Biology subjects

Rugg-Gunn, P.

Publications and source records attributed to Rugg-Gunn, P..

11 recordsLinked to original sources

bioRxiv2025

XIST Drives X-Chromosome Inactivation and Safeguards Female Extraembryonic Cells in Humans

Dosage compensation of sex chromosomes through X-chromosome inactivation (XCI) is required for mice extra-embryonic tissue growth and embryo development. The species specificity in mechanisms and timing leading to XCI during early embryogenesis, however, left the key question of the interdependence between XCI and human development open. Here, we show that the differentiation of naive human pluripotent stem cells to trophoblast stem cells and extraembryonic mesoderm cells triggers XCI. The inactive X chromosome, however, displays an atypical chromatin state, lacking classical enrichment of heterochromatin markers and DNA methylation. We demonstrate that extraembryonic differentiation and XCI are kinetically and functionally linked. Using loss of function approaches, we prove that XIST is required for human XCI establishment. We also reveal that XCI is key for the survival of human female extraembryonic cells. Our work therefore links XCI to the formation of extraembryonic annexes, with important consequences for human reproductive biology. HIGHLIGHTSO_LINaive hPSCs to EXMCs and TSCs differentiation recapitulates human XCI C_LIO_LIThe Xi has an unusual chromatin status in human extraembryonic cells C_LIO_LIXIST is required for the establishment of human XCI C_LIO_LIXCI supports healthy development of human female extraembryonic cells C_LI

developmental biology↗

bioRxiv2025

Poising and connectivity of emergent human developmental enhancers in the transition from naive to primed pluripotency

In primed human pluripotent stem cells (hPSCs) resembling post-implantation epiblast, numerous lineage-specific enhancers assume the poised chromatin state, co-marked by H3K4me1 and Polycomb-associated H3K27me3 histone modifications. In contrast, poised enhancers (PEs) are scarce in naive hPSCs that model pre-implantation epiblast. PEs form abundant chromosomal contacts with developmental genes, but when these contacts emerge, how their formation relates to enhancer poising and their functional significance remains incompletely understood. Here, we devise high-resolution, PE-targeted Capture Hi-C to generate a comprehensive atlas of PE chromosomal contacts in the time course of hPSC transition from the naive to primed state. We find that enhancer poising emerges early in the transition, while the contacts show diverse dynamics that is only partially coupled to poising. PROTAC-induced degradation of Polycomb Repressive Complex 2 (PRC2) early in the transition weakens PE connectivity, while inhibition of its H3K27 methyltransferase activity does not, suggesting a non-catalytic role of Polycomb in supporting PE contacts. Notably, PE contacts persist after developmental activation or ectopic CRISPRa targeting and can mediate long-range gene induction. Together, these findings reveal the temporal and mechanistic principles of PE connectivity, highlighting a potential role of PE contacts in establishing gene expression patterns in human development.

genomics↗

bioRxiv2025

TrAEL-seq captures DNA replication dynamics in mammalian cells

Precise DNA replication is critical to the maintenance of genome stability, and the DNA replication machinery is a focal point of many current and upcoming chemotherapeutics. TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronised cells and does not require treatment with drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq including multiplexing of up to 6 samples that dramatically improves sample quality and throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication Initiation Zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, which is dominated by rapid fork movement in long replication regions (>1Mb). Short and long replication regions are not intrinsically different, and instead replication forks accelerate across the first [~]1 Mb of travel such that forks progress faster in the middle of regions lying between widely spaced Initiation Zones. We propose that this is a natural consequence of fewer replication forks being active later in S phase when these distal regions are replicated and there being less competition for replication factors.

molecular biology↗

bioRxiv2023

Dbx2, an aging-related homeobox gene, inhibits the proliferation of adult neural progenitors

The subventricular zone (SVZ) of the adult mouse brain contains quiescent neural stem cells, which can be activated (aNSCs) to generate transit amplifying progenitors (TAPs), neuroblasts (NBs) and newborn neurons. Neurogenesis declines during aging, as the aged SVZ niche causes transcriptomic changes that promote NSC quiescence and decrease proliferating neural/stem progenitor cells (NSPCs). The transcription factors mediating these changes, however, remain unclear. We previously found that the homeobox gene Dbx2 is upregulated in aged SVZ NSPCs and inhibits NSPC culture growth. Here, we report that Dbx2 is repressed by Epidermal Growth Factor Receptor signaling, which promotes NSPC proliferation and decreases in the aged SVZ. We show that Dbx2 inhibits NSPC proliferation by hindering the G2/M transition and elucidate the transcriptomic networks modulated by Dbx2, highlighting its role in the downregulation of the cell cycle molecular pathways. Accordingly, Dbx2 function is negatively correlated with the transcriptional signatures of proliferative NSPCs (aNSCs, TAPs and early NBs). These results point to Dbx2 as a molecular node relaying the anti-neurogenic input of the aged niche to the NSPC transcriptome.

