulations are challenging, systems approach has become a successful means to investigate physiologically relevant contexts. This review focuses on the recent findings in lymphocyte biology, from their development, differentiation into subsets, and heterogeneity in their functions, enabled by these systems approaches. Furthermore, we review examples of the application of findings from systems approach studies and discuss how now to leave the rich dataset in the curse of high dimensionality., Competing Interests: Conflict of interest statement S.C.B. is a consultant and shareholders Ionpath Inc. that commercializes MIBI technology. S.C.B. is an inventor on, and receive royalties for patents relating to MIBI technology. W.J.G. is named as an inventor on patents describing ATAC-seq methods. 10X Genomics has licensed intellectual property on which W.J.G. is listed as an inventor. W.J.G. holds options in 10X Genomics and is a consultant for Ultima Genomics and Guardant Health. W.J.G. is a scientific co-founder of Protillion Biosciences., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Severe pain is a widespread health problem in need of novel treatment approaches. In the current study we used real water to give virtual objects (i.e., animated virtual water) more realistic physical properties (wet liquid qualities). Healthy volunteers aged 18-34 participated in a within-subject randomized study comparing participants' worst pain during brief thermal stimuli with (1) No Immersive Virtual Reality (VR), versus (2) during VR + no tactile feedback versus (3) VR + real water (with tactile feedback from co-located real objects). Tactile feedback significantly decreased pain intensity (VR analgesia, p < 0.01), compared to VR with no tactile feedback, and compared to No VR (baseline). Tactile feedback made the virtual water feel significantly more real, increased participant's sense of presence, and both VR conditions were distracting (significantly reduced accuracy on an attention demanding task). As a non-pharmacologic analgesic, mixed reality reduced pain by 35% in the current study, comparable to the analgesia from a moderate dose of hydromorphone in previous published experimental studies. Tactile feedback also significantly increased avatar embodiment, the participants illusion of ownership of the virtual hands, which has potential to improve the effectiveness of avatar therapy for chronic pain in future studies. Mixed reality should be tested as treatment in pain patients., (© 2023. The Author(s).)

Understanding spinal cord assembly is essential to elucidate how motor behavior is controlled and how disorders arise. The human spinal cord is exquisitely organized, and this complex organization contributes to the diversity and intricacy of motor behavior and sensory processing. But how this complexity arises at the cellular level in the human spinal cord remains unknown. Here we transcriptomically profiled the midgestation human spinal cord with single-cell resolution and discovered remarkable heterogeneity across and within cell types. Glia displayed diversity related to positional identity along the dorso-ventral and rostro-caudal axes, while astrocytes with specialized transcriptional programs mapped into white and gray matter subtypes. Motor neurons clustered at this stage into groups suggestive of alpha and gamma neurons. We also integrated our data with multiple existing datasets of the developing human spinal cord spanning 22 weeks of gestation to investigate the cell diversity over time. Together with mapping of disease-related genes, this transcriptomic mapping of the developing human spinal cord opens new avenues for interrogating the cellular basis of motor control in humans and guides human stem cell-based models of disease., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts., (© 2023. The Author(s).)

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact "motifs," whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of "exceptions to the rule" highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.

The vast majority of human pancreatic ductal adenocarcinomas (PDACs) harbor TP53 mutations, underscoring p53's critical role in PDAC suppression. PDAC can arise when pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), giving rise to premalignant pancreatic intraepithelial neoplasias (PanINs), which finally progress to PDAC. The occurrence of TP53 mutations in late-stage PanINs has led to the idea that p53 acts to suppress malignant transformation of PanINs to PDAC. However, the cellular basis for p53 action during PDAC development has not been explored in detail. Here, we leverage a hyperactive p53 variant-p53 53,54 -which we previously showed is a more robust PDAC suppressor than wild-type p53, to elucidate how p53 acts at the cellular level to dampen PDAC development. Using both inflammation-induced and KRAS G12D -driven PDAC models, we find that p53 53,54 both limits ADM accumulation and suppresses PanIN cell proliferation and does so more effectively than wild-type p53. Moreover, p53 53,54 suppresses KRAS signaling in PanINs and limits effects on the extracellular matrix (ECM) remodeling. While p53 53,54 has highlighted these functions, we find that pancreata in wild-type p53 mice similarly show less ADM, as well as reduced PanIN cell proliferation, KRAS signaling, and ECM remodeling relative to Trp53 -null mice. We find further that p53 enhances chromatin accessibility at sites controlled by acinar cell identity transcription factors. These findings reveal that p53 acts at multiple stages to suppress PDAC, both by limiting metaplastic transformation of acini and by dampening KRAS signaling in PanINs, thus providing key new understanding of p53 function in PDAC.

