scCircle-seq unveils the diversity and complexity of extrachromosomal circular DNAs in single cells.

Extrachromosomal circular DNAs (eccDNAs) have emerged as important intra-cellular mobile genetic elements that affect gene copy number and exert in trans regulatory roles within the cell nucleus. Here, we describe scCircle-seq, a method for profiling eccDNAs and unraveling their diversity and complexity in single cells. We implement and validate scCircle-seq in normal and cancer cell lines, demonstrating that most eccDNAs vary largely between cells and are stochastically inherited during cell division, although their genomic landscape is cell type-specific and can be used to accurately cluster cells of the same origin. eccDNAs are preferentially produced from chromatin regions enriched in H3K9me3 and H3K27me3 histone marks and are induced during replication stress conditions. Concomitant sequencing of eccDNAs and RNA from the same cell uncovers the absence of correlation between eccDNA copy number and gene expression levels, except for a few oncogenes, including MYC, contained within a large eccDNA in colorectal cancer cells. Lastly, we apply scCircle-seq to one prostate cancer and two breast cancer specimens, revealing cancer-specific eccDNA landscapes and a higher propensity of eccDNAs to form in amplified genomic regions. scCircle-seq is a scalable tool that can be used to dissect the complexity of eccDNAs across different cell and tissue types, and further expands the potential of eccDNAs for cancer diagnostics.

Compartmentalizing damaged DNA: A double-edged sword.

A recent publication in Nature by Arnould et al.1 describes a novel chromatin compartment, termed "damaged" or "D compartment," that facilitates the repair of DNA double-strand breaks but also increases the risk of potentially oncogenic translocation formation.

Enhancers dysfunction in the 3D genome of cancer cells.

Eukaryotic genomes are spatially organized inside the cell nucleus, forming a threedimensional (3D) architecture that allows for spatial separation of nuclear processes and for controlled expression of genes required for cell identity specification and tissue homeostasis. Hence, it is of no surprise that mis-regulation of genome architecture through rearrangements of the linear genome sequence or epigenetic perturbations are often linked to aberrant gene expression programs in tumor cells. Increasing research efforts have shed light into the causes and consequences of alterations of 3D genome organization. In this review, we summarize the current knowledge on how 3D genome architecture is dysregulated in cancer, with a focus on enhancer highjacking events and their contribution to tumorigenesis. Studying the functional effects of genome architecture perturbations on gene expression in cancer offers a unique opportunity for a deeper understanding of tumor biology and sets the basis for the discovery of novel therapeutic targets.

Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis.

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level HNRNPU downregulation leads to an increased fraction of neural progenitors in the maturing neuronal population. We conclude that the HNRNPU locus is involved in delayed commitment of neural progenitors to differentiate in cell types with hindbrain profile.

Separation of transcriptional repressor and activator functions in Drosophila HDAC3.

The histone deacetylase HDAC3 is associated with the NCoR/SMRT co-repressor complex, and its canonical function is in transcriptional repression, but it can also activate transcription. Here, we show that the repressor and activator functions of HDAC3 can be genetically separated in Drosophila. A lysine substitution in the N terminus (K26A) disrupts its catalytic activity and activator function, whereas a combination of substitutions (HEBI) abrogating the interaction with SMRTER enhances repressor activity beyond wild type in the early embryo. We conclude that the crucial functions of HDAC3 in embryo development involve catalytic-dependent gene activation and non-enzymatic repression by several mechanisms, including tethering of loci to the nuclear periphery.

A GC-centered view of 3D genome organization.

In the past two decades, our understanding of how the genome of mammalian cells is spatially organized in the three-dimensional (3D) space of the nucleus and how key nuclear processes are orchestrated in this space has drastically expanded. While genome organization has been extensively studied at the nanoscale, the higher-order arrangement of individual portions of the genome with respect to their intranuclear as well as reciprocal placement is less thoroughly characterized. Emerging evidence points to the existence of a complex radial arrangement of chromatin in the nucleus. However, what shapes this radial organization and whether it has any functional implications remain elusive. In this mini review, we first summarize our current knowledge on this rather overlooked aspect of mammalian genome organization. We then present a theoretical framework for explaining how the genome might be radially organized, focusing on the role of the guanine and cytosine density along the linear genome. Last, we discuss outstanding questions, hoping to inspire future experiments and spark interest in this topic within the 3D genome community.

Genomic Profiling Identifies Putative Pathogenic Alterations in NSCLC Brain Metastases.

