Biology subjects

Grimes, G.

Publications and source records attributed to Grimes, G..

11 recordsLinked to original sources

bioRxiv2026

Array-CNCC: precise aggregation and arrayed plating facilitate quantitative phenotyping of human cranial neural crest cells and craniofacial disease modelling

Facial development is highly sensitive to genetic and environmental perturbation, with craniofacial malformation associated with over one-third of congenital birth defects. The face arises during an early and largely inaccessible window of embryonic development, with a large contribution from transient and multipotent cranial neural crest cells (CNCCs). Assessment of the molecular and cellular mechanisms driving normal and disordered human facial development therefore relies greatly on the use of in vitro cellular models. Here, we adapted a neurosphere-based CNCC differentiation protocol to facilitate robust quantification of early specification and migration events. Introduction of single-cell aggregation with arrayed plating enabled standardisation of neurosphere size, growth and patterning. Inclusion of fibronectin coating enhanced the efficiency of neurosphere attachment and synchronicity of CNCC migration timing. To demonstrate application of the Array-CNCC method, we developed a strategy for mosaic co-culture, which can facilitate differentiation of wildtype untreated cells directly alongside cells exposed to distinct drug treatments or genetic alterations. Finally, we present a screening approach which we use to test the impact of distinct extracellular matrix components on neurosphere morphology, CNCC migration and gene expression. Together, the Array-CNCC method is highly amenable to quantitative phenotyping and screening approaches, enabling enhanced craniofacial disease modelling with both cellular and molecular readouts.

developmental biology↗

bioRxiv2025

ATRX Deficiency Drives Aberrant Type I Interferon Signalling Through cGAS-Dependent Transcriptional Dysregulation

The X-linked -thalassaemia intellectual disability syndrome (ATRX) protein is a chromatin remodeller involved in transcriptional regulation and genome stability. While the importance of ATRX in development and malignancy is well recognised, its role in innate immunity is less well defined. In two unrelated patients demonstrating cerebral white matter disease, learning difficulties and a persistent upregulation of interferon stimulated gene expression in whole blood, we identified the same Y1758C missense substitution in ATRX. Using patient-derived cells, engineered fibroblasts and neuronal models, we show that this substitution, and other loss of function mutations in ATRX, result in enhanced type I interferon signalling through a cGAS-dependent mechanism uncoupled from the DNA sensing activity of cGAS. Loss of ATRX function leads to alterations in the chromatin distribution of DAXX and H3.3, with cGAS essential for the changes in nucleosome composition and gene expression mediated by ATRX deficiency. Thus, our study highlights a previously unrecognized link between ATRX dysfunction and inflammation involving a non-canonical role of cGAS.

immunology↗

bioRxiv2023

Mutational scanning reveals oncogenic CTNNB1 mutations have diverse effects on signalling and clinical traits

CTNNB1, the gene encoding {beta}-catenin, is a frequent target for oncogenic mutations activating the canonical Wnt signalling pathway, typically via missense mutations within a degron hotspot motif in exon 3. Here, we combine saturation genome editing with a fluorescent reporter assay to quantify signalling phenotypes for all 342 missense mutations in the mutation hotspot, including 74 recurrent mutations reported in over 6000 tumours. Our data define the genetic requirements for {beta}-catenin degron function and reveal diverse levels of signal activation among known driver mutations. Tumorigenesis in different human tissues involves selection for CTNNB1 mutations spanning distinct ranges of effect size. In hepatocellular carcinoma, mutations that activate {beta}-catenin relatively weakly are associated with worse prognosis compared to stronger activating mutations, despite greater immune cell infiltration in the tumour microenvironment. Our work therefore provides a resource to understand mutational diversity within a pan-cancer mutation hotspot, with potential implications for targeted therapy.

cancer biology↗

bioRxiv2023

Genetic variation at 11q23.1 confers colorectal cancer risk by dysregulation of colonic tuft cell transcriptional activator POU2AF2

