ORCID Profile
0000-0002-5034-0869
Current Organisation
University of Oxford
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Publisher: Cold Spring Harbor Laboratory
Date: 15-05-2021
DOI: 10.1101/2021.05.14.444178
Abstract: The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increased in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells was associated with the formation of tissue-specific sub-TADs over Runx1 , a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletions of promoter-proximal CTCF sites at the sub-TAD boundaries had no obvious effects on E-P interactions but led to partial loss of domain structure, mildly affected gene expression, and delayed hematopoietic development. Together, our analyses of gene regulation at a large multi-promoter developmental gene revealed that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.
Publisher: Cold Spring Harbor Laboratory
Date: 16-06-2020
DOI: 10.1101/2020.06.15.151837
Abstract: Tracking and understanding data quality, analysis and reproducibility are critical concerns in the biological sciences. This is especially true in genomics where Next Generation Sequencing (NGS) based technologies such as ChIP-seq, RNA-seq and ATAC-seq are generating a flood of genome-scale data. These data-types are extremely high level and complex with single experiments capable of mapping ten to hundreds of thousands of biologically meaningful events across the genome. However, such data are usually processed with automated tools and pipelines, generating tabular outputs and static visualizations. These are difficult to interact with and require substantial bioinformatic skills to manipulate and query. Similarly, interpretation is normally made at a high level without the ability to visualise the underlying data in detail and so the complexity and quality of the real underlying biological signal is lost. Also genomics datasets require integration with other genomics datasets to be properly interpreted and this integration with multiple tracks again requires substantial bioinformatics skills and is difficult to visualise across multiple pertinent datasets. Conventional genome browsers do allow for the detailed visualisation of multiple tracks but are limited to browsing single locations and do not allow for interactions with the dataset as a whole. MLV has been developed to allow users to fluidly interact with genomics datasets at multiple scales, from complete metadata labelled and clustered populations to detailed representations of in idual elements. It has inbuilt tools to integrate signals across multiple datasets and to perform dimensionality reduction and clustering analysis based on the extracted signal, allowing for the high-level analysis of complex datasets while maintaining visualisation of the fine grain structure of the data. MLV’s ability to visualise clustering within the data combined with efficient tools for large-scale tagging of in idual elements makes it a unique tool for the generation of annotated datasets for modern machine learning approaches. Multi Locus View (MLV) is a web based tool for the visualisation, analysis and annotation of Next Generation Sequencing data sets. The user is able to browse the raw data, cluster, and combine the data with other analysis. Intuitive filtering and visualisation then enables the user to quickly locate and annotate regions of interest. User datasets can then be shared with other users or made public for quick assessment from the academic community. MLV is publically available at mlv.molbiol.ox.ac.uk and the source code is available at github.com/Hughes-Genome-Group/mlv
Publisher: Springer Science and Business Media LLC
Date: 29-03-2023
Publisher: Research Square Platform LLC
Date: 28-01-2021
DOI: 10.21203/RS.3.PEX-1244/V1
Abstract: NuTi Capture-C is a Chromosome Conformation Capture (3C) approach, which can very efficiently identify chromatin interactions at target viewpoints at high resolution. The addition of high-throughput sequencing adaptors prior to enrichment allows for multiplexing of replicates and comparison s les. This method is an improvement on the previous NG Capture-C 1 method in that modifications have been made to the in situ 3C method to improve nuclear integrity (Nuclear 3C). Additionally, capture has been optimised to viewpoint complexity through titration, maximising on target sequence specificity. The experiment will take several weeks and provide reproducible interaction profiles for tens to thousands of viewpoints of interest.
