Multifaceted roles of cohesin in regulating transcriptional loops.

Cohesin is required for chromatin loop formation. However, its precise role in regulating gene transcription remains largely unknown. We investigated the relationship between cohesin and RNA Polymerase II (RNAPII) using single-molecule mapping and live-cell imaging methods in human cells. Cohesin-mediated transcriptional loops were highly correlated with those of RNAPII and followed the direction of gene transcription. Depleting RAD21, a subunit of cohesin, resulted in the loss of long-range (>100 kb) loops between distal (super-)enhancers and promoters of cell-type-specific genes. By contrast, the short-range (<50 kb) loops were insensitive to RAD21 depletion and connected genes that are mostly housekeeping. This result explains why only a small fraction of genes are affected by the loss of long-range chromatin interactions due to cohesin depletion. Remarkably, RAD21 depletion appeared to up-regulate genes located in early initiation zones (EIZ) of DNA replication, and the EIZ signals were amplified drastically without RAD21. Our results revealed new mechanistic insights of cohesin's multifaceted roles in establishing transcriptional loops, preserving long-range chromatin interactions for cell-specific genes, and maintaining timely order of DNA replication.

An allelic series of spontaneous Rorb mutant mice exhibit a gait phenotype, changes in retina morphology and behavior, and gene expression signatures associated with the unfolded protein response.

The Retinoid-related orphan receptor beta (RORß) gene encodes a developmental transcription factor and has 2 predominant isoforms created through alternative first exon usage; one specific to the retina and another present more broadly in the central nervous system, particularly regions involved in sensory processing. RORß belongs to the nuclear receptor family and plays important roles in cell fate specification in the retina and cortical layer formation. In mice, loss of RORß causes disorganized retina layers, postnatal degeneration, and production of immature cone photoreceptors. Hyperflexion or "high-stepping" of rear limbs caused by reduced presynaptic inhibition by Rorb-expressing inhibitory interneurons of the spinal cord is evident in RORß-deficient mice. RORß variants in patients are associated with susceptibility to various neurodevelopmental conditions, primarily generalized epilepsies, but including intellectual disability, bipolar, and autism spectrum disorders. The mechanisms by which RORß variants confer susceptibility to these neurodevelopmental disorders are unknown but may involve aberrant neural circuit formation and hyperexcitability during development. Here we report an allelic series in 5 strains of spontaneous Rorb mutant mice with a high-stepping gait phenotype. We show retinal abnormalities in a subset of these mutants and demonstrate significant differences in various behavioral phenotypes related to cognition. Gene expression analyses in all 5 mutants reveal a shared over-representation of the unfolded protein response and pathways related to endoplasmic reticulum stress, suggesting a possible mechanism of susceptibility relevant to patients.

ChIATAC is an efficient strategy for multi-omics mapping of 3D epigenomes from low-cell inputs.

Connecting genes to their cis-regulatory elements has been enabled by genome-wide mapping of chromatin interactions using proximity ligation in ChIA-PET, Hi-C, and their derivatives. However, these methods require millions of input cells for high-quality data and thus are unsuitable for many studies when only limited cells are available. Conversely, epigenomic profiling via transposase digestion in ATAC-seq requires only hundreds to thousands of cells to robustly map open chromatin associated with transcription activity, but it cannot directly connect active genes to their distal enhancers. Here, we combine proximity ligation in ChIA-PET and transposase accessibility in ATAC-seq into ChIATAC to efficiently map interactions between open chromatin loci in low numbers of input cells. We validate ChIATAC in Drosophila cells and optimize it for mapping 3D epigenomes in human cells robustly. Applying ChIATAC to primary human T cells, we reveal mechanisms that topologically regulate transcriptional programs during T cell activation.

Single-cell multimodal glioma analyses identify epigenetic regulators of cellular plasticity and environmental stress response.

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.

FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes.

The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC's long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a "mixed" neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the "neuronal" genes are bound by both SOX2 and AP1.

Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription.

Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer.

Reduced subgenomic RNA expression is a molecular indicator of asymptomatic SARS-CoV-2 infection.

