Biology subjects

Ponting, C. P.

Publications and source records attributed to Ponting, C. P..

15 recordsLinked to original sources

bioRxiv2025

Systematic morphological profiling of human and mouse miRNAs in 24M single cells

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression whose contributions to cell biology remain underexplored. Here, we used Cell Painting to quantify the morphological effect of 2,565 human and 1,900 mouse miRNA mimics on 24 million cells across five cell lines. To do so, we developed a novel single-cell morphological profiling analysis framework, involving stringent batch correction, feature selection, and hit calling. With this, we discovered that 9% of human and 15% of mouse miRNA mimics significantly alter cell morphology in at least one cell line. Eighteen miRNAs caused significant changes in multiple cell lines, including eight orthologous miRNAs that altered morphology in both human and mouse cells. Among the replicating miRNAs were human and mouse miR-155-5p, which affected morphology in at least one replicate of four cell lines. As expected, miRNAs with identical seed sequences induced more similar morphological changes than miRNAs with different seeds. Also, morphological changes were associated with cytotoxicity and annotation confidence. This comprehensive single-cell morphological resource will help elucidate the cellular functions of human and mouse miRNAs.

genomics↗

bioRxiv2025

Indistinguishable mitochondrial phenotypes after exposure of healthy myoblasts to myalgic encephalomyelitis or control serum

Myalgic Encephalomyelitis (ME) / Chronic Fatigue Syndrome is a disease of uncertain aetiology that affects up to 400,000 individuals in the UK. Exposure of cultured cells to the sera of people with ME has been proposed to cause phenotypic changes in these cells in vitro when compared to sera from healthy controls. ME serum factors causing these changes could inform the development of diagnostic tests. In this study, we performed a large-scale, pre-registered replication of an experiment from Fluge et al (2016) that reported an increase in maximal respiratory capacity in healthy myoblasts after treatment with serum from people with ME compared to serum from healthy controls. We replicated the original experiment with a larger sample size, using sera from 67 people with ME and 53 controls to treat healthy cultured myoblasts, and generated results from over 1,700 mitochondrial stress tests performed with a Seahorse Bioanalyser. We observed no significant differences between treatment with ME or healthy control sera for our primary outcome of interest, oxygen consumption rate at maximal respiratory capacity. Results from our study provide strong evidence against the hypothesis that ME blood factors differentially affect healthy myoblast mitochondrial phenotypes in vitro.

biochemistry↗

bioRxiv2025

baal-nf identifies motif-disrupting variants that decrease transcription factor binding affinity

Human traits vary in part due to genetically-determined change of transcription factor (TF) binding affinity within gene regulatory regions. However, few trait-causal variants or mechanisms are known. Here we propose 1,960 variants as strong candidates for causally altering human traits. They were discovered by baal-nf which uses ChIP-sequencing data to identify allelic imbalance at heterozygous sites ( allele-specific binding sites; ASBs) within affinity-concordant positions within TF- and/or co-factor binding motifs. These variants are evolutionarily conserved, and enriched for trait associations and gene expression QTLs. baal-nf and these high-quality ASBs now allow trait variation due to altered TF binding to be investigated.

genomics↗

bioRxiv2023

High order expression dependencies finely resolve cryptic states and subtypes in single cell data

AO_SCPLOWBSTRACTC_SCPLOWSingle cells are typically typed by clustering in reduced dimensional transcriptome space. Here we introduce Stator, a novel method, workflow and app that reveals cell types, subtypes and states without relying on local proximity of cells in gene expression space. Rather, Stator derives higher-order gene expression dependencies from a sparse gene-by-cell expression matrix. From these dependencies the method multiply labels the same single cell according to type, sub-type and state (activation, differentiation or cell cycle sub-phase). By applying the method to data from mouse embryonic brain, and human healthy or diseased liver, we show how Stator first recapitulates other methods cell type labels, and then reveals combinatorial gene expression markers of cell type, state, and disease at higher resolution. By allowing multiple state labels for single cells we reveal cell type fates of embryonic progenitor cells and liver cancer states associated with patient survival.

molecular biology↗

bioRxiv2023

The histone chaperone activity of SPT2 controls chromatin structure and function in Metazoa

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans orthologue of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at these loci. We also show that human SPT2 controls the incorporation of new H3.3 into chromatin. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