developmental biology↗

bioRxiv2022

NANOGP1, a tandem duplicate of NANOG, exhibits partial functional conservation in human naive pluripotent stem cells

Gene duplication events are important drivers of evolution by providing genetic material for new gene functions. They also create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the key transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naive-state pluripotent stem cells. Sequence analysis and epitope-tagging of the endogenous locus revealed that NANOGP1 is protein-coding with an intact homeobox domain. NANOGP1 has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways including point mutations in the homeodomain. NANOGP1 is a strong inducer of naive pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naive pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development. Summary statementEstablishing that NANOGP1 has retained partial functional conservation with its ancestral copy NANOG sheds light on the role of gene duplication and subfunctionalisation in human pluripotency and development.

developmental biology↗

bioRxiv2020

Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single stranded DNA 3 ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3 ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3 ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1{Delta} mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

molecular biology↗

bioRxiv2020

Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1. Mammalian heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1 droplets in vitro, and scaffold heterochromatin into dynamic condensates in ESCs, leading to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatins biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide new insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates.

molecular biology↗

bioRxiv2020

Illuminating chromatin compaction in live cells and fixed tissues using SiR-DNA fluorescence lifetime

The global compaction state of chromatin in a nucleus is an important component of cell identity that has been difficult to measure. We have developed a quantitative method to measure the chromatin compaction state in both live and fixed cells, without the need for genetic modification, using the fluorescence lifetime of SiR-DNA dye. After optimising this method using live cancer cell lines treated to induce chromatin compaction or decompaction, we observed chromatin compaction in differentiating epithelial cells in fixed tissue sections, as well as local decompaction foci that may represent transcription factories. In addition, we shed new light on chromatin decompaction during embryonic stem cell transition out of their naive pluripotent state. This method will be useful to studies of nuclear architecture, and may be easy, cheap, and accessible enough to serve as a general assay of stem-ness.

cell biology↗

bioRxiv2019

Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

SO_SCPCAPUMMARYC_SCPCAPA complex and poorly understood interplay between 3D genome organisation, transcription factors and chromatin state underpins cell identity. To gain a systems-level understanding of this interplay, we generated a high-resolution atlas of annotated chromatin interactions in naive and primed human pluripotent stem cells and developed a network-graph approach to examine the atlas at multiple spatial scales. Investigating chromatin interactions as a network uncovered highly connected hubs that changed substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merged to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. Importantly, we identified state-specific differences in enhancer activity and interactivity that corresponded with widespread reconfiguration of transcription factor binding and target gene expression. These findings provide multilayered insights into the gene regulatory control of human pluripotency and our systems-based network approach could be applied broadly to uncover new principles of 3D genome organisation.

genomics↗

bioRxiv2019

Single cell multi-omics profiling reveals a hierarchical epigenetic landscape during mammalian germ layer specification

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNA-sequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers.\n\nHighlightsO_LIFirst map of mouse gastrulation using comprehensive single cell triple-omic analysis.\nC_LIO_LIExit from pluripotency is associated with a global repressive epigenetic landscape, driven by a sharp gain of DNA methylation and a gradual decrease of chromatin accessibility.\nC_LIO_LIDNA methylation and chromatin accessibility changes in enhancers, but not in promoters, are associated with germ layer formation.\nC_LIO_LIMesoderm and endoderm enhancers become open and demethylated upon lineage commitment.\nC_LIO_LIEctoderm enhancers are primed in the early epiblast and protected from the global repressive dynamics, supporting a default model of ectoderm commitment in vivo.\nC_LI

developmental biology↗

bioRxiv2018

Genome-scale oscillations in DNA methylation during exit from pluripotency

Pluripotency is accompanied by the erasure of parental epigenetic memory with naive pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, coexpression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modelling, we show that this variability is associated with coherent, genome-scale, oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide fresh insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.\n\nHighlightsO_LICo-expression of DNMT3s and TETs drive genome-scale oscillations of DNA methylation\nC_LIO_LIOscillation amplitude is greatest at a CpG density characteristic of enhancers\nC_LIO_LICell synchronisation reveals oscillation period and link with primary transcripts\nC_LIO_LIMultiomic single-cell profiling provides evidence for oscillatory dynamics in vivo\nC_LI

genomics↗