Introduction: Brain metastases (BM) severely affect the prognosis and quality of life of patients with NSCLC. Recently, molecularly targeted agents were found to have promising activity against BM in patients with NSCLC whose primary tumors carry "druggable" mutations. Nevertheless, it remains critical to identify specific pathogenic alterations that drive NSCLC-BM and that can provide novel and more effective therapeutic targets. Methods: To identify potentially targetable pathogenic alterations in NSCLC-BM, we profiled somatic copy number alterations (SCNAs) in 51 matched pairs of primary NSCLC and BM samples from 33 patients with lung adenocarcinoma and 18 patients with lung squamous cell carcinoma. In addition, we performed multiregion copy number profiling on 15 BM samples and whole-exome sequencing on 40 of 51 NSCLC-BM pairs. Results: BM consistently had a higher burden of SCNAs compared with the matched primary tumors, and SCNAs were typically homogeneously distributed within BM, suggesting BM do not undergo extensive evolution once formed. By comparing focal SCNAs in matched NSCLC-BM pairs, we identified putative BM-driving alterations affecting multiple cancer genes, including several potentially targetable alterations in genes such as CDK12, DDR2, ERBB2, and NTRK1, which we validated in an independent cohort of 84 BM samples. Finally, we identified putative pathogenic alterations in multiple cancer genes, including genes involved in epigenome editing and 3D genome organization, such as EP300, CTCF, and STAG2, which we validated by targeted sequencing of an independent cohort of 115 BM samples. Conclusions: Our study represents the most comprehensive genomic characterization of NSCLC-BM available to date, paving the way to functional studies aimed at assessing the potential of the identified pathogenic alterations as clinical biomarkers and targets.

FRET-FISH probes chromatin compaction at individual genomic loci in single cells.

Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.

An atlas of endogenous DNA double-strand breaks arising during human neural cell fate determination.

Endogenous DNA double-strand breaks (DSBs) occurring in neural cells have been implicated in the pathogenesis of neurodevelopmental disorders (NDDs). Currently, a genomic map of endogenous DSBs arising during human neurogenesis is missing. Here, we applied in-suspension Breaks Labeling In Situ and Sequencing (sBLISS), RNA-Seq, and Hi-C to chart the genomic landscape of DSBs and relate it to gene expression and genome architecture in 2D cultures of human neuroepithelial stem cells (NES), neural progenitor cells (NPC), and post-mitotic neural cells (NEU). Endogenous DSBs were enriched at the promoter and along the gene body of transcriptionally active genes, at the borders of topologically associating domains (TADs), and around chromatin loop anchors. NDD risk genes harbored significantly more DSBs in comparison to other protein-coding genes, especially in NEU cells. We provide sBLISS, RNA-Seq, and Hi-C datasets for each differentiation stage, and all the scripts needed to reproduce our analyses. Our datasets and tools represent a unique resource that can be harnessed to investigate the role of genome fragility in the pathogenesis of NDDs.

The era of 3D and spatial genomics.

Over a decade ago the advent of high-throughput chromosome conformation capture (Hi-C) sparked a new era of 3D genomics. Since then the number of methods for mapping the 3D genome has flourished, enabling an ever-increasing understanding of how DNA is packaged in the nucleus and how the spatiotemporal organization of the genome orchestrates its vital functions. More recently, the next generation of spatial genomics technologies has begun to reveal how genome sequence and 3D genome organization vary between cells in their tissue context. We summarize how the toolkit for charting genome topology has evolved over the past decade and discuss how new technological developments are advancing the field of 3D and spatial genomics.

Dangerous RNA links pave the way to glioblastoma.

A new study by Deforzh et al. (2022) demonstrates how two long non-coding RNAs (lncRNAs) link a distal enhancer to the HOXD3/D4/miR-10b gene promoter, leading to transcriptional activation of the therapeutic target miR-10b in glioblastoma multiforme.

Simultaneous visualization of DNA loci in single cells by combinatorial multi-color iFISH.

Single-molecule DNA fluorescence in situ hybridization (FISH) techniques enable studying the three-dimensional (3D) organization of the genome at the single cell level. However, there is a major unmet need for open access, high quality, curated and reproducible DNA FISH datasets. Here, we describe a dataset obtained by applying our recently developed iFISH method to simultaneously visualize 16 small (size range: 62-73 kilobases, kb) DNA loci evenly spaced on chromosome 2 in human cells, in a single round of hybridization. We show how combinatorial color coding can be used to precisely localize multiple loci in 3D within single cells, and how inter-locus distances scale inversely with chromosome contact frequencies determined by high-throughput chromosome conformation capture (Hi-C). We provide raw images and 3D coordinates for nearly 10,000 FISH dots. Our dataset provides a free resource that can facilitate studies of 3D genome organization in single cells and can be used to develop automatic FISH analysis algorithms.