Publisher: Cold Spring Harbor Laboratory
Date: 27-01-2021
DOI: 10.1101/2021.01.25.428108
Abstract: ATAC-seq, ChIP-seq, and DNase-seq have revolutionized molecular biology by allowing researchers to identify important DNA-encoded elements genome-wide. Regions where these elements are found appear as peaks in the analog signal of an assay’s coverage track, and despite the ease with which humans can visually categorize these regions, meaningful peak calls from whole genome datasets require complex analytical techniques. Current methods focus on statistical tests to classify peaks, reducing the information-dense peak shapes to simply maximum height, and discounting that background signals do not completely follow any known probability distribution for significance testing. Deep learning has been shown to be highly accurate for image recognition, on par or exceeding human ability, providing an opportunity to reimagine and improve peak calling. We present the peak calling framework LanceOtron, which combines multifaceted enrichment measurements with deep learning image recognition techniques for assessing peak shape. In benchmarking transcription factor binding, chromatin modification, and open chromatin datasets, LanceOtron outperforms the long-standing, gold-standard peak caller MACS2 through its improved selectivity and near perfect sensitivity. In addition to command line accessibility, a graphical web application was designed to give any researcher the ability to generate optimal peak calls and interactive visualizations in a single step.
Publisher: Oxford University Press (OUP)
Date: 22-07-2022
DOI: 10.1093/BIOINFORMATICS/BTAC525
Abstract: Genome sequencing experiments have revolutionized molecular biology by allowing researchers to identify important DNA-encoded elements genome wide. Regions where these elements are found appear as peaks in the analog signal of an assay’s coverage track, and despite the ease with which humans can visually categorize these patterns, the size of many genomes necessitates algorithmic implementations. Commonly used methods focus on statistical tests to classify peaks, discounting that the background signal does not completely follow any known probability distribution and reducing the information-dense peak shapes to simply maximum height. Deep learning has been shown to be highly accurate for many pattern recognition tasks, on par or even exceeding human capabilities, providing an opportunity to reimagine and improve peak calling. We present the peak calling framework LanceOtron, which combines deep learning for recognizing peak shape with multifaceted enrichment calculations for assessing significance. In benchmarking ATAC-seq, ChIP-seq and DNase-seq, LanceOtron outperforms long-standing, gold-standard peak callers through its improved selectivity and near-perfect sensitivity. A fully featured web application is freely available from LanceOtron.molbiol.ox.ac.uk, command line interface via python is pip installable from PyPI at roject/lanceotron/, and source code and benchmarking tests are available at github.com/LHentges/LanceOtron. Supplementary data are available at Bioinformatics online.
Publisher: Springer Science and Business Media LLC
Date: 22-01-2021
DOI: 10.1038/S41467-020-20809-6
Abstract: Chromosome conformation capture (3C) provides an adaptable tool for studying erse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two in idual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/0008-5472.22425273.V1
Abstract: supplementary figures and materials
Publisher: Springer Science and Business Media LLC
Date: 27-06-2023
Publisher: American Society for Microbiology
Date: 04-2014
DOI: 10.1128/EC.00040-14
Abstract: The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX 33 RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in in idual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs in idually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus , yeast, and mammals, indicating an ancient origin.
Publisher: Cold Spring Harbor Laboratory
Date: 04-08-2019
DOI: 10.1101/724005
Abstract: Understanding 3D genome structure requires high throughput, genome-wide approaches. However, assays for all vs. all chromatin interaction mapping are expensive and time consuming, which severely restricts their usage for large-scale mutagenesis screens or for mapping the impact of sequence variants. Computational models sophisticated enough to grasp the determinants of chromatin folding provide a unique window into the functional determinants of 3D genome structure as well as the effects of genome variation. A chromatin interaction predictor should work at the base pair level but also incorporate large-scale genomic context to simultaneously capture the large scale and intricate structures of chromatin architecture. Similarly, to be a flexible and generalisable approach it should also be applicable to data it has not been explicitly trained on. To develop a model with these properties, we designed a deep neuronal network (deepC) that utilizes transfer learning to accurately predict chromatin interactions from DNA sequence at megabase scale. The model generalizes well to unseen chromosomes and works across cell types, Hi-C data resolutions and a range of sequencing depths. DeepC integrates DNA sequence context on an unprecedented scale, bridging the different levels of resolution from base pairs to TADs. We demonstrate how this model allows us to investigate sequence determinants of chromatin folding at genome-wide scale and to predict the importance of regulatory elements and the impact of sequence variations.