Background: It is estimated that up to 80% of infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are asymptomatic and asymptomatic patients can still effectively transmit the virus and cause disease. While much of the effort has been placed on decoding single nucleotide variation in SARS-CoV-2 genomes, considerably less is known about their transcript variation and any correlation with clinical severity in human hosts, as defined here by the presence or absence of symptoms. Methods: To assess viral genomic signatures of disease severity, we conducted a systematic characterization of SARS-CoV-2 transcripts and genetic variants in 81 clinical specimens collected from symptomatic and asymptomatic individuals using multi-scale transcriptomic analyses including amplicon-seq, short-read metatranscriptome and long-read Iso-seq. Results: Here we show a highly coordinated and consistent pattern of sgRNA expression from individuals with robust SARS-CoV-2 symptomatic infection and their expression is significantly repressed in the asymptomatic infections. We also observe widespread inter- and intra-patient variants in viral RNAs, known as quasispecies frequently found in many RNA viruses. We identify unique sets of deletions preferentially found primarily in symptomatic individuals, with many likely to confer changes in SARS-CoV-2 virulence and host responses. Moreover, these frequently occurring structural variants in SARS-CoV-2 genomes serve as a mechanism to further induce SARS-CoV-2 proteome complexity. Conclusions: Our results indicate that differential sgRNA expression and structural mutational burden are highly correlated with the clinical severity of SARS-CoV-2 infection. Longitudinally monitoring sgRNA expression and structural diversity could further guide treatment responses, testing strategies, and vaccine development.

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BACKGROUND: Understanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production. RESULTS: We analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds. CONCLUSION: In this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism.

Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development.

Lineage-specific gene expression is modulated by a balance between transcriptional activation and repression during animal development. Knowledge about enhancer-centered transcriptional activation has advanced considerably, but silencers and their roles in normal development remain poorly understood. Here, we performed chromatin interaction analyses of Polycomb repressive complex 2 (PRC2), a key inducer of transcriptional gene silencing, to uncover silencers, their molecular identity and associated chromatin connectivity. Systematic analysis of cis-regulatory silencer elements reveals their chromatin features and gene-targeting specificity. Deletion of certain PRC2-bound silencers in mice results in transcriptional derepression of their interacting genes and pleiotropic developmental phenotypes, including embryonic lethality. While some PRC2-bound elements function as silencers in pluripotent cells, they can transition into active tissue-specific enhancers during development, highlighting their regulatory versatility. Our study characterizes the molecular profile of silencers and their associated chromatin architectures, and suggests the possibility of targeted reactivation of epigenetically silenced genes.

Comparative Molecular Life History of Spontaneous Canine and Human Gliomas.

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.

Ultra-long Read Sequencing for Whole Genomic DNA Analysis.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 µg of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Mapping the Global Chromatin Connectivity Network for Sox2 Function in Neural Stem Cell Maintenance.

The SOX2 transcription factor is critical for neural stem cell (NSC) maintenance and brain development. Through chromatin immunoprecipitation (ChIP) and chromatin interaction analysis (ChIA-PET), we determined genome-wide SOX2-bound regions and Pol II-mediated long-range chromatin interactions in brain-derived NSCs. SOX2-bound DNA was highly enriched in distal chromatin regions interacting with promoters and carrying epigenetic enhancer marks. Sox2 deletion caused widespread reduction of Pol II-mediated long-range interactions and decreased gene expression. Genes showing reduced expression in Sox2-deleted cells were significantly enriched in interactions between promoters and SOX2-bound distal enhancers. Expression of one such gene, Suppressor of Cytokine Signaling 3 (Socs3), rescued the self-renewal defect of Sox2-ablated NSCs. Our work identifies SOX2 as a major regulator of gene expression through connections to the enhancer network in NSCs. Through the definition of such a connectivity network, our study shows the way to the identification of genes and enhancers involved in NSC maintenance and neurodevelopmental disorders.

Multiplex chromatin interactions with single-molecule precision.

The genomes of multicellular organisms are extensively folded into 3D chromosome territories within the nucleus1. Advanced 3D genome-mapping methods that combine proximity ligation and high-throughput sequencing (such as chromosome conformation capture, Hi-C)2, and chromatin immunoprecipitation techniques (such as chromatin interaction analysis by paired-end tag sequencing, ChIA-PET)3, have revealed topologically associating domains4 with frequent chromatin contacts, and have identified chromatin loops mediated by specific protein factors for insulation and regulation of transcription5-7. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C8 potentially overcomes this issue, this method may be limited by the sparsity of data that is inherent to current single-cell assays. Recent advances in microfluidics have opened opportunities for droplet-based genomic analysis9 but this approach has not yet been adapted for chromatin interaction analysis. Here we describe a strategy for multiplex chromatin-interaction analysis via droplet-based and barcode-linked sequencing, which we name ChIA-Drop. We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with single-molecule precision, which has not been possible using methods based on population-level pairwise contacts. By applying ChIA-Drop to Drosophila cells, we show that chromatin topological structures predominantly consist of multiplex chromatin interactions with high heterogeneity; ChIA-Drop also reveals promoter-centred multivalent interactions, which provide topological insights into transcription.

Linked-read Sequencing Analysis Reveals Tumor-specific Genome Variation Landscapes in Neurofibromatosis Type 2 (NF2) Patients.

