A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart.

The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

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.

Genome-wide maps of nuclear lamina interactions in single human cells.

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.

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.

Deconwolf enables high-performance deconvolution of widefield fluorescence microscopy images.

Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.

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.

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.

Human skin long noncoding RNA WAKMAR1 regulates wound healing by enhancing keratinocyte migration.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

Spatially resolved transcriptomics and beyond.

Considerable progress in sequencing technologies makes it now possible to study the genomic and transcriptomic landscape of single cells. However, to better understand the complexity of multicellular organisms, we must devise ways to perform high-throughput measurements while preserving spatial information about the tissue context or subcellular localization of analysed nucleic acids. In this Innovation article, we summarize pioneering technologies that enable spatially resolved transcriptomics and discuss how these methods have the potential to extend beyond transcriptomics to encompass spatially resolved genomics, proteomics and possibly other omic disciplines.

A versatile genome-scale PCR-based pipeline for high-definition DNA FISH.

We developed a cost-effective genome-scale PCR-based method for high-definition DNA FISH (HD-FISH). We visualized gene loci with diffraction-limited resolution, chromosomes as spot clusters and single genes together with transcripts by combining HD-FISH with single-molecule RNA FISH. We provide a database of over 4.3 million primer pairs targeting the human and mouse genomes that is readily usable for rapid and flexible generation of probes.

Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing.

We present a genome-wide approach to map DNA double-strand breaks (DSBs) at nucleotide resolution by a method we termed BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). We validated and tested BLESS using human and mouse cells and different DSBs-inducing agents and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs and complex genome-wide DSB landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and we identified >2,000 nonuniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions, with a specificity and resolution unachievable by current techniques.

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.

High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue.

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.

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.

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.

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.

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.

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.