Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and spatial proximity.

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.

Imaging extrachromosomal DNA (ecDNA) in cancer.

Extrachromosomal DNA (ecDNA) are circular regions of DNA that are found in many cancers. They are an important means of oncogene amplification, and correlate with treatment resistance and poor prognosis. Consequently, there is great interest in exploring and targeting ecDNA vulnerabilities as potential new therapeutic targets for cancer treatment. However, the biological significance of ecDNA and their associated regulatory control remains unclear. Light microscopy has been a central tool in the identification and characterisation of ecDNA. In this review we describe the different cellular models available to study ecDNA, and the imaging tools used to characterise ecDNA and their regulation. The insights gained from quantitative imaging are discussed in comparison with genome sequencing and computational approaches. We suggest that there is a crucial need for ongoing innovation using imaging if we are to achieve a full understanding of the dynamic regulation and organisation of ecDNA and their role in tumourigenesis.

Unique activities of two overlapping PAX6 retinal enhancers.

Enhancers play a critical role in development by precisely modulating spatial, temporal, and cell type-specific gene expression. Sequence variants in enhancers have been implicated in diseases; however, establishing the functional consequences of these variants is challenging because of a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development, with a regulatory landscape containing multiple enhancers driving the expression in the eye. Whether these enhancers perform additive, redundant or distinct functions is unknown. Here, we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers, HS5 and NRE. Using a unique combination of live imaging and single-cell RNA sequencing in dual enhancer-reporter zebrafish embryos, we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping, these enhancers have distinct activities in different cell types and therefore likely nonredundant functions. This work demonstrates that unique cell type-specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo.

Ultra-long-range interactions between active regulatory elements.

Contacts between enhancers and promoters are thought to relate to their ability to activate transcription. Investigating factors that contribute to such chromatin interactions is therefore important for understanding gene regulation. Here, we have determined contact frequencies between millions of pairs of cis-regulatory elements from chromosome conformation capture data sets and analyzed a collection of hundreds of DNA-binding factors for binding at regions of enriched contacts. This analysis revealed enriched contacts at sites bound by many factors associated with active transcription. We show that active regulatory elements, independent of cohesin and polycomb, interact with each other across distances of tens of megabases in vertebrate and invertebrate genomes and that interactions correlate and change with activity. However, these ultra-long-range interactions are not dependent on RNA polymerase II transcription or individual transcription cofactors. Using simulations, we show that a model of chromatin and multivalent binding factors can give rise to long-range interactions via bridging-induced clustering. We propose that long-range interactions between cis-regulatory elements are driven by at least three distinct processes: cohesin-mediated loop extrusion, polycomb contacts, and clustering of active regions.

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops.

Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci-at different length scales-can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.

Mena regulates nesprin-2 to control actin-nuclear lamina associations, trans-nuclear membrane signalling and gene expression.

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes, play key roles in fundamental cellular processes, including the sensing and transduction of mechanical cues. Here, we investigate systems-level changes in the integrin adhesome in patient-derived cutaneous squamous cell carcinoma cells and identify the actin regulatory protein Mena as a key node in the adhesion complex network. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We uncover an unexpected role for Mena at the nuclear membrane, where it controls nuclear architecture, chromatin repositioning and gene expression. Our findings identify an adhesion protein that regulates gene transcription via direct signalling across the nuclear envelope.

Expansion of ventral foregut is linked to changes in the enhancer landscape for organ-specific differentiation.

Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase in cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation has not been considered as having a specific role. Here we exploit human embryonic stem cell-derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion-dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage-inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion; it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. Expansion of lineage-specific intermediates may therefore be an important step in achieving high-fidelity in vitro differentiation.

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering in glioblastoma stem cells.

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size, and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional hubs, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harbouring distinct ecDNA species (EGFR, CDK4, PDGFRA). We find no evidence that ecDNA routinely cluster with one another or closely interact with transcriptional hubs. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy is similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that it is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with each other or with high concentrations of the transcriptional machinery.

Cohesin is required for long-range enhancer action at the Shh locus.

The regulatory landscapes of developmental genes in mammals can be complex, with enhancers spread over many hundreds of kilobases. It has been suggested that three-dimensional genome organization, particularly topologically associating domains formed by cohesin-mediated loop extrusion, is important for enhancers to act over such large genomic distances. By coupling acute protein degradation with synthetic activation by targeted transcription factor recruitment, here we show that cohesin, but not CTCF, is required for activation of the target gene Shh by distant enhancers in mouse embryonic stem cells. Cohesin is not required for activation directly at the promoter or by an enhancer located closer to the Shh gene. Our findings support the hypothesis that chromatin compaction via cohesin-mediated loop extrusion allows for genes to be activated by enhancers that are located many hundreds of kilobases away in the linear genome and suggests that cohesin is dispensable for enhancers located more proximally.