HYPOTHESIS: We hypothesize that genomic variants including deletions, insertions, inversions, and tandem duplications beyond the changes in tumor suppressor NF2 gene affect gene expression of tumor-specific pathways in vestibular schwannomas (VS) patients with Neurofibromatosis type 2 (NF2), thus contributing to their clinical behavior. BACKGROUND: Genomic variation could reconfigure transcription in NF2 transformation process. Therefore, genome-wide high-resolution characterization of structural variants (SV) landscapes in NF2 tumors can expand our understanding of the genes regulating the clinical phenotypes in NF2-associated VS. METHODS: We performed whole-genome haplotype-specific structural variation analysis using synthetic linked reads generated through microfluidics-based barcoding of high molecular weight DNA followed by high-coverage Illumina paired-end whole-genome sequencing from 10 patients' tumors of different growth rates and their matching blood samples. RESULTS: NF2 tumor-specific deletions and large SVs were detected and can be classified based on their association with tumor growth rates. Through detailed annotation of these mutations, we uncover common alleles affected by these deletions and large SVs that can be associated with signaling pathways implicated in cell proliferation and tumorigenesis. CONCLUSION: The genomic variation landscape of NF2-related VS was investigated through whole-genome linked-read sequencing. Large SVs, in addition to deletions, were identified and may serve as modulators of clinical behavior.

Sox2 conditional mutation in mouse causes ataxic symptoms, cerebellar vermis hypoplasia, and postnatal defects of Bergmann glia.

Sox2 is a transcription factor active in the nervous system, within different cell types, ranging from radial glia neural stem cells to a few specific types of differentiated glia and neurons. Mutations in the human SOX2 transcription factor gene cause various central nervous system (CNS) abnormalities, involving hippocampus and eye defects, as well as ataxia. Conditional Sox2 mutation in mouse, with different Cre transgenes, previously recapitulated different essential features of the disease, such as hippocampus and eye defects. In the cerebellum, Sox2 is active from early embryogenesis in the neural progenitors of the cerebellar primordium; Sox2 expression is maintained, postnatally, within Bergmann glia (BG), a differentiated cell type essential for Purkinje neurons functionality and correct motor control. By performing Sox2 Cre-mediated ablation in the developing and postnatal mouse cerebellum, we reproduced ataxia features. Embryonic Sox2 deletion (with Wnt1Cre) leads to reduction of the cerebellar vermis, known to be commonly related to ataxia, preceded by deregulation of Otx2 and Gbx2, critical regulators of vermis development. Postnatally, BG is progressively disorganized, mislocalized, and reduced in mutants. Sox2 postnatal deletion, specifically induced in glia (with GLAST-CreERT2), reproduces the BG defect, and causes (milder) ataxic features. Our results define a role for Sox2 in cerebellar function and development, and identify a functional requirement for Sox2 within postnatal BG, of potential relevance for ataxia in mouse mutants, and in human patients.

Picky comprehensively detects high-resolution structural variants in nanopore long reads.

Acquired genomic structural variants (SVs) are major hallmarks of cancer genomes, but they are challenging to reconstruct from short-read sequencing data. Here we exploited the long reads of the nanopore platform using our customized pipeline, Picky ( https://github.com/TheJacksonLaboratory/Picky ), to reveal SVs of diverse architecture in a breast cancer model. We identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses, and uncovered repetitive DNA as the major source of variation. Examination of genome-wide breakpoints at nucleotide resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with the propensity for interchromosomal connectivity and was found to be enriched in promoters and transcribed regions of the genome. Furthermore, we observed an over-representation of reciprocal translocations from chromosomal double-crossovers through phased SVs. We demonstrate that Picky analysis is an effective tool for comprehensive detection of SVs in cancer genomes from long-read data.

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species.

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

Genome Sequence of Roseovarius sp. Strain MCTG156(2b) Isolated from a Phytoplankton Net Trawl on the Scottish West Coast.

Roseovarius sp. strain MCTG156(2b) was isolated from a phytoplankton net sample collected on the west coast of Scotland and was selected based on its ability to degrade polycyclic aromatic hydrocarbons. Here, we present the genome sequence of this strain, which is 5,113,782 bp, with 5,142 genes and an average G+C content of 60.7%.

Genome Sequence of Oceanicola sp. Strain MCTG156(1a), Isolated from a Scottish Coastal Phytoplankton Net Sample.

Oceanicola sp. strain MCTG156(1a) was isolated from a phytoplankton net sample collected on the west coast of Scotland and selected based on its ability to degrade polycyclic aromatic hydrocarbons. Here, we present the genome sequence of this strain, which comprises 3,881,122 bp with 3,949 genes and an average G+C content of 62.7%.

Absence of genome reduction in diverse, facultative endohyphal bacteria.

Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.