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During oncogene-induced senescence there are striking changes in the organisation of heterochromatin in the nucleus. This is accompanied by activation of a pro-inflammatory gene expression programme - the senescence associated secretory phenotype (SASP) - driven by transcription factors such as NF-κB. The relationship between heterochromatin re-organisation and the SASP has been unclear. Here we show that TPR, a protein of the nuclear pore complex basket required for heterochromatin re-organisation during senescence, is also required for the very early activation of NF-κB signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-κB nuclear import. We show that TPR is required for the activation of innate immune signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. We show that HMGA1 is also required for cytoplasmic chromatin fragment formation. Together these data suggest that re-organisation of heterochromatin is involved in altered structural integrity of the nuclear periphery during senescence, and that this can lead to activation of cytoplasmic nucleic acid sensing, NF-κB signalling, and activation of the SASP., Competing Interests: BB, YK, SB, TC, AQ, CB, JA, WB The authors declare that no competing interests exist., (© 2024, Bartlett et al.)

Background: Genome-wide association studies (GWAS) have revealed a multitude of candidate genetic variants affecting the risk of developing complex traits and diseases. However, the highlighted regions are typically in the non-coding genome, and uncovering the functional causative single nucleotide variants (SNVs) is challenging. Prioritization of variants is commonly based on genomic annotation with markers of active regulatory elements, but current approaches still poorly predict functional variants. To address this, we systematically analyze six markers of active regulatory elements for their ability to identify functional variants., Results: We benchmark against molecular quantitative trait loci (molQTL) from assays of regulatory element activity that identify allelic effects on DNA-binding factor occupancy, reporter assay expression, and chromatin accessibility. We identify the combination of DNase footprints and divergent enhancer RNA (eRNA) as markers for functional variants. This signature provides high precision, but with a trade-off of low recall, thus substantially reducing candidate variant sets to prioritize variants for functional validation. We present this as a framework called FINDER-Functional SNV IdeNtification using DNase footprints and eRNA., Conclusions: We demonstrate the utility to prioritize variants using leukocyte count trait and analyze variants in linkage disequilibrium with a lead variant to predict a functional variant in asthma. Our findings have implications for prioritizing variants from GWAS, in development of predictive scoring algorithms, and for functionally informed fine mapping approaches., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gómez Acuña et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023 Uttley et al.)

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., (© 2023 Friman et al.; Published by Cold Spring Harbor Laboratory Press.)

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., (© 2023 Williamson et al.)

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., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

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., Competing Interests: KP, EF, SB, PD, VG, AH, GM, PB, SB, SP, WB No competing interests declared, (© 2022, Purshouse et al.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., Competing Interests: SB, DK, KU, AM, ND, WB No competing interests declared, (© 2021, Bhatia et al.)

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., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Athmane N et al.)

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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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.

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.

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.

Corneal resistance factor (CRF) is altered during corneal diseases progression. Genome-wide-association studies (GWAS) indicated potential CRF and disease genetics overlap. Here, we characterise 135 CRF loci following GWAS in 76029 UK Biobank participants. Enrichment of extra-cellular matrix gene-sets, genetic correlation with corneal thickness (70% (SE = 5%)), reported keratoconus risk variants at 13 loci, all support relevance to corneal stroma biology. Fine-mapping identifies a subset of 55 highly likely causal variants, 91% of which are non-coding. Genomic features enrichments, using all associated variants, also indicate prominent regulatory causal role. We newly established open chromatin landscapes in two widely-used human cornea immortalised cell lines using ATAC-seq. Variants associated with CRF were significantly enriched in regulatory regions from the corneal stroma-derived cell line and enrichment increases to over 5 fold for variants prioritised by fine-mapping-including at GAS7, SMAD3 and COL6A1 loci. Our analysis generates many hypotheses for future functional validation of aetiological mechanisms.

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system., (© 2020 Boyle et al.; Published by Cold Spring Harbor Laboratory Press.)

