Biology subjects

Wingett, S.

Publications and source records attributed to Wingett, S..

7 recordsLinked to original sources

bioRxiv2024

Repression of CADM1 transcription by HPV type 18 is mediated by three-dimensional rearrangement of promoter-enhancer interactions

Upon infection, human papillomavirus (HPV) manipulates host cell gene expression to create an environment that is supportive of a productive and persistent infection. The virus-induced changes to the host cells transcriptome are thought to contribute to carcinogenesis. Here, we show by RNA-sequencing that oncogenic HPV18 episome replication in primary human foreskin keratinocytes (HFKs) drives host transcriptional changes that are consistent between multiple HFK donors. We have previously shown that HPV18 episome replication in HFKs results in post-transcriptional stabilisation of the host chromatin insulation protein CTCF. Since CTCF is an important regulator of host cell transcription via coordination of epigenetic boundaries and long-range chromosomal interactions, we hypothesised that HPV18-induced stabilisation of CTCF may contribute to host transcription reprogramming. Analysis of CTCF binding in the host cell genome by ChIP-Seq revealed that while the total number of CTCF binding sites is not altered by the virus, there are a sub-set of CTCF binding sites that are either enriched or depleted of CTCF. Many of these altered sites are clustered within regulatory elements of differentially expressed genes, including the tumour suppressor gene cell adhesion molecule 1 (CADM1), which supresses epithelial cell growth and invasion. We show that HPV18 establishment results in reduced CTCF binding at the CADM1 promoter and upstream enhancer. Loss of CTCF binding is coincident with epigenetic repression of CADM1, in the absence of CpG hypermethylation, while adjacent genes including the transcriptional regulator ZBTB16 are activated. These data indicate that the CADM1 locus is subject to topological rearrangement following HPV18 establishment. We tested this hypothesis using 4C-Seq (circular chromosome confirmation capture-sequencing) and show that HPV18 establishment causes a loss of long-range chromosomal interactions between the CADM1 transcriptional start site and the upstream transcriptional enhancer. These data show that HPV18 manipulates host cell promoter-enhancer interactions to drive transcriptional reprogramming that may contribute to HPV-induced disease progression. AUTHOR SUMMARYInfection with oncogenic HPV is the cause of numerous cancer types, which generally arise after persistent HPV infection. Upon infection, HPV alters the gene expression profile of infected cells to facilitate virus replication and persistence. Multiple mechanisms of HPV-induced host cell reprogramming have been previously suggested. Here, we show that HPV infection induces rearrangement of specific genomic loci by altering the chromatin binding of the host cell protein CTCF, an important regulator of chromatin architecture. Loss of CTCF binding to a cluster of binding sites at the CADM1 locus on chromosome 11 is coincident with epigenetic reprogramming and disruption of long-range chromatin interactions, resulting in transcriptional repression of CADM1. Our data show that repression of CADM1 is an early event in HPV-driven disease, preceding hypermethylation of the CADM1 transcriptional promoter that is frequently observed in HPV-driven cancers, demonstrating a novel mechanism of HPV-induced host cell transcriptional reprogramming.

microbiology↗

bioRxiv2023

Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.

cell biology↗

bioRxiv2023

Dynamic DNA methylation turnover at the exit of pluripotency epigenetically primes gene regulatory elements for hematopoietic lineage specification

Epigenetic mechanisms govern developmental cell fate decisions, but how DNA methylation coordinates with chromatin structure and three-dimensional DNA folding to enact cell-type specific gene expression programmes remains poorly understood. Here, we use mouse embryonic stem and epiblast-like cells deficient for 5-methyl cytosine or its oxidative derivatives (5-hydroxy-, 5-formyl- and 5-carboxy-cytosine) to dissect the gene regulatory mechanisms that control cell lineage specification at the exit of pluripotency. Genetic ablation of either DNA methyltransferase (Dnmt) or Ten-eleven-translocation (Tet) activity yielded largely distinct sets of dysregulated genes, revealing divergent transcriptional defects upon perturbation of individual branches of the DNA cytosine methylation cycle. Unexpectedly, we found that disrupting DNA methylation or oxidation interferes with key enhancer features, including chromatin accessibility, enhancer-characteristic histone modifications, and long-range chromatin interactions with putative target genes. In addition to affecting transcription of select genes in pluripotent stem cells, we observe impaired enhancer priming, including a loss of three-dimensional interactions, at regulatory elements associated with key lineage-specifying genes that are required later in development, as we demonstrate for the key hematopoietic genes Klf1 and Lyl1. Consistently, we observe impaired transcriptional activation of blood genes during embryoid body differentiation of knockout cells. Our findings identify a novel role for the dynamic turnover of DNA methylation at the exit of pluripotency to establish and maintain chromatin states that epigenetically prime enhancers for later activation during developmental cell diversification. HighlightsO_LIWe perform a detailed epigenetic characterisation of the mouse embryonic stem cell (ESC) to epiblast-like cell (EpiLC) transition in wild type, Tet triple-knockout (TKO) and Dnmt TKO lines and develop a novel clustering approach to interrogate the data. C_LIO_LITet TKO reduces H3K4me1 and H3K27ac levels across enhancer elements upon pluripotency exit whilst Dnmt TKO affects only H3K4me1 levels, suggesting a novel role for oxidative derivatives in H3K4me1 deposition. C_LIO_LITet TKO and Dnmt TKO affect enhancer priming in EpiLCs which is associated with failure to upregulate hematopoietic genes upon differentiation. C_LIO_LILong-range chromosomal interactions between primed enhancers and their target genes are weakened in both Dnmt and Tet TKO. C_LI