molecular biology↗

bioRxiv2022

Dispensing with unnecessary assumptions in population genetics analysis

Parametric assumptions in population genetics analysis - including linearity, sources of population stratification and additivity of variance as part of a Gaussian noise - are often made, yet their (approximate) validity depends on variant and traits of interest, as well as genetic ancestry and population dependence structure of the sample cohort. We present a unified statistical workflow, called TarGene, for targeted estimation of effect sizes, as well as two-point and higher-order epistatic interactions of genomic variants on polygenic traits, which dispenses with these unnecessary assumptions. Our approach is founded on Targeted Learning, a framework for estimation that integrates mathematical statistics, machine learning and causal inference. TarGene maximises power whilst simultaneously maximising control over false discoveries by: (i) guaranteeing optimal bias-variance trade-off, (ii) taking into account potential covariate non-linearities, sources of population stratification and dependence structure, and (iii) detecting genetic non-linearities. The necessity of this model-independent approach is demonstrated via extensive simulations. We validate the effectiveness of our method by reproducing previously verified effect sizes on UK Biobank data, whilst simultaneously discovering non-linear effect sizes of additional allelic copies on trait or disease, in a PheWAS study involving 781 traits. Specifically, we demonstrate genetic non-linearity at the FTO locus is significant for 54 traits in this study. We further find three pairs of epistatic loci associated with skin color that have been previously reported to be associated with hair color. Finally, we illustrate how TarGene can be used to investigate higher-order interactions using three variants linked to the vitamin D receptor complex. TarGene provides a platform for comparative analyses across biobanks, or integration of multiple biobanks and heterogeneous populations to simultaneously increase power and control for type I errors, whilst taking into account population stratification and complex dependence structures.

genetics↗

bioRxiv2022

Ependymal cell maturation is heterogeneous and ongoing in the mouse spinal cord and dynamically regulated in response to injury

The spinal cord neural stem cell potential resides within the ependymal cells lining the central canal. These cells are, however, heterogeneous, and we know little about the biological diversity this represents. Here we use single-cell RNA-sequencing to profile adult mouse spinal cord ependymal cells. We uncover transcriptomes of known subtypes and a new mature ependymal cell state, that becomes more prominent with age. Comparison of ependymal cell transcriptomes from the brain and spinal cord revealed that ongoing cell maturation distinguishes spinal cord ependymal cells from their postmitotic brain counterparts. Using an ex vivo model of spinal cord injury, we show that ependymal cell maturation is reversible but also highly regulated. We revisit ependymal cell identities in adult human spinal cord and uncover evidence for their maturation and surprising ventralisation with age. This first in-depth characterisation of spinal cord ependymal cells paves the way to manipulation of distinct ependymal subtypes, provides insights into ependymal cell maturation dynamics and informs strategies for coaxing ependymal cell-driven spinal cord repair.

developmental biology↗

bioRxiv2022

Genetic control of KRAB-ZFP genes explains distal CpG-site methylation which associates with human disease phenotypes.

Kruppel-associated box zinc finger proteins (KRAB-ZFPs) are the largest gene family of transcriptional regulators in higher vertebrates. We have developed a method for inferring these factors DNA binding-sites in vivo. We achieved this by combining the genome-wide association of methylation at 573,027 CpG-sites (meQTL) in blood and KRAB-ZFP RNA expression changes (eQTL). This methods efficacy is clearly demonstrated by showing that the CpG-sites whose methylation is affected by KRAB-ZFP expression changes occur preferentially near KRAB-ZFP binding-sites in the HEK293T cell line. We found such an enrichment to be significant for 31% of factors tested. In addition, we found that binding-sites of many KRAB-ZFPs are significantly enriched (or sometimes depleted) in disease-associated CpG-sites. In total, 9% (11 of 125) of the traits tested, and 14% (32 of 222) of the factors (KRAB-ZFPs and TIF1-beta) tested, were enriched or depleted in one or more trait-factor pairing. Rheumatoid arthritis and human immunodeficiency virus (HIV) infection were associated with the largest number of KRAB-ZFP enrichments. There have been variable reports on the effects on HIV infection dynamics of transcription intermediary factor 1-beta (TIF1-beta, also known as KAP1 or TRIM28), a nuclear corepressor for KRAB-ZFPs. We provide evidence that KRAB-ZFP-independent effects of TIF1-beta are responsible for the decreased methylation of CpG-sites within its binding-sites that is observed in HIV infection. In conclusion, KRAB-ZFPs often affect CpG-site methylation within the proximity of their binding-sites, and CpG-sites within the binding-sites of KRAB-ZFPs are enriched for association with many traits, including rheumatoid arthritis and HIV infection.

genomics↗

bioRxiv2021

Refining the domain architecture model of the replication origin firing factor Treslin/TICRR

Faithful genome duplication requires appropriately controlled replication origin firing. The metazoan Treslin/TICRR origin firing factor and its yeast orthologue Sld3 are regulation hubs of origin firing. They share the Sld3-Treslin domain (STD) and the adjacent TopBP1/Dpb11 interaction domain (TDIN). We report a revised domain architecture model of Treslin/TICRR. Complementary protein sequence analyses uncovered Ku70-homologous {beta}-barrel folds in the Treslin/TICRR middle domain (M domain) and in Sld3. Thus, the Sld3-homologous Treslin/TICRR core comprises its three central domains, M domain, STD and TDIN. This Sld3-core is flanked by non-conserved terminal domains, the CIT (conserved in Treslins) and the C-terminus. We also identified Ku70-like {beta}-barrels in MTBP and Sld7. Our binding experiments showed that the Treslin {beta}-barrel mediates interaction with the MTBP {beta}-barrel, reminiscent of the homotypic Ku70-Ku80 dimerization. This binding mode is conserved in the Sld3-Sld7 dimer. We used Treslin/TICRR domain mutants to show that all Sld3-core domains and the non-conserved terminal domains fulfil important functions during origin firing in human cells. Thus, metazoa-specific and widely conserved molecular processes cooperate during origin firing in metazoa.