Single-cell technology has become an indispensable tool in cardiovascular research since its first introduction in 2009. Here, we highlight the recent remarkable progress in using single-cell technology to study transcriptomic and epigenetic heterogeneity in cardiac disease and development. We then introduce the key concepts in single-cell multi-omics modalities that apply to cardiovascular research. Lastly, we discuss some of the trending concepts in single-cell technology that are expected to propel cardiovascular research to the next phase of single-cell research., (© 2023. Springer Nature Limited.)

The ability to analyze the transcriptomic and epigenomic states of individual single cells has in recent years transformed our ability to measure and understand biological processes. Recent advancements have focused on increasing sensitivity and throughput to provide richer and deeper biological insights at the cellular level. The next frontier is the development of multiomic methods capable of analyzing multiple features from the same cell, such as the simultaneous measurement of the transcriptome and the chromatin accessibility of candidate regulatory elements. In this chapter, we discuss and describe SHARE-seq (Simultaneous high-throughput ATAC, and RNA expression with sequencing) for carrying out simultaneous chromatin accessibility and transcriptome measurements in single cells, together with the experimental and analytical considerations for achieving optimal results., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In eukaryotes, these CREs are generally characterized by nucleosomal depletion and thus higher physical accessibility of DNA. Many methods exploit this property to map regions of high average accessibility, and thus putative active CREs, in bulk. However, these techniques do not provide information about coordinated patterns of accessibility along the same DNA molecule, nor do they map the absolute levels of occupancy/accessibility. SMF (Single-Molecule Footprinting) fills these gaps by leveraging recombinant DNA cytosine methyltransferases (MTase) to mark accessible locations on individual DNA molecules. In this chapter, we discuss current methods and important considerations for performing SMF experiments., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Immune aging combines cellular defects in adaptive immunity with the activation of pathways causing a low-inflammatory state. Here we examined the influence of age on the kinetic changes in the epigenomic and transcriptional landscape induced by T cell receptor (TCR) stimulation in naive CD4 + T cells. Despite attenuated TCR signaling in older adults, TCR activation accelerated remodeling of the epigenome and induced transcription factor networks favoring effector cell differentiation. We identified increased phosphorylation of STAT5, at least in part due to aberrant IL-2 receptor and lower HELIOS expression, as upstream regulators. Human HELIOS-deficient, naive CD4 + T cells, when transferred into human-synovium-mouse chimeras, infiltrated tissues more efficiently. Inhibition of IL-2 or STAT5 activity in T cell responses of older adults restored the epigenetic response pattern to the one seen in young adults. In summary, reduced HELIOS expression in non-regulatory naive CD4 + T cells in older adults directs T cell fate decisions toward inflammatory effector cells that infiltrate tissue., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Active cis-regulatory elements (cREs) in eukaryotes are characterized by nucleosomal depletion and, accordingly, higher accessibility. This property has turned out to be immensely useful for identifying cREs genome-wide and tracking their dynamics across different cellular states and is the basis of numerous methods taking advantage of the preferential enzymatic cleavage/labeling of accessible DNA. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has emerged as the most versatile and widely adaptable method and has been widely adopted as the standard tool for mapping open chromatin regions. Here, we discuss the current optimal practices and important considerations for carrying out ATAC-seq experiments, primarily in the context of mammalian systems., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD., Competing Interests: Declaration of interests W.J.G. is named as an inventor on patents describing ATAC-seq methods. 10X Genomics has licensed intellectual property on which W.J.G. is listed as an inventor. W.J.G. holds options in 10X Genomics and is a consultant for Ultima Genomics and Guardant Health. W.J.G. is a scientific co-founder of Protillion Biosciences. A.S. is an employee of Insitro and is a consultant at Myokardia. A.K. is a consulting Fellow with Illumina, a member of the SAB of OpenTargets (GSK), PatchBio, SerImmune and a scientific co-founder of RavelBio. M.A., L.S., A. Banerjee, and K.F. are employees of Illumina. J.C.W. is a co-founder of Khloris Biosciences but has no competing interests, as the work presented here is completely independent. The other authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)