Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.

The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen.

The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.

Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response.

Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild-type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.

Neuronal HSF-1 coordinates the propagation of fat desaturation across tissues to enable adaptation to high temperatures in C. elegans.

To survive elevated temperatures, ectotherms adjust the fluidity of membranes by fine-tuning lipid desaturation levels in a process previously described to be cell autonomous. We have discovered that, in Caenorhabditis elegans, neuronal heat shock factor 1 (HSF-1), the conserved master regulator of the heat shock response (HSR), causes extensive fat remodeling in peripheral tissues. These changes include a decrease in fat desaturase and acid lipase expression in the intestine and a global shift in the saturation levels of plasma membrane's phospholipids. The observed remodeling of plasma membrane is in line with ectothermic adaptive responses and gives worms a cumulative advantage to warm temperatures. We have determined that at least 6 TAX-2/TAX-4 cyclic guanosine monophosphate (cGMP) gated channel expressing sensory neurons, and transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) are required for signaling across tissues to modulate fat desaturation. We also find neuronal hsf-1 is not only sufficient but also partially necessary to control the fat remodeling response and for survival at warm temperatures. This is the first study to show that a thermostat-based mechanism can cell nonautonomously coordinate membrane saturation and composition across tissues in a multicellular animal.

Detecting chromosomal interactions in Capture Hi-C data with CHiCAGO and companion tools.

Capture Hi-C is widely used to obtain high-resolution profiles of chromosomal interactions involving, at least on one end, regions of interest such as gene promoters. Signal detection in Capture Hi-C data is challenging and cannot be adequately accomplished with tools developed for other chromosome conformation capture methods, including standard Hi-C. Capture Hi-C Analysis of Genomic Organization (CHiCAGO) is a computational pipeline developed specifically for Capture Hi-C analysis. It implements a statistical model accounting for biological and technical background components, as well as bespoke normalization and multiple testing procedures for this data type. Here we provide a step-by-step guide to the CHiCAGO workflow that is aimed at users with basic experience of the command line and R. We also describe more advanced strategies for tuning the key parameters for custom experiments and provide guidance on data preprocessing and downstream analysis using companion tools. In a typical experiment, CHiCAGO takes ~2-3 h to run, although pre- and postprocessing steps may take much longer.

The global and promoter-centric 3D genome organization temporally resolved during a circadian cycle.

BACKGROUND: Circadian gene expression is essential for organisms to adjust their physiology and anticipate daily changes in the environment. The molecular mechanisms controlling circadian gene transcription are still under investigation. In particular, how chromatin conformation at different genomic scales and regulatory elements impact rhythmic gene expression has been poorly characterized. RESULTS: Here we measure changes in the spatial chromatin conformation in mouse liver using genome-wide and promoter-capture Hi-C alongside daily oscillations in gene transcription. We find topologically associating domains harboring circadian genes that switch assignments between the transcriptionally active and inactive compartment at different hours of the day, while their boundaries stably maintain their structure over time. To study chromatin contacts of promoters at high resolution over time, we apply promoter capture Hi-C. We find circadian gene promoters displayed a maximal number of chromatin contacts at the time of their peak transcriptional output. Furthermore, circadian genes, as well as contacted and transcribed regulatory elements, reach maximal expression at the same timepoints. Anchor sites of circadian gene promoter loops are enriched in DNA binding sites for liver nuclear receptors and other transcription factors, some exclusively present in either rhythmic or stable contacts. Finally, by comparing the interaction profiles between core clock and output circadian genes, we show that core clock interactomes are more dynamic compared to output circadian genes. CONCLUSION: Our results identify chromatin conformation dynamics at different scales that parallel oscillatory gene expression and characterize the repertoire of regulatory elements that control circadian gene transcription through rhythmic or stable chromatin configurations.

Proximity RNA-seq: A Sequencing Method to Identify Co-localization of RNA.