molecular biology↗

bioRxiv2020

Ageing compromises mouse thymus function and remodels epithelial cell differentiation

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

immunology↗

bioRxiv2019

Resolving the fibrotic niche of human liver cirrhosis using single-cell transcriptomics

Currently there are no effective antifibrotic therapies for liver cirrhosis, a major killer worldwide. To obtain a cellular resolution of directly-relevant pathogenesis and to inform therapeutic design, we profile the transcriptomes of over 100,000 primary human single cells, yielding molecular definitions for the major non-parenchymal cell types present in healthy and cirrhotic human liver. We uncover a novel scar-associated TREM2+CD9+ macrophage subpopulation with a fibrogenic phenotype, that has a distinct differentiation trajectory from circulating monocytes. In the endothelial compartment, we show that newly-defined ACKR1+ and PLVAP+ endothelial cells expand in cirrhosis and are topographically located in the fibrotic septae. Multi-lineage ligand-receptor modelling of specific interactions between the novel scar-associated macrophages, endothelial cells and collagen-producing myofibroblasts in the fibrotic niche, reveals intra-scar activity of several major pathways which promote hepatic fibrosis. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides the conceptual framework required to discover rational therapeutic targets in liver cirrhosis.

immunology↗

bioRxiv2019

Proteome-by-phenome Mendelian Randomisation detects 38 proteins with causal roles in human diseases and traits

Target identification remains a crucial challenge in drug development. To enable unbiased detection of proteins and pathways that have a causal role in disease pathogenesis or progression, we propose proteome-by-phenome Mendelian Randomisation (P2MR). We first detected genetic variants associated with plasma concentration of 249 proteins. We then used 64 replicated variants in two-sample Mendelian Randomisation to quantify evidence of a causal role for each protein across 846 phenotypes: this yielded 509 robust protein-outcome links. P2MR provides substantial promise for drug target prioritisation. We provide confirmatory evidence for a causal role for the proteins encoded at multiple cardiovascular disease risk loci (FGF5, IL6R, LPL, LTA), and discovered that intestinal fatty acid binding protein (FABP2) contributes to disease pathogenesis. Additionally, we find and replicate evidence for a causal role of tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS1; SIRPA) in schizophrenia. Our results provide specific prediction of the effects of changes of plasma protein concentration on complex phenotypes in humans.

genomics↗

bioRxiv2018

Identification of functional long non-coding RNAs in C. elegans

BackgroundFunctional characterisation of the compact genome of the model organism Caenorhabditis elegans remains incomplete despite its sequencing twenty years ago. The last decade of research has seen a tremendous increase in the number of non-coding RNAs identified in various organisms. While we have mechanistic understandings of small non-coding RNA pathways, long non-coding RNAs represent a diverse class of active transcripts whose function remains less well characterised.\n\nResultsBy analysing hundreds of published transcriptome datasets, we annotated 3,397 potential lncRNAs including 146 multi-exonic loci that showed increased nucleotide conservation and GC content relative to other non-coding regions. Using CRISPR / Cas9 genome editing we generated deletion mutants for ten long non-coding RNA loci. Using automated microscopy for in-depth phenotyping, we show that six of the long non-coding RNA loci are required for normal development and fertility. Using RNA interference mediated gene knock-down, we provide evidence that for two of the long non-coding RNA loci, the observed phenotypes are dependent on the corresponding RNA transcripts.\n\nConclusionsOur results highlight that a large section of the non-coding regions of the C. elegans genome remain unexplored. Based on our in vivo analysis of a selection of high-confidence lncRNA loci, we expect that a significant proportion of these high-confidence regions is likely to have biological function at either the genomic or the transcript level.

genetics↗

bioRxiv2017

Paupar LncRNA Promotes KAP1 Dependent Chromatin Changes And Regulates Subventricular Zone Neurogenesis

Many long non-coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and molecular mechanisms of action remain poorly understood. Paupar, a CNS expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of genome-wide transcriptional regulatory elements. We show here that Paupar transcript directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through formation of a DNA binding ribonucleoprotein complex containing Paupar, KAP1 and the PAX6 transcription factor. Paupar-KAP1 genome-wide co-occupancy reveals a 4-fold enrichment of overlap between Paupar and KAP1 bound sequences. Furthermore, both Paupar and Kap1 loss of function in vivo accelerates lineage progression in the mouse postnatal subventricular zone (SVZ) stem cell niche and disrupts olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans-acting modes of lncRNA-mediated epigenetic regulation, the mechanisms of KAP1 genomic recruitment and identify Paupar and Kap1 as regulators of SVZ neurogenesis.

neuroscience↗

bioRxiv2017

The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

cell biology↗