High-throughput PRIME-editing screens identify functional DNA variants in the human genome.

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and single-base resolution manner remains challenging. Here, we develop a pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp MYC enhancer via PRIME-mediated single-base resolution analysis. Next, we applied PRIME to functionally characterize 1,304 genome-wide association study (GWAS)-identified non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate that PRIME is capable of characterizing genetic variants at single-base resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Multicenter integrated analysis of noncoding CRISPRi screens.

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.

Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.

The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.

An atlas of gene regulatory elements in adult mouse cerebrum.

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures1. Recent surveys of mouse and human brains with single-cell transcriptomics2-6 and high-throughput imaging technologies7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans.

Phase separation of YAP-MAML2 differentially regulates the transcriptome.

Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Cell-type-specific 3D epigenomes in the developing human cortex.

Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.

An atlas of dynamic chromatin landscapes in mouse fetal development.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

Author Correction: An atlas of dynamic chromatin landscapes in mouse fetal development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

THUNDER: A reference-free deconvolution method to infer cell type proportions from bulk Hi-C data.

Hi-C data provide population averaged estimates of three-dimensional chromatin contacts across cell types and states in bulk samples. Effective analysis of Hi-C data entails controlling for the potential confounding factor of differential cell type proportions across heterogeneous bulk samples. We propose a novel unsupervised deconvolution method for inferring cell type composition from bulk Hi-C data, the Two-step Hi-c UNsupervised DEconvolution appRoach (THUNDER). We conducted extensive simulations to test THUNDER based on combining two published single-cell Hi-C (scHi-C) datasets. THUNDER more accurately estimates the underlying cell type proportions compared to reference-free methods (e.g., TOAST, and NMF) and is more robust than reference-dependent methods (e.g. MuSiC). We further demonstrate the practical utility of THUNDER to estimate cell type proportions and identify cell-type-specific interactions in Hi-C data from adult human cortex tissue samples. THUNDER will be a useful tool in adjusting for varying cell type composition in population samples, facilitating valid and more powerful downstream analysis such as differential chromatin organization studies. Additionally, THUNDER estimated contact profiles provide a useful exploratory framework to investigate cell-type-specificity of the chromatin interactome while experimental data is still rare.

High-throughput CRISPRi and CRISPRa technologies in 3D genome regulation for neuropsychiatric diseases.

Advances in genomics have led to the identification of many risk loci with hundreds of genes and thousands of DNA variants associated with neuropsychiatric disorders. A significant barrier to understanding the genetic underpinnings of complex diseases is the lack of functional characterization of risk genes and variants in biological systems relevant to human health and connecting disease-associated variants to pathological phenotypes. Characterizing gene and DNA variant functions requires genetic perturbations followed by molecular and cellular assays of neurobiological phenotypes. However, generating null or mutant alleles is low throughput, making it impossible to characterize disease-associated variants in large quantities efficiently. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens can be leveraged to dissect the biological consequences of the tested genes and variants in their native context. Nevertheless, testing non-coding variants associated with complex diseases remains non-trivial. In this review, we first discuss the current challenges of interpreting the function of the non-coding genome and approaches to prioritizing disease-associated variants in the context of the 3D epigenome. Second, we provide a brief overview of high-throughput CRISPRi and CRISPRa screening strategies applicable for characterizing non-coding sequences in appropriate biological systems. Lastly, we discuss the promising prospects of using CRISPR-based technologies to dissect DNA sequences associated with neuropsychiatric diseases.

CharID: a two-step model for universal prediction of interactions between chromatin accessible regions.

Open chromatin regions (OCRs) allow direct interaction between cis-regulatory elements and trans-acting factors. Therefore, predicting all potential OCR-mediated loops is essential for deciphering the regulation mechanism of gene expression. However, existing loop prediction tools are restricted to specific anchor types. Here, we present CharID (Chromatin Accessible Region Interaction Detector), a two-step model that combines neural network and ensemble learning to predict OCR-mediated loops. In the first step, CharID-Anchor, an attention-based hybrid CNN-BiGRU network is constructed to discriminate between the anchor and nonanchor OCRs. In the second step, CharID-Loop uses gradient boosting decision tree with chromosome-split strategy to predict the interactions between anchor OCRs. The performance was assessed in three human cell lines, and CharID showed superior prediction performance compared with other algorithms. In contrast to the methods designed to predict a particular type of loops, CharID can detect varieties of chromatin loops not limited to enhancer-promoter loops or architectural protein-mediated loops. We constructed the OCR-mediated interaction network using the predicted loops and identified hub anchors, which are highlighted by their proximity to housekeeping genes. By analyzing loops containing SNPs associated with cardiovascular disease, we identified an SNP-gene loop indicating the regulation mechanism of the GFOD1. Taken together, CharID universally predicts diverse chromatin loops beyond other state-of-the-art methods, which are limited by anchor types, and experimental techniques, which are limited by sensitivities drastically decaying with the genomic distance of anchors. Finally, we hosted Peaksniffer, a user-friendly web server that provides online prediction, query and visualization of OCRs and associated loops.

Transcriptional network orchestrating regional patterning of cortical progenitors.

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

Pathological hemodynamic changes and leukocyte transmigration disrupt the blood-spinal cord barrier after spinal cord injury.