Publisher: Cold Spring Harbor Laboratory
Date: 18-02-2020
DOI: 10.1101/2020.02.17.952572
Abstract: DNA folding within nuclei is a highly ordered process, with implications for gene regulation and development. An array of chromosome conformation capture (3C) methods have been developed to investigate how DNA is packaged within nuclei and to interrogate specific interactions. While these methods use different approaches to examine target loci (many-versus-all) or the entire genome (all-versus-all), they all rely on the core principle of endonuclease digestion and proximity-based ligation to re-arrange genomic order to reflect the three-dimensional nuclear conformation. This sequence reorganization creates novel chimeric DNA fragments which require specialist bioinformatic tools to analyze and visualize. Despite this need for specialist bioinformatic skills, the core biological importance of genome folding has seen widespread methodological uptake. To service the needs of experimentalists using the many-versus-all Capture-C family of methods we have developed CaptureCompendium a toolkit of software to simplify the design, analysis and presentation of 3C experiments.
Publisher: Wiley
Date: 07-05-2014
DOI: 10.1111/MMI.12620
Abstract: Transcription factors containing DNA binding domains generally regulate transcription by direct interaction with DNA. For most transcription factors, including the fungal Zn(II)2Cys6 zinc binuclear cluster transcription factors, the DNA binding motif is essential for function. However, Aspergillus nidulans TamA and the related Saccharomyces cerevisiae Dal81p protein contain Zn(II)2Cys6 motifs shown to be dispensable for function. TamA acts at several promoters as a coactivator of the global nitrogen GATA transcription factor AreA. We now show that TamA is the major transcriptional activator of gdhA, encoding the key nitrogen metabolism enzyme NADP-glutamate dehydrogenase. Moreover, activation of gdhA by TamA occurs primarily by a mechanism requiring the TamA DNA binding motif. We show that the TamA DNA binding motif is required for DNA binding of FLAG-epitope-tagged TamA to the gdhA promoter. We identify a conserved promoter element required for TamA activation, and show that TamA and AreA are reciprocally required for full binding at the gdhA promoter under conditions where AreA is inactive at most promoters but active at gdhA. Therefore TamA has dual functions as a DNA-binding transcription factor and a non-DNA-binding coactivator. Dual DNA-binding and coactivator functions provide an additional level of combinatorial control to mediate gene-specific expression.
Publisher: The Company of Biologists
Date: 2017
DOI: 10.1242/DEV.147322
Abstract: The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable and only the VPE is required for optimal Eomes expression in vivo. Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions embryonic stem cells (ESC), prior to gene activation. The locus resides in a large (500kb) pre-formed compartment in ESC and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a, and four additional putative enhancers display increased chromatin accessibility in DE associated with Smad2/3 binding coincident with transcriptional activation. In contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus erse regulatory mechanisms govern activation of lineage specifying TFs during early development.
Publisher: Springer Science and Business Media LLC
Date: 28-08-2015
DOI: 10.1038/SREP13592
Abstract: Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger . We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.
