Publications by authors named "Damien J Downes"

26 Publications

  • Page 1 of 1

Reactivation of a developmentally silenced embryonic globin gene.

Nat Commun 2021 07 21;12(1):4439. Epub 2021 Jul 21.

MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

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.
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http://dx.doi.org/10.1038/s41467-021-24402-3DOI Listing
July 2021

A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker.

Nat Commun 2021 06 21;12(1):3806. Epub 2021 Jun 21.

MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

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.
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http://dx.doi.org/10.1038/s41467-021-23980-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217497PMC
June 2021

Duplication and Functional Divergence of Branched-Chain Amino Acid Biosynthesis Genes in Aspergillus nidulans.

mBio 2021 06 22;12(3):e0076821. Epub 2021 Jun 22.

Department of Plant Pathology, Kansas State University, Manhattan, Kansas, USA.

Fungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding β-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both β-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)Cys transcription factor LeuB. 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. While BCAA biosynthesis is well characterized in yeasts, it is poorly understood in filamentous fungal pathogens. Developing a thorough understanding of both the genes encoding the metabolic enzymes for BCAA biosynthesis and how their expression is regulated will inform target selection for antifungal drug development.
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http://dx.doi.org/10.1128/mBio.00768-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262921PMC
June 2021

Defining genome architecture at base-pair resolution.

Nature 2021 07 9;595(7865):125-129. Epub 2021 Jun 9.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

In higher eukaryotes, many genes are regulated by enhancers that are 10-10 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
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http://dx.doi.org/10.1038/s41586-021-03639-4DOI Listing
July 2021

Multi Locus View: an extensible web-based tool for the analysis of genomic data.

Commun Biol 2021 05 25;4(1):623. Epub 2021 May 25.

MRC WIMM Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

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 https://mlv.molbiol.ox.ac.uk .
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http://dx.doi.org/10.1038/s42003-021-02097-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8149710PMC
May 2021

Enhancers predominantly regulate gene expression during differentiation via transcription initiation.

Mol Cell 2021 03 3;81(5):983-997.e7. Epub 2021 Feb 3.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK; MRC WIMM Centre for Computational Biology, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. Electronic address:

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 transcriptional enhancers influence these events to control gene expression. Here, we present an approach that evaluates the level and change in promoter-proximal transcription (initiation and pausing) in the context of differential gene expression, genome-wide. This combinatorial approach shows that in primary cells, control of gene expression during differentiation is achieved predominantly via changes in transcription initiation rather than via release of Pol II pausing. Using genetically engineered mouse models, deleted for functionally validated enhancers of the α- and β-globin loci, we confirm that these elements regulate Pol II recruitment and/or initiation to modulate gene expression. Together, our data show that gene expression during differentiation is regulated predominantly at the level of initiation and that enhancers are key effectors of this process.
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http://dx.doi.org/10.1016/j.molcel.2021.01.002DOI Listing
March 2021

High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale.

Nat Commun 2021 01 22;12(1):531. Epub 2021 Jan 22.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse 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 individual 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.
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http://dx.doi.org/10.1038/s41467-020-20809-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822813PMC
January 2021

Recapitulation of erythropoiesis in congenital dyserythropoietic anaemia type I (CDA-I) identifies defects in differentiation and nucleolar abnormalities.

Haematologica 2020 10 29;Online ahead of print. Epub 2020 Oct 29.

Weatherall Institute of Molecular Medicine, Oxford University, Oxford.

The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic 'spongy' heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.
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http://dx.doi.org/10.3324/haematol.2020.260158DOI Listing
October 2020

Hypoxia Induces Transcriptional and Translational Downregulation of the Type I IFN Pathway in Multiple Cancer Cell Types.

Cancer Res 2020 12 28;80(23):5245-5256. Epub 2020 Oct 28.

Department of Medical Oncology, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

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/paracrine effects on tumor growth.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611234PMC
December 2020

Genetic and functional insights into CDA-I prevalence and pathogenesis.

J Med Genet 2021 03 9;58(3):185-195. Epub 2020 Jun 9.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

Background: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes and . Little is understood about either protein and it is unclear in which cellular pathways they participate.

Methods: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by . Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation.

Results: We identify six novel mutations and one novel mutation in and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells.