Motivation: Hi-C is currently the method of choice to investigate the global 3D organization of the genome. A major limitation of Hi-C is the sequencing depth required to robustly detect loops in the data. A popular approach used to mitigate this issue, even in single-cell Hi-C data, is genome-wide averaging (piling-up) of peaks, or other features, annotated in high-resolution datasets, to measure their prominence in less deeply sequenced data. However, current tools do not provide a computationally efficient and versatile implementation of this approach., Results: Here, we describe coolpup.py-a versatile tool to perform pile-up analysis on Hi-C data. We demonstrate its utility by replicating previously published findings regarding the role of cohesin and CTCF in 3D genome organization, as well as discovering novel details of Polycomb-driven interactions. We also present a novel variation of the pile-up approach that can aid the statistical analysis of looping interactions. We anticipate that coolpup.py will aid in Hi-C data analysis by allowing easy to use, versatile and efficient generation of pile-ups., Availability and Implementation: Coolpup.py is cross-platform, open-source and free (MIT licensed) software. Source code is available from https://github.com/Phlya/coolpuppy and it can be installed from the Python Packaging Index., (© The Author(s) 2020. Published by Oxford University Press.)

Background: Thousands of mammalian promoters are defined by co-enrichment of the histone tail modifications H3K27me3 (repressive) and H3K4me3 (activating) and are thus termed bivalent. It was previously observed that bivalent genes in human ES cells (hESC) are frequent targets for hypermethylation in human cancers, and depletion of DNA methylation in mouse embryonic stem cells has a marked impact on H3K27me3 distribution at bivalent promoters. However, only a fraction of bivalent genes in stem cells are targets of hypermethylation in cancer, and it is currently unclear whether all bivalent promoters are equally sensitive to DNA hypomethylation and whether H3K4me3 levels play a role in the interplay between DNA methylation and H3K27me3., Results: We report the sub-classification of bivalent promoters into two groups-promoters with a high H3K27me3:H3K4me3 (hiBiv) ratio or promoters with a low H3K27me3:H3K4me3 ratio (loBiv). HiBiv are enriched in canonical Polycomb components, show a higher degree of local intrachromosomal contacts and are highly sensitive to DNA hypomethylation in terms of H3K27me3 depletion from broad Polycomb domains. In contrast, loBiv promoters are enriched in non-canonical Polycomb components, show lower intrachromosomal contacts and are less sensitive to DNA hypomethylation at the same genomic resolution. Multiple systems reveal that hiBiv promoters are more depleted of Polycomb complexes than loBiv promoters following a reduction in DNA methylation, and we demonstrate that H3K27me3 re-accumulates at promoters when DNA methylation is restored. In human cancer, we show that hiBiv promoters lose H3K27me3 and are more susceptible to DNA hypermethylation than loBiv promoters., Conclusion: We conclude that bivalency as a general term to describe mammalian promoters is an over-simplification and our sub-classification has revealed novel insights into the interplay between the largely antagonistic presence of DNA methylation and Polycomb systems at bivalent promoters. This approach redefines molecular pathologies underlying disease in which global DNA methylation is aberrant or where Polycomb mutations are present.

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Topologically associating domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD - creating internal deletions, deleting CTCF sites, and deleting and inverting sequences at TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used to investigate the changes in chromatin conformation that result from these manipulations. Our data suggest that these substantial alterations in TAD structure have no readily detectable effect on Shh expression patterns or levels of Shh expression during development - except where enhancers are deleted - and result in no detectable phenotypes. Only in the case of a larger deletion at one TAD boundary could ectopic influence of the Shh limb enhancer be detected on a gene ( Mnx1 ) in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Over the past few years, interest in chromatin and its evolution has grown. To further advance these interests, we organized a workshop with the support of The Company of Biologists to debate the current state of knowledge regarding the origin and evolution of chromatin. This workshop led to prospective views on the development of a new field of research that we term 'EvoChromo'. In this short Spotlight article, we define the breadth and expected impact of this new area of scientific inquiry on our understanding of both chromatin and evolution., (© 2019. Published by The Company of Biologists Ltd.)

The human genome is not randomly organised, with respect to both the linear organisation of the DNA sequence along chromosomes and to the spatial organisation of chromosomes in the cell nucleus. Here I discuss how these patterns of sequence organisation were first discovered by molecular biologists and how they relate to the patterns revealed decades earlier by cytogeneticists and manifest as chromosome bands.