Publisher: Springer Science and Business Media LLC
Date: 24-07-2017
DOI: 10.1038/NCB3573
Publisher: Microbiology Society
Date: 12-2013
Abstract: NADP-dependent glutamate dehydrogenase (NADP-GDH) is a key enzyme in the assimilation of alternative nitrogen nutrient sources through ammonium in fungi. In Aspergillus nidulans, NADP-GDH is encoded by gdhA. Several transcription factors are known to regulate gdhA expression, including AreA, the major transcription activator of nitrogen metabolic genes, and TamA, a co-activator of AreA. TamA also interacts with LeuB, the regulator of leucine biosynthesis. We have investigated the effects of leucine biosynthesis on gdhA regulation, and found that leucine regulates the levels of NADP-GDH activity and gdhA expression. We show, using mutants with perturbed levels of α-isopropylmalate (α-IPM), that this leucine biosynthesis intermediate affects gdhA regulation. Leucine regulation of gdhA requires a functional LeuB with an intact Zn(II)2Cys6 DNA-binding domain. By analysing the prevalence of putative LeuB DNA-binding sites in promoters of gdhA orthologues we predict broad conservation of leucine regulation of NADP-GDH expression within ascomycetes except in the fusaria and fission yeasts. Using promoter mutations in gdhA-lacZ reporter genes we identified two sites of action for LeuB within the A. nidulans gdhA promoter. These two sites lack sequence identity, with one site conforming to the predicted LeuB DNA-binding site consensus motif, whereas the second site is a novel regulatory sequence element conserved in Aspergillus gdhA promoters. These data suggest that LeuB regulates NADP-GDH expression in response to leucine levels, which may act as an important sensor of nitrogen availability.
Publisher: Cold Spring Harbor Laboratory
Date: 02-03-2020
DOI: 10.1101/2020.03.02.953745
Abstract: Chromosome conformation capture (3C) provides an adaptable tool for studying erse biological questions. Current 3C methods provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at up to several hundred loci. All 3C methods are affected to varying degrees by inefficiency, bias and noise. As such, generation of reproducible high-resolution interaction profiles has not been achieved at scale. To overcome this barrier, we systematically tested and improved upon current methods. We show that isolation of 3C libraries from intact nuclei, as well as shortening and titration of enrichment oligonucleotides used in high-resolution methods reduces noise and increases on-target sequencing. We combined these technical modifications into a new method Nuclear-Titrated (NuTi) Capture-C, which provides a -fold increase in informative sequencing content over current Capture-C protocols. Using NuTi Capture-C we target 8,061 promoters in triplicate, demonstrating that this method generates reproducible high-resolution genome-wide 3C interaction profiles at scale.
Publisher: Springer Science and Business Media LLC
Date: 25-05-2021
DOI: 10.1038/S42003-021-02097-Y
Abstract: Tracking and understanding data quality, analysis and reproducibility are critical concerns in the biological sciences. This is especially true in genomics where next generation sequencing (NGS) based technologies such as ChIP-seq, RNA-seq and ATAC-seq are generating a flood of genome-scale data. However, such data are usually processed with automated tools and pipelines, generating tabular outputs and static visualisations. Interpretation is normally made at a high level without the ability to visualise the underlying data in detail. Conventional genome browsers are limited to browsing single locations and do not allow for interactions with the dataset as a whole. Multi Locus View (MLV), a web-based tool, has been developed to allow users to fluidly interact with genomics datasets at multiple scales. The user is able to browse the raw data, cluster, and combine the data with other analysis and annotate the data. User datasets can then be shared with other users or made public for quick assessment from the academic community. MLV is publically available at mlv.molbiol.ox.ac.uk .
Publisher: Oxford University Press (OUP)
Date: 03-02-2021
DOI: 10.1093/NAR/GKAB053
Abstract: The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate in idually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.
Publisher: American Society for Microbiology
Date: 29-06-2021
Abstract: Branched-chain amino acid (BCAA) biosynthesis is important for pathogenic fungi to successfully cause disease in human and plant hosts. The enzymes for their production are absent from humans and, therefore, provide potential antifungal targets.
Publisher: Elsevier BV
Date: 03-2021
Publisher: Springer Science and Business Media LLC
Date: 09-02-2022
DOI: 10.1038/S41467-022-28376-8
Abstract: The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increase in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells is associated with the formation of tissue-specific sub-TADs over Runx1 , a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletion of promoter-proximal CTCF sites at the sub-TAD boundaries has no obvious effects on E-P interactions but leads to partial loss of domain structure, mildly affects gene expression, and delays hematopoietic development. Together, our analysis of gene regulation at a large multi-promoter developmental gene reveals that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.