Common genetic variation at 11q23.1 is associated with colorectal cancer (CRC) risk, and exerts local (cis) expression quantitative trait locus (cis-eQTL) effects on POU2AF2, COLCA1 and POU2AF3 genes. However, complex linkage disequilibrium and correlated expression at the 11q23.1 locus has thus far hindered elucidation of the mechanisms by which genetic variants impart CRC risk. Here, we establish that rs3087967 is the likely causal eQTL at this locus, co-localising with expression of POU2AF2 and CRC risk. Furthermore, we show trans-eQTL effects on 21 distant target genes, which are highly enriched for Tuft cell markers. Analysis of available scRNAseq, ChIPseq and scATACseq data implicates POU2AF2 as the primary controller of the tuft cell specific trans-genes through POU2F3-correlated genetic regulation. Immunofluorescence demonstrates that the rs3087967 risk genotype (T) is associated with lower tuft cell abundance in human colonic epithelium. CRISPR-mediated deletion of the 11q23.1 risk locus in the mouse germline exacerbated the ApcMin/+ mouse phenotype upon abrogation of Pou2af2 expression specifically. Taken together, we implicate a key protective role of tuft cells in the large bowel and the importance of mis-regulation of POU2AF2 as the prime tuft cell transcriptional activator at this locus.

genetics↗

bioRxiv2023

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops

Polycomb-repressive complex 1 (PRC1) has a strong influence on 3D genome organization, mediating local chromatin compaction as well as localized and chromosome-wide clustering of target loci. Several subunits of PRC1 have been shown to 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-hexandiol, which disrupts 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 combined with deep sequencing analyses, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci - at megabase and tens of megabase length scales - can be reversibly disrupted by the addition and subsequent removal of 1,6-hexandiol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to the reduction of PRC1 binding following 1,6-hexandiol treatment as the addition of 2,5-hexandiol 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, characteristic of liquid-liquid phase separation, have a role in polycomb-mediated genome organization.

molecular biology↗

bioRxiv2022

Exploring the association between Body Mass Index, Sex and Gene Expression in human colorectal epithelium

IntroductionColorectal cancer (CRC) is the second most common cause of cancer death globally. Genome-wide association studies have established that cancer risk mediated through common genetic variants can be linked to variation in gene expression. Since obesity and male sex impart substantially elevated CRC risk, we studied transcriptional profiles of normal colorectal mucosa using RNA sequencing to better understand the relationship of these risk factors with gene expression levels. MethodsNormal colorectal mucosa was sampled from 365 participants (208 males, 157 females) either during surgery (n=103) or through endoscopic biopsy (n=262) from cancer patients and patients with other unrelated conditions. In total, 238 samples were used for our discovery dataset and 380 samples were obtained for the validation of our findings. The transcription analysis was done using paired-end total RNA sequencing. Data processing and gene filtering followed the Genotype-Tissue Expression (GTEx) Project pipeline v8. Differential Expression Analysis (DEA) was performed on normalised counts to evaluate effects of sex and body mass index on the total gene expression, as well as possible confounding effects of cancer presence on the gene expression in normal colorectal tissue. ResultsFollowing filtering, there were 15,465 genes available for analysis. DEA identified two genes that were significantly associated with sex and five associated with body mass index. However, whilst these nominal signals are of interest, none of the genes associated with sex remained significant in a replication dataset. Due to the missing BMI information, replication of DEA by BMI was not possible. ConclusionWe found no systematic differences in gene expression in normal colorectal epithelium between males and females, nor did we find a strong association between gene expression and BMI. Although sample size may limit our analysis, the results suggest no or limited confounding effects of BMI and sex on gene expression in normal colorectal mucosa samples.

genomics↗

bioRxiv2022

Factors influencing patterns of gene expression in large bowel mucosa

AimVariation in genomic sequences that control gene expression in target tissues is increasingly recognized as the underlying basis of disease susceptibility, especially cancer. This is particularly relevant to common genetic variation impacting on cancer risk. Exogenous factors may also influence gene expression, potentially leaving a transcriptomic signature of environmental risk factors in normal tissues. Therefore, understanding endogenous and exogenous influences over gene expression patterns in normal tissue is critical to the study of functional genomics and its relationship to disease susceptibility. Here, we investigated demographic and sampling variables that could impact on gene expression in normal colorectal mucosa. MethodWe prospectively collected normal mucosa from 424 patients undergoing colorectal surgery or outpatient assessment through surgical stripping of normal mucosa from resected colorectal specimens or rectal biopsy. Gene expression was assessed using Illumina HT-12 microarrays and analysed against demographic (age, gender, BMI) and sampling factors (general anaesthesia, cleansing bowel agents, sample site, time to RNA preservation) using adjusted linear regression modelling. ResultsAge, gender, smoking status, sampling under anaesthetic, sample site and sampling method were associated with differential gene expression in the adjusted model. BMI or use of cleansing bowel preparation did not impact gene expression. Age was associated with differential expression of 16 genes and significant enrichment in pathways relevant to tumourigenesis, including immune process, cell proliferation, adhesion and death. Sample site was associated with differential expression of 2515 genes, with 1102 genes more highly expressed in the proximal colon (proximal to splenic flexure). Gender and sampling under anaesthetic were associated with differential expression of 99 and 851 genes respectively. Increased time to RNA preservation (45-90 minutes) was associated with enrichment of pathways consistent with tissue ischaemia including response to wounding, apoptotic process, and response to oxygen levels. ConclusionsDemographic and sampling factors influence gene expression patterns in normal colorectal mucosa, often with a large magnitude of effect. Meanwhile, greater time to RNA preservation is associated with patterns of gene expression consistent with tissue ischaemia which questions the generalisability of assessment of gene expression patterns generated from post-mortem studies. These results highlight the importance of fully adjusted expression analyses and may indicate mechanisms underlying age, gender and site-specific differences in CRC incidence, progression and outcome.