During oncogene-induced senescence (OIS), heterochromatin is lost from the nuclear periphery and forms internal senescence-associated heterochromatin foci (SAHFs). We show that an increased nuclear pore density during OIS is responsible for SAHF formation. In particular, the nucleoporin TPR is necessary for both formation and maintenance of SAHFs. Loss of SAHFs does not affect cell cycle arrest but abrogates the senescence-associated secretory phenotype-a program of inflammatory cytokine gene activation. Our results uncover a previously unknown role of nuclear pores in heterochromatin reorganization in mammalian nuclei and demonstrate the importance of heterochromatin organization for a specific gene activation program., (© 2019 Boumendil et al.; Published by Cold Spring Harbor Laboratory Press.)

Due to recent advances in experimental and theoretical approaches, the dynamic three-dimensional organization (3D) of the nucleus has become a very active area of research in life sciences. We now understand that the linear genome is folded in ways that may modulate how genes are expressed during the basic functioning of cells. Importantly, it is now possible to build 3D models of how the genome folds within the nucleus and changes over time (4D). Because genome folding influences its function, this opens exciting new possibilities to broaden our understanding of the mechanisms that determine cell fate. However, the rapid evolution of methods and the increasing complexity of data can result in ambiguity and reproducibility challenges, which may hamper the progress of this field. Here, we describe such challenges ahead and provide guidelines to think about strategies for shared standardized validation of experimental 4D nucleome data sets and models.

Polycomb repressive complexes (PRCs) 1 and 2 are essential chromatin regulators of cell identity. PRC1, a dominant executer of Polycomb-mediated control, functions as multiple sub-complexes that possess catalytic-dependent H2AK119 mono-ubiquitination (H2AK119ub) and catalytic-independent activities. Here, we show that, despite its well-established repressor functions, PRC1 binds to both silent and active genes. Through in vivo loss-of-function studies, we show that global PRC1 function is essential for skin development and stem cell (SC) specification, whereas PRC1 catalytic activity is dispensable. Further dissection demonstrated that both canonical and non-canonical PRC1 complexes bind to repressed genes, marked by H2AK119ub and PRC2-mediated H3K27me3. Interestingly, loss of canonical PRC1, PRC1 catalytic activity, or PRC2 leads to expansion of mechanosensitive Merkel cells in neonatal skin. Non-canonical PRC1 complexes, however, also bind to and promote expression of genes critical for skin development and SC formation. Together, our findings highlight PRC1's diverse roles in executing a precise developmental program., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the version of this article initially published, Wendy Bickmore and Madapura Pradeepa were incorrectly not indicated as corresponding authors. The error has been corrected in the HTML and PDF versions of the paper.

We found that the clinical phenotype associated with BRD4 haploinsufficiency overlapped with that of Cornelia de Lange syndrome (CdLS), which is most often caused by mutation of NIPBL. More typical CdLS was observed with a de novo BRD4 missense variant, which retained the ability to coimmunoprecipitate with NIPBL, but bound poorly to acetylated histones. BRD4 and NIPBL displayed correlated binding at super-enhancers and appeared to co-regulate developmental gene expression.

Background: Enhancers are modular regulatory elements that are central to the spatial and temporal regulation of gene expression. Bidirectional transcription initiating at enhancers has been proposed to mark active enhancers and as such has been utilized to experimentally identify active enhancers de novo., Results: Here, we show that bidirectional transcription initiation is a pervasive feature of accessible chromatin, including at enhancers, promoters, and other DNase hypersensitive regions not marked with canonical histone modification profiles. Transcription is less predictive for enhancer activity than epigenetic modifications such as H3K4me1 or the accessibility of DNA when measured both in enhancer assays and at endogenous loci. The stability of enhancer initiated transcripts does not influence measures of enhancer activity and we cannot detect evidence of purifying selection on the resulting enhancer RNAs within the human population., Conclusions: Our results indicate that bidirectional transcription initiation from accessible chromatin is not sufficient for, nor specific to, enhancer activity. Transcription initiating at enhancers may be a frequent by-product of promiscuous RNA polymerase initiation at accessible chromatin and is unlikely to generally play a functional role in enhancer activity.