BACKGROUND: Blood-spinal cord barrier (BSCB) disruption is a key event after spinal cord injury (SCI), which permits unfavorable blood-derived substances to enter the neural tissue and exacerbates secondary injury. However, limited mechanical impact is usually followed by a large-scale BSCB disruption in SCI. How the BSCB disruption is propagated along the spinal cord in the acute period of SCI remains unclear. Thus, strategies for appropriate clinical treatment are lacking. METHODS: A SCI contusion mouse model was established in wild-type and LysM-YFP transgenic mice. In vivo two-photon imaging and complementary studies, including immunostaining, capillary western blotting, and whole-tissue clearing, were performed to monitor BSCB disruption and verify relevant injury mechanisms. Clinically applied target temperature management (TTM) to reduce the core body temperature was tested for the efficacy of attenuating BSCB disruption. RESULTS: Barrier leakage was detected in the contusion epicenter within several minutes and then gradually spread to more distant regions. Membrane expression of the main tight junction proteins remained unaltered at four hours post-injury. Many junctional gaps emerged in paracellular tight junctions at the small vessels from multiple spinal cord segments at 15 min post-injury. A previously unnoticed pathological hemodynamic change was observed in the venous system, which likely facilitated gap formation and barrier leakage by exerting abnormal physical force on the BSCB. Leukocytes were quickly initiated to transverse through the BSCB within 30 min post-SCI, actively facilitating gap formation and barrier leakage. Inducing leukocyte transmigration generated gap formation and barrier leakage. Furthermore, pharmacological alleviation of pathological hemodynamic changes or leukocyte transmigration reduced gap formation and barrier leakage. TTM had very little protective effects on the BSCB in the early period of SCI other than partially alleviating leukocyte infiltration. CONCLUSIONS: Our data show that BSCB disruption in the early period of SCI is a secondary change, which is indicated by widespread gap formation in tight junctions. Pathological hemodynamic changes and leukocyte transmigration contribute to gap formation, which could advance our understanding of BSCB disruption and provide new clues for potential treatment strategies. Ultimately, TTM is inadequate to protect the BSCB in early SCI.

Engineered Extracellular Vesicles for Delivery of an IL-1 Receptor Antagonist Promote Targeted Repair of Retinal Degeneration.

Retinal degeneration (RD) is an irreversible blinding disease that seriously affects patients' daily activities and mental health. Targeting hyperactivated microglia and regulating polarization are promising strategies for treating the disease. Mesenchymal stem cell (MSC) transplantation is proven to be an effective treatment due to its immunomodulatory and regenerative properties. However, the low efficiency of cell migration and integration of MSCs remains a major obstacle to clinical use. The goal of this study is to develop a nanodelivery system that targets hyperactivated microglia and inhibits their release of proinflammatory factors, to achieve durable neuroprotection. This approach is to engineer extracellular vesicles (EVs) isolated from MSC, modify them with a cyclic RGD (cRGD) peptide on their surface, and load them with an antagonist of the IL-1 receptor, anakinra. Comparing with non-engineered EVs, it is observed that engineered cRGD-EVs exhibit an increased targeting efficiency against hyperactivated microglia and strongly protected photoreceptors in experimental RD cells and animal models. This study provides a strategy to improve drug delivery to degenerated retinas and offers a promising approach to improve the treatment of RD through targeted modulation of the immune microenvironment via engineered cRGD-EVs.

SMNN: batch effect correction for single-cell RNA-seq data via supervised mutual nearest neighbor detection.

Batch effect correction has been recognized to be indispensable when integrating single-cell RNA sequencing (scRNA-seq) data from multiple batches. State-of-the-art methods ignore single-cell cluster label information, but such information can improve the effectiveness of batch effect correction, particularly under realistic scenarios where biological differences are not orthogonal to batch effects. To address this issue, we propose SMNN for batch effect correction of scRNA-seq data via supervised mutual nearest neighbor detection. Our extensive evaluations in simulated and real datasets show that SMNN provides improved merging within the corresponding cell types across batches, leading to reduced differentiation across batches over MNN, Seurat v3 and LIGER. Furthermore, SMNN retains more cell-type-specific features, partially manifested by differentially expressed genes identified between cell types after SMNN correction being biologically more relevant, with precision improving by up to 841.0%.

Accuracy, Sensitivity, and Specificity of the LLIS and ULL27 in Detecting Breast Cancer-Related Lymphedema.

INTRODUCTION: Breast cancer-related lymphedema occurs in up to 30% of women following axillary lymph node dissection (ALND) and less commonly following sentinel lymph node biopsy. To quantify disability in these patients, patient-reported outcome measures (PROMs) have proven useful; however, given the overlap of symptoms between ALND and lymphedema, examination of their accuracy, sensitivity, and specificity in detecting lymphedema in breast cancer patients undergoing ALND is needed. METHODS: The Lymphedema Life Impact Scale (LLIS) and the Upper Limb Lymphedema 27 scale (ULL27) were administered to patients who had undergone ALND at least 2 years prior and either did or did not develop lymphedema. Survey responses and the degree of disability were compared to generate receiver operator characteristic (ROC) curves, and the sensitivity and specificity of PROMs to diagnose lymphedema were analyzed. RESULTS: Both PROMs were highly accurate, sensitive, and specific for detecting lymphedema. The LLIS had an accuracy of 97%, sensitivity of 100%, and specificity of 84.8% at a cutoff of ≥ 5.88 overall percent impairment score (higher scores indicate worse disability). The ULL27 had an accuracy of 93%, sensitivity of 88.6%, and specificity of 90.9% at a cutoff of ≤ 83.3 global score (lower scores indicate worse disability). CONCLUSIONS: The LLIS and the ULL27 appear to be highly specific for lymphedema and capable of differentiating it from symptoms resulting from ALND alone. Our findings suggest that use of these questionnaires with a threshold may be effective for diagnosing lymphedema, potentially reducing the need for frequent clinic visits and time-consuming measurements.