Publisher: Elsevier BV
Date: 04-2022
Publisher: American Society of Hematology
Date: 28-01-2021
Publisher: Springer Science and Business Media LLC
Date: 17-06-2022
DOI: 10.1038/S41467-022-31194-7
Abstract: The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.
Publisher: Bio-Protocol, LLC
Date: 2017
Publisher: Research Square Platform LLC
Date: 28-01-2021
DOI: 10.21203/RS.3.PEX-1246/V1
Abstract: Through-out erythropoiesis cells undergo several morphological and regulatory changes. In mouse, these changes can be characterized through isolation of specific intermediate populations based on the surface levels of Ter119 (Glycophorin A-associated protein/Ly-76) and CD71 (Transferrin receptor 1) 1-2 . This method takes roughly one day and can be used to generate sufficient cells for most high-throughput sequencing techniques with low input adaptations.
Publisher: MDPI AG
Date: 08-2018
DOI: 10.3390/MPS1030029
Abstract: The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the lification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.
Publisher: Cold Spring Harbor Laboratory
Date: 21-10-2019
DOI: 10.1101/812123
Abstract: Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). Yet, despite years of studies we still do not know the protein copy number of TFs in the nucleus. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first Gene Regulatory Network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that in the nucleus, corepressors are dramatically more abundant than coactivators at the protein, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.
Publisher: Cold Spring Harbor Laboratory
Date: 16-11-2019
DOI: 10.1101/844191
Abstract: Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo , while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/0008-5472.22425273
Abstract: supplementary figures and materials
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/0008-5472.C.6511992
Abstract: Abstract Hypoxia is a common phenomenon in solid tumors and is strongly linked to hallmarks of cancer. Recent evidence has shown that hypoxia promotes local immune suppression. Type I IFN supports cytotoxic T lymphocytes by stimulating the maturation of dendritic cells and enhancing their capacity to process and present antigens. However, little is known about the relationship between hypoxia and the type I IFN pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA) followed by IFNα/β secretion and transcriptional activation of IFN-stimulated genes (ISG). In this study, we determined the effects of hypoxia on the type I IFN pathway in breast cancer and the mechanisms involved. In cancer cell lines and xenograft models, mRNA and protein expressions of the type I IFN pathway were downregulated under hypoxic conditions. This pathway was suppressed at each level of signaling, from the dsRNA sensors RIG-I and MDA5, the adaptor MAVS, transcription factors IRF3, IRF7, and STAT1, and several ISG including RIG-I, IRF7, STAT1, and ADAR-p150. Importantly, IFN secretion was reduced under hypoxic conditions. HIF1α- and HIF2α-mediated regulation of gene expression did not explain most of the effects. However, ATAC-seq data revealed in hypoxia that peaks with STAT1 and IRF3 motifs had decreased accessibility. Collectively, these results indicate that hypoxia leads to an overall downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in an HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumors. Significance: These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine aracrine effects on tumor growth. /
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/0008-5472.C.6511992.V1
Abstract: Abstract Hypoxia is a common phenomenon in solid tumors and is strongly linked to hallmarks of cancer. Recent evidence has shown that hypoxia promotes local immune suppression. Type I IFN supports cytotoxic T lymphocytes by stimulating the maturation of dendritic cells and enhancing their capacity to process and present antigens. However, little is known about the relationship between hypoxia and the type I IFN pathway, which comprises the sensing of double-stranded RNA and DNA (dsRNA/dsDNA) followed by IFNα/β secretion and transcriptional activation of IFN-stimulated genes (ISG). In this study, we determined the effects of hypoxia on the type I IFN pathway in breast cancer and the mechanisms involved. In cancer cell lines and xenograft models, mRNA and protein expressions of the type I IFN pathway were downregulated under hypoxic conditions. This pathway was suppressed at each level of signaling, from the dsRNA sensors RIG-I and MDA5, the adaptor MAVS, transcription factors IRF3, IRF7, and STAT1, and several ISG including RIG-I, IRF7, STAT1, and ADAR-p150. Importantly, IFN secretion was reduced under hypoxic conditions. HIF1α- and HIF2α-mediated regulation of gene expression did not explain most of the effects. However, ATAC-seq data revealed in hypoxia that peaks with STAT1 and IRF3 motifs had decreased accessibility. Collectively, these results indicate that hypoxia leads to an overall downregulation of the type I IFN pathway due to repressed transcription and lower chromatin accessibility in an HIF1/2α-independent manner, which could contribute to immunosuppression in hypoxic tumors. Significance: These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine aracrine effects on tumor growth. /
Publisher: Springer Science and Business Media LLC
Date: 02-2022
Publisher: Springer Science and Business Media LLC
Date: 21-07-2021
DOI: 10.1038/S41467-021-24402-3
Abstract: The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.