TADs do not stay in the loop.

In a recent issue of Science, Gabriele et al. have, for the first time, quantified the dynamics of a topologically associating domain (TAD) in live cells by coupling super-resolution imaging and computational modelling, concluding that a TAD spends most of its life in a "partially extruded state" and that CTCF-CTCF loops are rare.

MUC4 is not expressed in cell lines used for live cell imaging.

Background: The ability to visualise specific mammalian gene loci in living cells is important for understanding the dynamic processes linked to transcription. However, some of the tools used to target mammalian genes for live cell imaging, such as dCas9, have been reported to themselves impede processes linked to transcription. The MUC4 gene is a popular target for live cell imaging studies due to the repetitive nature of sequences within some exons of this gene. Methods: We set out to compare the impact of dCas9 and TALE-based imaging tools on MUC4 expression, including in human cell lines previously reported as expressing MUC4. Results: We were unable to detect MUC4 mRNA in these cell lines. Moreover, analysis of publicly available data for histone modifications associated with transcription, and data for transcription itself, indicate that neither MUC4, nor any of the mucin gene family are significantly expressed in the cell lines where dCas9 targeting has been reported to repress MUC4 and MUC1 expression, or in the cell lines where dCas13 has been used to report MUC4 RNA detection in live cells. Conclusions: Methods for visualising specific gene loci and gene transcripts in live human cells are very challenging. Our data suggest that care should be given to the choice of the most appropriate cell lines for these analyses and that orthogonal methods of assaying gene expression be carefully compared.

Quantitative spatial and temporal assessment of regulatory element activity in zebrafish.

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.

Linking the FTO obesity rs1421085 variant circuitry to cellular, metabolic, and organismal phenotypes in vivo.

Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates Irx3 and Irx5 gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot-dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet-induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant's action.

Extensive pleiotropism and allelic heterogeneity mediate metabolic effects of IRX3 and IRX5.

Whereas coding variants often have pleiotropic effects across multiple tissues, noncoding variants are thought to mediate their phenotypic effects by specific tissue and temporal regulation of gene expression. Here, we investigated the genetic and functional architecture of a genomic region within the FTO gene that is strongly associated with obesity risk. We show that multiple variants on a common haplotype modify the regulatory properties of several enhancers targeting IRX3 and IRX5 from megabase distances. We demonstrate that these enhancers affect gene expression in multiple tissues, including adipose and brain, and impart regulatory effects during a restricted temporal window. Our data indicate that the genetic architecture of disease-associated loci may involve extensive pleiotropy, allelic heterogeneity, shared allelic effects across tissues, and temporally restricted effects.

Cornelia de Lange syndrome-associated mutations cause a DNA damage signalling and repair defect.

Cornelia de Lange syndrome is a multisystem developmental disorder typically caused by mutations in the gene encoding the cohesin loader NIPBL. The associated phenotype is generally assumed to be the consequence of aberrant transcriptional regulation. Recently, we identified a missense mutation in BRD4 associated with a Cornelia de Lange-like syndrome that reduces BRD4 binding to acetylated histones. Here we show that, although this mutation reduces BRD4-occupancy at enhancers it does not affect transcription of the pluripotency network in mouse embryonic stem cells. Rather, it delays the cell cycle, increases DNA damage signalling, and perturbs regulation of DNA repair in mutant cells. This uncovers a role for BRD4 in DNA repair pathway choice. Furthermore, we find evidence of a similar increase in DNA damage signalling in cells derived from NIPBL-deficient individuals, suggesting that defective DNA damage signalling and repair is also a feature of typical Cornelia de Lange syndrome.

Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction.

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture.

From bedside to bench: regulation of host factors in SARS-CoV-2 infection.

The zoonotic coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), which causes COVID-19 (coronavirus disease-2019), has resulted in a pandemic. This has led to an urgent need to understand the molecular determinants of SARS-CoV-2 infection, factors associated with COVID-19 heterogeneity and severity, and therapeutic options for these patients. In this review, we discuss the role of host factors in SARS-CoV-2 infection and describe variations in host factor expression as mechanisms underlying the symptoms and severity of COVID-19. We focus on two host factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), implicated in SARS-CoV-2 infection. We also discuss genetic variants associated with COVID-19 severity revealed in selected patients and based on genome-wide association studies (GWASs). Furthermore, we highlight important advances in cell and chromatin biology, such as single-cell RNA and chromatin sequencing and chromosomal conformation assays, as methods that may aid in the discovery of viral-host interactions in COVID-19. Understanding how regulation of host factor genes varies in physiological and pathological states might explain the heterogeneity observed in SARS-CoV-2 infection, help identify pathways for therapeutic development, and identify patients most likely to progress to severe COVID-19.