RNA gradients: Shapers of 3D genome architecture.

A growing body of evidence points to a role of nuclear RNAs (nucRNAs) in shaping the three-dimensional (3D) architecture of the genome within the nucleus of a eukaryotic cell. nucRNAs are non-homogeneously distributed within the nucleus where they can form global and local gradients that might contribute to instructing the formation and coordinating the function of different types of 3D genome structures. In this article, we highlight the available literature supporting a role of nucRNAs as 3D genome shapers and propose that nucRNA gradients are key mediators of genome structure and function.

piRNAs initiate transcriptional silencing of spermatogenic genes during C. elegans germline development.

Eukaryotic genomes harbor invading transposable elements that are silenced by PIWI-interacting RNAs (piRNAs) to maintain genome integrity in animal germ cells. However, whether piRNAs also regulate endogenous gene expression programs remains unclear. Here, we show that C. elegans piRNAs trigger the transcriptional silencing of hundreds of spermatogenic genes during spermatogenesis, promoting sperm differentiation and function. This silencing signal requires piRNA-dependent small RNA biogenesis and loading into downstream nuclear effectors, which correlates with the dynamic reorganization of two distinct perinuclear biomolecular condensates present in germ cells. In addition, the silencing capacity of piRNAs is temporally counteracted by the Argonaute CSR-1, which targets and licenses spermatogenic gene transcription. The spatial and temporal overlap between these opposing small RNA pathways contributes to setting up the timing of the spermatogenic differentiation program. Thus, our work identifies a prominent role for piRNAs as direct regulators of endogenous transcriptional programs during germline development and gamete differentiation.

Somatic Copy Number Alterations in Human Cancers: An Analysis of Publicly Available Data From The Cancer Genome Atlas.

Somatic copy number alterations (SCNAs) are a pervasive trait of human cancers that contributes to tumorigenesis by affecting the dosage of multiple genes at the same time. In the past decade, The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) initiatives have generated and made publicly available SCNA genomic profiles from thousands of tumor samples across multiple cancer types. Here, we present a comprehensive analysis of 853,218 SCNAs across 10,729 tumor samples belonging to 32 cancer types using TCGA data. We then discuss current models for how SCNAs likely arise during carcinogenesis and how genomic SCNA profiles can inform clinical practice. Lastly, we highlight open questions in the field of cancer-associated SCNAs.

COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance.

While mass-scale vaccination campaigns are ongoing worldwide, genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the emergence and global spread of viral variants of concern (VOC). Here, we present a streamlined workflow-COVseq-which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We benchmark COVseq against a standard library preparation method (NEBNext) on 29 SARS-CoV-2 positive samples, reaching 95.4% of concordance between single-nucleotide variants detected by both methods. Application of COVseq to 245 additional SARS-CoV-2 positive samples demonstrates the ability of the method to reliably detect emergent VOC as well as its compatibility with downstream phylogenetic analyses. A cost analysis shows that COVseq could be used to sequence thousands of samples at less than 15 USD per sample, including library preparation and sequencing costs. We conclude that COVseq is a versatile and scalable method that is immediately applicable for SARS-CoV-2 genomic surveillance and easily adaptable to other pathogens such as influenza viruses.

Genome-wide detection of DNA double-strand breaks by in-suspension BLISS.

sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.

Nuclear gene proximity and protein interactions shape transcript covariations in mammalian single cells.

Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.

GPSeq reveals the radial organization of chromatin in the cell nucleus.

With the exception of lamina-associated domains, the radial organization of chromatin in mammalian cells remains largely unexplored. Here we describe genomic loci positioning by sequencing (GPSeq), a genome-wide method for inferring distances to the nuclear lamina all along the nuclear radius. GPSeq relies on gradual restriction digestion of chromatin from the nuclear lamina toward the nucleus center, followed by sequencing of the generated cut sites. Using GPSeq, we mapped the radial organization of the human genome at 100-kb resolution, which revealed radial patterns of genomic and epigenomic features and gene expression, as well as A and B subcompartments. By combining radial information with chromosome contact frequencies measured by Hi-C, we substantially improved the accuracy of whole-genome structure modeling. Finally, we charted the radial topography of DNA double-strand breaks, germline variants and cancer mutations and found that they have distinctive radial arrangements in A and B subcompartments. We conclude that GPSeq can reveal fundamental aspects of genome architecture.