RNA localization is an important regulatory layer of gene expression and cell functioning. The protocol guides through the Proximity RNA-seq method, in which RNA molecules are sequenced in their spatial, cellular context to derive RNA co-localization and transcriptome organization. Transcripts in individual subcellular particles from chemically crosslinked cells are tagged with the same, unique DNA barcode in water-in-oil emulsion droplets. First, single DNA barcodes are PCR amplified and immobilized on single, small magnetic beads in droplets. Subsequently, 3' ends of bead-bound barcode copies are tailed with random pentadecamers. Then beads are encapsulated again into droplets together with crosslinked subcellular particles containing RNA. Reverse transcription using random pentadecamers as primers is performed in droplets, which optimally contain one bead and one particle, in order to tag RNAs co-localized to the same particle. Sequencing such cDNA molecules identifies the RNA molecule and the barcode. Subsequent analysis of transcripts that share the same barcode, i.e., co-barcoding, reveals RNA co-localization and interactions. The technique is not restricted to pairs of RNAs but can as well detect groups of transcripts and estimates local RNA density or connectivity for individual transcripts. We provide here a detailed protocol to perform and analyze Proximity RNA-seq on cell nuclei to study spatial, nuclear RNA organization.

Active and repressed biosynthetic gene clusters have spatially distinct chromosome states.

While colocalization within a bacterial operon enables coexpression of the constituent genes, the mechanistic logic of clustering of nonhomologous monocistronic genes in eukaryotes is not immediately obvious. Biosynthetic gene clusters that encode pathways for specialized metabolites are an exception to the classical eukaryote rule of random gene location and provide paradigmatic exemplars with which to understand eukaryotic cluster dynamics and regulation. Here, using 3C, Hi-C, and Capture Hi-C (CHi-C) organ-specific chromosome conformation capture techniques along with high-resolution microscopy, we investigate how chromosome topology relates to transcriptional activity of clustered biosynthetic pathway genes in Arabidopsis thaliana Our analyses reveal that biosynthetic gene clusters are embedded in local hot spots of 3D contacts that segregate cluster regions from the surrounding chromosome environment. The spatial conformation of these cluster-associated domains differs between transcriptionally active and silenced clusters. We further show that silenced clusters associate with heterochromatic chromosomal domains toward the periphery of the nucleus, while transcriptionally active clusters relocate away from the nuclear periphery. Examination of chromosome structure at unrelated clusters in maize, rice, and tomato indicates that integration of clustered pathway genes into distinct topological domains is a common feature in plant genomes. Our results shed light on the potential mechanisms that constrain coexpression within clusters of nonhomologous eukaryotic genes and suggest that gene clustering in the one-dimensional chromosome is accompanied by compartmentalization of the 3D chromosome.

RNA proximity sequencing data and analysis pipeline from a human neuroblastoma nuclear transcriptome.

We have previously developed and described a method for measuring RNA co-locations within cells, called Proximity RNA-seq, which promises insights into RNA expression, processing, storage and translation. Here, we describe transcriptome-wide proximity RNA-seq datasets obtained from human neuroblastoma SH-SY5Y cell nuclei. To aid future users of this method, we also describe and release our analysis pipeline, CloseCall, which maps cDNA to a custom transcript annotation and allocates cDNA-linked barcodes to barcode groups. CloseCall then performs Monte Carlo simulations on the data to identify pairs of transcripts, which are co-barcoded more frequently than expected by chance. Furthermore, derived co-barcoding frequencies for individual transcripts, dubbed valency, serve as proxies for RNA density or connectivity for that given transcript. We outline how this pipeline was applied to these sequencing datasets and openly share the processed data outputs and access to a virtual machine that runs CloseCall. The resulting data specify the spatial organization of RNAs and builds hypotheses for potential regulatory relationships between RNAs.

RNA proximity sequencing reveals the spatial organization of the transcriptome in the nucleus.

The global, three-dimensional organization of RNA molecules in the nucleus is difficult to determine using existing methods. Here we introduce Proximity RNA-seq, which identifies colocalization preferences for pairs or groups of nascent and fully transcribed RNAs in the nucleus. Proximity RNA-seq is based on massive-throughput RNA barcoding of subnuclear particles in water-in-oil emulsion droplets, followed by cDNA sequencing. Our results show RNAs of varying tissue-specificity of expression, speed of RNA polymerase elongation and extent of alternative splicing positioned at varying distances from nucleoli. The simultaneous detection of multiple RNAs in proximity to each other distinguishes RNA-dense from sparse compartments. Application of Proximity RNA-seq will facilitate study of the spatial organization of transcripts in the nucleus, including non-coding RNAs, and its functional relevance.

The adjuvant GLA-SE promotes human Tfh cell expansion and emergence of public TCRß clonotypes.