Publisher: Cold Spring Harbor Laboratory
Date: 24-04-2018
DOI: 10.1101/307405
Abstract: The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact within single cells is not understood. To address this we developed Tri-C, a new Chromosome Conformation Capture (3C) approach to identify concurrent chromatin interactions at in idual alleles within single cells. The heterogeneity of interactions observed between such cells shows that CTCF-mediated formation of chromatin domains and interactions within them are dynamic processes. Importantly, our analyses reveal higher-order structures involving simultaneous interactions between multiple enhancers and promoters within in idual cells. This provides a structural basis for understanding how multiple cis -elements act together to establish robust regulation of gene expression.
Publisher: Cold Spring Harbor Laboratory
Date: 06-2018
Abstract: Changes in DNA methylation are among the best-documented epigenetic alterations accompanying organismal aging. However, whether and how altered DNA methylation is causally involved in aging have remained elusive. GADD45α (growth arrest and DNA damage protein 45A) and ING1 (inhibitor of growth family member 1) are adapter proteins for site-specific demethylation by TET (ten-eleven translocation) methylcytosine dioxygenases. Here we show that Gadd45a/Ing1 double-knockout mice display segmental progeria and phenocopy impaired energy homeostasis and lipodystrophy characteristic of Cebp ( CCAAT/enhancer-binding protein ) mutants. Correspondingly, GADD45α occupies C/EBPβ/δ-dependent superenhancers and, cooperatively with ING1, promotes local DNA demethylation via long-range chromatin loops to permit C/EBPβ recruitment. The results indicate that enhancer methylation can affect aging and imply that C/EBP proteins play an unexpected role in this process. Our study suggests a causal nexus between DNA demethylation, metabolism, and organismal aging.
Publisher: Springer Science and Business Media LLC
Date: 04-09-2017
DOI: 10.1038/S41467-017-00479-7
Abstract: β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem rogenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.
Publisher: American Association for Cancer Research (AACR)
Date: 12-2020
DOI: 10.1158/0008-5472.CAN-19-2306
Abstract: These findings characterize a new mechanism of immunosuppression by hypoxia via downregulation of the type I IFN pathway and its autocrine aracrine effects on tumor growth.
Publisher: Research Square Platform LLC
Date: 28-01-2021
DOI: 10.21203/RS.3.PEX-1245/V1
Abstract: 4sU-seq allows for measuring or nascent transcription which can reflect dynamic changes more accurately than traditional RNA-seq. Incorporation of the nucleoside analog 4-thiouridine into RNA allows the separation of nascent transcripts though reversible coupling to biotin and streptavdin bead pull-down. The protocol takes roughly two days to generate a sequencing ready library.