genomics↗

bioRxiv2022

Robust genetic analysis of the X-linked anophthalmic (Ie) mouse

Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a [~] 6Mb region on the X chromosome. Short and long read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism.

genetics↗

bioRxiv2022

Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, as well as variable expressivity of other ciliopathy features including cystic kidneys. Pcm1-/- multiciliated ependymal cells and PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromized early ciliogenesis. We show these molecular cascades are maintained in vivo, and we suggest that the cellular threshold to trigger ciliogenesis varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, inducing the departure of CP110 and CEP97 to initiate ciliogenesis.

cell biology↗

bioRxiv2021

In vivo modelling of patient genetic heterogeneity identifies concurrent Wnt and PI3K activity as a potent driver of invasive cholangiocarcinoma growth.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive and lethal malignancy of the bile ducts within the liver characterised by high levels of genetic heterogeneity. In the context of such genetic variability, determining which oncogenic mutations drive ICC growth has been difficult and developing modes of patient stratification and targeted therapies remains challenging. As a result, survival rates following a diagnosis with ICC have remained static since the late 1970s, whilst incidence of ICC has increased. Here, we performed the first functional in vivo study into the role that genetic heterogeneity plays in drivinga ICC via modelling of interactions between rare mutations with more common driver genes. By leveraging human ICC sequencing data to stratify and then model genetic heterogeneity in the mouse, we uncovered numerous novel tumour suppressors which, when lost, cooperate with the RAS oncoprotein to drive ICC growth. In this study, we specifically focus on a set of driver mutations that interact with KRAS to initiate aggressive, sarcomatoid-type ICC. We show that tumour growth of this cancer relies on both Wnt and PI3K signalling to drive proliferation and suppress apoptosis. Finally, we demonstrate that pharmacological co-inhibition of Wnt and PI3K in vivo substantially impedes the growth of ICC, regardless of mutational profile. As such, Wnt and PI3K activity should be considered as a signature by which patients can be stratified for treatment and inhibitors of these pathways should be levied as a treatment for patients diagnosed with ICC.

cancer biology↗

bioRxiv2020

Functional Predictors of Causative Cis-Regulatory Mutations in Mendelian Disease

Undiagnosed neurodevelopmental disease is significantly associated with rare variants in cis-regulatory elements (CRE) but demonstrating causality is challenging as target gene consequences may differ from a causative variant affecting the coding region. Here, we address this challenge by applying a procedure to discriminate likely diagnostic regulatory variants from those of neutral/low-penetrant effect. We identified six rare CRE variants using targeted and whole genome sequencing in 48 unrelated males with apparent X-linked intellectual disability (XLID) but without detectable coding region variants. These variants segregated appropriately in families and altered conserved bases in predicted CRE targeting known XLID genes. Three were unique and three were rare but too common to be plausibly causative for XLID. We compared the cis-regulatory activity of wild-type and mutant alleles in zebrafish embryos using dual-color fluorescent reporters. Two variants showed striking changes: one plausibly causative (FMR1CRE) and the other likely neutral/low-penetrant (TENM1CRE). These variants were "knocked-in" to mice and both altered embryonic neural expression of their target gene. Only Fmr1CRE mice showed disease-relevant behavioral defects. FMR1CRE is plausibly disease-associated resulting in complex misregulation of Fmr1/FMRP rather than loss-of-function. This is consistent both with absence of Fragile X syndrome in the probands and the observed electrophysiological anomalies in the FMR1CRE mouse brain. Although disruption of in vivo patterns of endogenous gene expression in disease-relevant tissues by CRE variants cannot be used as strong evidence for Mendelian disease association, in conjunction with extreme rarity in human populations and with relevant knock-in mouse phenotypes, such variants can become likely pathogenic.

genetics↗