The generation of protective humoral immunity after vaccination relies on the productive interaction between antigen-specific B cells and T follicular helper (Tfh) cells. Despite the central role of Tfh cells in vaccine responses, there is currently no validated way to enhance their differentiation in humans. From paired human lymph node and blood samples, we identify a population of circulating Tfh cells that are transcriptionally and clonally similar to germinal center Tfh cells. In a clinical trial of vaccine formulations, circulating Tfh cells were expanded in Tanzanian volunteers when an experimental malaria vaccine was adjuvanted in GLA-SE but not when formulated in Alum. The GLA-SE-formulated peptide was associated with an increase in the extrafollicular antibody response, long-lived antibody production, and the emergence of public TCRß clonotypes in circulating Tfh cells. We demonstrate that altering vaccine adjuvants is a rational approach for enhancing Tfh cells in humans, thereby supporting the long-lived humoral immunity that is required for effective vaccines.

Author Correction: Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks.

In the original version of the Article, the gene symbol for tissue factor pathway inhibitor was inadvertently given as 'TFP1' instead of 'TFPI'. This has now been corrected in both the PDF and HTML versions of the Article.

FastQ Screen: A tool for multi-genome mapping and quality control.

DNA sequencing analysis typically involves mapping reads to just one reference genome.  Mapping against multiple genomes is necessary, however, when the genome of origin requires confirmation. Mapping against multiple genomes is also advisable for detecting contamination or for identifying sample swaps which, if left undetected, may lead to incorrect experimental conclusions.  Consequently, we present FastQ Screen, a tool to validate the origin of DNA samples by quantifying the proportion of reads that map to a panel of reference genomes. FastQ Screen is intended to be used routinely as a quality control measure and for analysing samples in which the origin of the DNA is uncertain or has multiple sources.

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells.

BACKGROUND: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. RESULTS: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. CONCLUSIONS: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks.

Long-range chromosomal interactions bring distal regulatory elements and promoters together to regulate gene expression in biological processes. By performing promoter capture Hi-C (PCHi-C) on human embryonic stem cell-derived cardiomyocytes (hESC-CMs), we show that such promoter interactions are a key mechanism by which enhancers contact their target genes after hESC-CM differentiation from hESCs. We also show that the promoter interactome of hESC-CMs is associated with expression quantitative trait loci (eQTLs) in cardiac left ventricular tissue; captures the dynamic process of genome reorganisation after hESC-CM differentiation; overlaps genome-wide association study (GWAS) regions associated with heart rate; and identifies new candidate genes in such regions. These findings indicate that regulatory elements in hESC-CMs identified by our approach control gene expression involved in ventricular conduction and rhythm of the heart. The study of promoter interactions in other hESC-derived cell types may be of utility in functional investigation of GWAS-associated regions.

The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Capture Hi-C identifies putative target genes at 33 breast cancer risk loci.

Genome-wide association studies (GWAS) have identified approximately 100 breast cancer risk loci. Translating these findings into a greater understanding of the mechanisms that influence disease risk requires identification of the genes or non-coding RNAs that mediate these associations. Here, we use Capture Hi-C (CHi-C) to annotate 63 loci; we identify 110 putative target genes at 33 loci. To assess the support for these target genes in other data sources we test for associations between levels of expression and SNP genotype (eQTLs), disease-specific survival (DSS), and compare them with somatically mutated cancer genes. 22 putative target genes are eQTLs, 32 are associated with DSS and 14 are somatically mutated in breast, or other, cancers. Identifying the target genes at GWAS risk loci will lead to a greater understanding of the mechanisms that influence breast cancer risk and prognosis.

Long-Range Enhancer Interactions Are Prevalent in Mouse Embryonic Stem Cells and Are Reorganized upon Pluripotent State Transition.

Transcriptional enhancers, including super-enhancers (SEs), form physical interactions with promoters to regulate cell-type-specific gene expression. SEs are characterized by high transcription factor occupancy and large domains of active chromatin, and they are commonly assigned to target promoters using computational predictions. How promoter-SE interactions change upon cell state transitions, and whether transcription factors maintain SE interactions, have not been reported. Here, we used promoter-capture Hi-C to identify promoters that interact with SEs in mouse embryonic stem cells (ESCs). We found that SEs form complex, spatial networks in which individual SEs contact multiple promoters, and a rewiring of promoter-SE interactions occurs between pluripotent states. We also show that long-range promoter-SE interactions are more prevalent in ESCs than in epiblast stem cells (EpiSCs) or Nanog-deficient ESCs. We conclude that SEs form cell-type-specific interaction networks that are partly dependent on core transcription factors, thereby providing insights into the gene regulatory organization of pluripotent cells.