Publisher: Cold Spring Harbor Laboratory
Date: 19-09-2022
DOI: 10.1101/2022.09.19.508549
Abstract: Resolving causal genes for type 2 diabetes at loci implicated by genome-wide association studies (GWAS) requires integrating functional genomic data from relevant cell types. Chromatin features in endocrine cells of the pancreatic islet are particularly informative and recent studies leveraging chromosome conformation capture (3C) with Hi-C based methods have elucidated regulatory mechanisms in human islets. However, these genome-wide approaches are less sensitive and afford lower resolution than methods that target specific loci. To gauge the extent to which targeted 3C further resolves chromatin-mediated regulatory mechanisms at GWAS loci, we generated interaction profiles at 23 loci using next-generation (NG) Capture-C in a human beta cell model (EndoC-βH1) and contrasted these maps with Hi-C maps in EndoC-βH1 cells and human islets and a promoter capture Hi-C map in human islets. We found improvements in assay sensitivity of up to 33-fold and resolved 4.8X more chromatin interactions. At a subset of 18 loci with 25 co-localised GWAS and eQTL signals, NG Capture-C interactions implicated effector transcripts at five additional genetic signals relative to promoter capture Hi-C through physical contact with gene promoters. Therefore, high resolution chromatin interaction profiles at selectively targeted loci can complement genome- and promoter-wide maps.
Publisher: Springer Science and Business Media LLC
Date: 09-06-2021
DOI: 10.1038/S41586-021-03639-4
Abstract: In higher eukaryotes, many genes are regulated by enhancers that are 10
Publisher: Oxford University Press (OUP)
Date: 12-2014
Abstract: 4-Nitroquinoline 1-oxide (4-NQO) is a highly carcinogenic chemical that induces mutations in bacteria, fungi, and animals through the formation of bulky purine adducts. 4-NQO has been used as a mutagen for genetic screens and in both the study of DNA damage and DNA repair. In the model eukaryote Aspergillus nidulans, 4-NQO−based genetic screens have been used to study erse processes, including gene regulation, mitosis, metabolism, organelle transport, and septation. Early work during the 1970s using bacterial and yeast mutation tester strains concluded that 4-NQO was a guanine-specific mutagen. However, these strains were limited in their ability to determine full mutagenic potential, as they could not identify mutations at multiple sites, unlinked suppressor mutations, or G:C to C:G transversions. We have now used a whole genome resequencing approach with mutant strains generated from two independent genetic screens to determine the full mutagenic spectrum of 4-NQO in A. nidulans. Analysis of 3994 mutations from 38 mutant strains reveals that 4-NQO induces substitutions in both guanine and adenine residues, although with a 19-fold preference for guanine. We found no association between mutation load and mutagen dose and observed no sequence bias in the residues flanking the mutated purine base. The mutations were distributed randomly throughout most of the genome. Our data provide new evidence that 4-NQO can potentially target all base pairs. Furthermore, we predict that current practices for 4-NQO−induced mutagenesis are sufficient to reach gene saturation for genetic screens with feasible identification of causative mutations via whole genome resequencing.
Publisher: Springer Science and Business Media LLC
Date: 21-06-2021
DOI: 10.1038/S41467-021-23980-6
Abstract: Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.
Publisher: Cold Spring Harbor Laboratory
Date: 24-10-2019
DOI: 10.1101/813618
Abstract: Genome-wide association studies (GWAS) have identified over 150,000 links between common genetic variants and human traits or complex diseases. Over 80% of these associations map to polymorphisms in non-coding DNA. Therefore, the challenge is to identify disease-causing variants, the genes they affect, and the cells in which these effects occur. We have developed a platform using ATAC-seq, DNaseI footprints, NG Capture-C and machine learning to address this challenge. Applying this approach to red blood cell traits identifies a significant proportion of known causative variants and their effector genes, which we show can be validated by direct in vivo modelling.