Fertilization triggers assembly of higher-order chromatin structure from a condensed maternal and a naïve paternal genome to generate a totipotent embryo. Chromatin loops and domains have been detected in mouse zygotes by single-nucleus Hi-C (snHi-C), but not bulk Hi-C. It is therefore unclear when and how embryonic chromatin conformations are assembled. Here, we investigated whether a mechanism of cohesin-dependent loop extrusion generates higher-order chromatin structures within the one-cell embryo. Using snHi-C of mouse knockout embryos, we demonstrate that the zygotic genome folds into loops and domains that critically depend on Scc1-cohesin and that are regulated in size and linear density by Wapl. Remarkably, we discovered distinct effects on maternal and paternal chromatin loop sizes, likely reflecting differences in loop extrusion dynamics and epigenetic reprogramming. Dynamic polymer models of chromosomes reproduce changes in snHi-C, suggesting a mechanism where cohesin locally compacts chromatin by active loop extrusion, whose processivity is controlled by Wapl. Our simulations and experimental data provide evidence that cohesin-dependent loop extrusion organizes mammalian genomes over multiple scales from the one-cell embryo onward., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Glucocorticoids act by binding to the glucocorticoid receptor (GR), which binds to specific motifs within enhancers of target genes to activate transcription. Previous studies have suggested that GRs can promote interactions between gene promoters and distal elements within target loci. In contrast, we demonstrate here that glucocorticoid addition to mouse bone-marrow-derived macrophages produces very rapid chromatin unfolding detectable by fluorescence in situ hybridization (FISH) at loci associated with GR binding. Rapid chromatin decompaction was generally not dependent on transcription at those loci that are known to be inducible in both mouse and human macrophages and was sustained for up to 5 days following ligand removal. Chromatin decompaction was not dependent upon persistent GR binding, which decayed fully after 24 hr. We suggest that sustained large-scale chromatin reorganization forms an important part of the response to glucocorticoid and might contribute to glucocorticoid sensitivity and resistance., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the premature ageing disease Hutchinson-Gilford progeria syndrome (HGPS), the underlying genetic defect in the lamin A gene leads to accumulation at the nuclear lamina of progerin-a mutant form of lamin A that cannot be correctly processed. This has been reported to result in defects in the DNA damage response and in DNA repair, leading to the hypothesis that, as in normal ageing and in other progeroid syndromes caused by mutation of genes of the DNA repair and DNA damage response pathways, increased DNA damage may be responsible for the premature ageing phenotypes in HGPS patients. However, this hypothesis is based upon the study of markers of the DNA damage response, rather than measurement of DNA damage per se or the consequences of unrepaired DNA damage-mutation. Here, using a mutation reporter cell line, we directly compared the inherent and induced mutation rates in cells expressing wild-type lamin A or progerin. We find no evidence for an elevated mutation rate in progerin-expressing cells. We conclude that the cellular defect in HGPS cells does not lie in the repair of DNA damage per se.

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator-PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip-located at the 5' end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5' Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5' end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5' Hoxa genes and that Hottip RNA binds to the 5' end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis.

Repo-Man is a protein phosphatase 1 (PP1) targeting subunit that regulates mitotic progression and chromatin remodelling. After mitosis, Repo-Man/PP1 remains associated with chromatin but its function in interphase is not known. Here we show that Repo-Man, via Nup153, is enriched on condensed chromatin at the nuclear periphery and at the edge of the nucleopore basket. Repo-Man/PP1 regulates the formation of heterochromatin, dephosphorylates H3S28 and it is necessary and sufficient for heterochromatin protein 1 binding and H3K27me3 recruitment. Using a novel proteogenomic approach, we show that Repo-Man is enriched at subtelomeric regions together with H2AZ and H3.3 and that depletion of Repo-Man alters the peripheral localization of a subset of these regions and alleviates repression of some polycomb telomeric genes. This study shows a role for a mitotic phosphatase in the regulation of the epigenetic landscape and gene expression in interphase.

The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh-Shh-brain-enhancer-6 (SBE6)-that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development., (© 2016 The Authors.)

Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene's developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision., Competing Interests: The authors declare that no competing interests exist.

Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2 nes ) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4 + CD8 + T-cell stage from which tumors initiate. Premalignant CD4 + CD8 + T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis., (© 2016 Woodward et al.; Published by Cold Spring Harbor Laboratory Press.)