molecular biology↗

bioRxiv2019

A neuronal thermostat controls membrane fluidity in C. elegans

An organisms ability to adapt to heat can be key to its survival. Cells adapt to temperature shifts by adjusting lipid desaturation levels and the fluidity of membranes in a process that is thought to be controlled cell autonomously. We have discovered that subtle, step-wise increments in ambient temperature can lead to the conserved heat shock response being activated in head neurons of C. elegans. This response is exactly opposite to the expression of the lipid desaturase FAT-7 in the worms gut. We find that the over-expression of the master regulator of this response, Hsf-1, in head neurons, causes extensive fat remodeling to occur across tissues. These changes include a decrease in FAT-7 expression and a shift in the levels of unsaturated fatty acids in the plasma membrane. These shifts are in line with membrane fluidity requirements to survive in warmer temperatures. We have identified that the cGMP receptor, TAX-2/TAX-4, as well as TGF-{beta}/BMP signaling, as key players in the transmission of neuronal stress to peripheral tissues. This is the first study to suggest that a thermostat-based mechanism can centrally coordinate membrane fluidity in response to warm temperatures across tissues in multicellular animals.

genetics↗

bioRxiv2018

DNA methylation oscillation defines classes of enhancers

Understanding the regulatory landscape of human cells requires the integration of genomic and epigenomic maps, capturing combinatorial levels of cell type-specific and invariant activity states.\n\nHere, we segmented whole-genome bisulfite sequencing-derived methylomes into consecutive blocks of co-methylation (COMETs) to obtain spatial variation patterns of DNA methylation (DNAm oscillations) integrated with histone modifications and promoter-enhancer interactions derived from promoter capture Hi-C (PCHi-C) sequencing of the same purified blood cells.\n\nMapping DNAm oscillations onto regulatory genome annotation revealed that enhancers are enriched for DNAm hyper-oscillations (>30-fold), where multiple machine learning models support DNAm as predictive of enhancer location. Based on this analysis, we report overall predictive power of 99% for DNAm oscillations, 77.3% for DNaseI, 41% for CGIs, 20% for UMRs and 0% for LMRs, demonstrating the power of DNAm oscillations over other methods for enhancer prediction. Methylomes of activated and non-activated CD4+ T cells indicate that DNAm oscillations exist in both states irrespective of activation; hence they can be used to determine the location of latent enhancers.\n\nOur approach advances the identification of tissue-specific regulatory elements and outperforms previous approaches defining enhancer classes based on DNA methylation.

genomics↗

bioRxiv2017

Heterogeneous chromatin mobility derived from chromatin states is a determinant of genome organisation in S. cerevisiae

Spatial organisation of the genome is essential for regulating gene activity, yet the mechanisms that shape this three-dimensional organisation in eukaryotes are far from understood. Here, we combine bioinformatic determination of chromatin states during normal growth and heat shock, and computational polymer modelling of genome structure, with quantitative microscopy and Hi-C to demonstrate that differential mobility of yeast chromosome segments leads to spatial self-organisation of the genome. We observe that more than forty percent of chromatin-associated proteins display a poised and heterogeneous distribution along the chromosome, creating a heteropolymer. This distribution changes upon heat shock in a concerted, state-specific manner. Simulating yeast chromosomes as heteropolymers, in which the mobility of each segment depends on its cumulative protein occupancy, results in functionally relevant structures, which match our experimental data. This thermodynamically driven self-organisation achieves spatial clustering of poised genes and mechanistically contributes to the directed relocalisation of active genes to the nuclear periphery upon heat shock.\n\nOne Sentence SummaryUnequal protein occupancy and chromosome segment mobility drive 3D organisation of the genome.

systems biology↗

bioRxiv2016

Cell cycle dynamics of chromosomal organisation at single-cell resolution

Chromosomes in proliferating metazoan cells undergo dramatic structural metamorphoses every cell cycle, alternating between a highly condensed mitotic structure facilitating chromosome segregation, and a decondensed interphase structure accommodating transcription, gene silencing and DNA replication. These cyclical structural transformations have been evident under the microscope for over a century, but their molecular-level analysis is still lacking. Here we use single-cell Hi-C to study chromosome conformations in thousands of individual cells, and discover a continuum of cis-interaction profiles that finely position individual cells along the cell cycle. We show that chromosomal compartments, topological domains (TADs), contact insulation and long-range loops, all defined by ensemble Hi-C maps, are governed by distinct cell cycle dynamics. In particular, DNA replication correlates with build-up of compartments and reduction in TAD insulation, while loops are generally stable from G1 through S and G2. Analysing whole genome 3D structural models using haploid cell data, we discover a radial architecture of chromosomal compartments with distinct epigenomic signatures. Our single-cell data creates an essential new paradigm for the re-interpretation of chromosome conformation maps through the prism of the cell cycle.

genomics↗