Publisher: Cold Spring Harbor Laboratory
Date: 07-2020
DOI: 10.1101/2020.07.01.182089
Abstract: Mammalian genomes are sub ided into large (50-2000 kb) regions of chromatin referred to as Topologically Associating Domains (TADs or sub-TADs). Chromatin within an in idual TAD contacts itself more frequently than with regions in surrounding TADs thereby directing enhancer-promoter interactions. In many cases, the borders of TADs are defined by convergently orientated boundary elements associated with CCCTC-binding factor (CTCF), which stabilises the cohesin complex on chromatin and prevents its translocation. This delimits chromatin loop extrusion which is thought to underlie the formation of TADs. However, not all CTCF-bound sites act as boundaries and, importantly, not all TADs are flanked by convergent CTCF sites. Here, we examined the CTCF binding sites within a ∼70 kb sub-TAD containing the duplicated mouse α-like globin genes and their five enhancers (5’-R1-R2-R3-Rm-R4-α1-α2-3’). The 5’ border of this sub-TAD is defined by a pair of CTCF sites. Surprisingly, we show that deletion of the CTCF binding sites within and downstream of the α-globin locus leaves the sub-TAD largely intact. The predominant 3’ border of the sub-TAD is defined by a steep reduction in contacts: this corresponds to the transcribed α2-globin gene rather than the CTCF sites at the 3’-end of the sub-TAD. Of interest, the almost identical α1- and α2-globin genes interact differently with the enhancers, resulting in preferential expression of the proximal α1-globin gene which behaves as a partial boundary between the enhancers and the distal α2-globin gene. Together, these observations provide direct evidence that actively transcribed genes can behave as boundary elements. Mammalian genomes are complex, organised 3D structures, partitioned into Topologically Associating Domains (TADs): chromatin regions that preferentially self-interact. These chromatin interactions are thought to be driven by a mechanism that continuously extrudes chromatin loops, forming structures delimited by chromatin boundary elements and reflecting the activity of enhancers and promoters. Boundary elements bind architectural proteins such as CCCTC-binding factor (CTCF). Previously, an overlap between the functional roles of enhancers and promoters has been shown. However, whether there is overlap between enhancers romoters and boundary elements is not known. Here, we show that actively transcribed genes can also behave as boundary elements, similar to CTCF boundaries. In both cases, multi-protein complexes bound to these regions may stall the process of chromatin loop extrusion.
Publisher: Cold Spring Harbor Laboratory
Date: 30-08-2019
DOI: 10.1101/744367
Abstract: We employ and extensively characterise an ex vivo culture system to study terminal erythroid maturation of CD34 + progenitors from the peripheral blood of normal in iduals and patients with Congenital Dyserythropoietic Anaemia type 1 (CDA-I). Using morphological analysis, FACS analysis and the proteomic approach CyTOF, we analysed patient-derived erythroblasts stage-matched with those from healthy donors during the expansion phase and into early differentiation. In patient cells, aspects of disordered erythropoiesis manifest midway through differentiation, including increased proliferation and changes in the DNA accessibility profile. We also show that cultured erythroblasts from CDA-I patients recapitulate the pathognomic feature of this erythroid disorder with up to 40% of the cells having abnormal ‘spongy’ chromatin morphology by electron microscopy, as well as upregulation of GDF15, a marker of ineffective erythropoiesis. In the tertiary phase of culture, patient cells show significantly less enucleation and there is persistence of earlier erythroid precursors. Furthermore, the enucleation defect appears to be more severe in patients with mutations in C15orf41 , as compared to the other known causative gene CDAN1 , indicating a genotype henotype correlation in CDA-I. Such erythroblasts are a valuable resource for investigating the pathogenesis of this disease and provide the opportunity for streamlining diagnosis for CDA-I patients and ultimately other forms of unexplained anaemia.
Publisher: Springer Science and Business Media LLC
Date: 11-2021
Publisher: Oxford University Press (OUP)
Date: 23-11-2017
DOI: 10.1093/NAR/GKX1194
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Damien Downes.