Conclusion: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
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http://dx.doi.org/10.1136/jmedgenet-2020-106880DOI Listing
March 2021

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

Mol Cell 2020 06 23;78(5):960-974.e11. Epub 2020 Apr 23.

Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON K1H8L6, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H8L6, Canada. Electronic address:

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. 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, co-repressors are dramatically more abundant than co-activators at the protein level, 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.
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http://dx.doi.org/10.1016/j.molcel.2020.03.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344268PMC
June 2020

DOT1L inhibition reveals a distinct subset of enhancers dependent on H3K79 methylation.

Nat Commun 2019 06 26;10(1):2803. Epub 2019 Jun 26.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Haematology Theme, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to demarcate potentially active enhancers. Here we reveal that putative enhancers marked with H3 lysine 79 (H3K79) di or trimethylation (me2/3) (which we name H3K79me2/3 enhancer elements or KEEs) can be found in multiple cell types. Mixed lineage leukemia gene (MLL) rearrangements (MLL-r) such as MLL-AF4 are a major cause of incurable acute lymphoblastic leukemias (ALL). Using the DOT1L inhibitor EPZ-5676 in MLL-AF4 leukemia cells, we show that H3K79me2/3 is required for maintaining chromatin accessibility, histone acetylation and transcription factor binding specifically at KEEs but not non-KEE enhancers. We go on to show that H3K79me2/3 is essential for maintaining enhancer-promoter interactions at a subset of KEEs. Together, these data implicate H3K79me2/3 as having a functional role at a subset of active enhancers in MLL-AF4 leukemia cells.
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http://dx.doi.org/10.1038/s41467-019-10844-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594956PMC
June 2019

High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates.

Methods Protoc 2018 Aug 1;1(3). Epub 2018 Aug 1.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DU, UK.

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 amplification 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.
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http://dx.doi.org/10.3390/mps1030029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481090PMC
August 2018

Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains.

Nat Genet 2018 12 29;50(12):1744-1751. Epub 2018 Oct 29.

MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

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 in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.
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http://dx.doi.org/10.1038/s41588-018-0253-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265079PMC
December 2018

Impaired DNA demethylation of C/EBP sites causes premature aging.

Genes Dev 2018 06 8;32(11-12):742-762. Epub 2018 Jun 8.

Institute of Molecular Biology (IMB), 55128 Mainz, Germany.

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 double-knockout mice display segmental progeria and phenocopy impaired energy homeostasis and lipodystrophy characteristic of () 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.
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http://dx.doi.org/10.1101/gad.311969.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049513PMC
June 2018

Low-input Capture-C: A Chromosome Conformation Capture Assay to Analyze Chromatin Architecture in Small Numbers of Cells.

Bio Protoc 2017 Dec;7(23)

Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.

Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This protocol describes two new low-input Capture-C approaches that can generate high-quality 3C interaction profiles from 10,000-20,000 cells, depending on the resolution used for analysis.
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http://dx.doi.org/10.21769/BioProtoc.2645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736099PMC
December 2017

Robust detection of chromosomal interactions from small numbers of cells using low-input Capture-C.

Nucleic Acids Res 2017 Dec;45(22):e184

Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.

Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000-20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C technique based on NanoString technology called C-String. By comparing C-String and Capture-C interaction profiles we show that the latter are not skewed by PCR amplification. Furthermore, we demonstrate that chromatin interactions detected by Capture-C do not depend on the degree of cross-linking by performing experiments with varying formaldehyde concentrations.
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http://dx.doi.org/10.1093/nar/gkx1194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728395PMC
December 2017

Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo.

Nat Cell Biol 2017 Aug 24;19(8):952-961. Epub 2017 Jul 24.

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK.

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.
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http://dx.doi.org/10.1038/ncb3573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540176PMC
August 2017

Functional characterisation of -regulatory elements governing dynamic expression in the early mouse embryo.

Development 2017 04 7;144(7):1249-1260. Epub 2017 Feb 7.

The Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK

The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The -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 VPE is required for optimal expression Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs 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 that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the target genes and is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.
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http://dx.doi.org/10.1242/dev.147322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399628PMC
April 2017

Spatial differentiation of gene expression in Aspergillus niger colony grown for sugar beet pulp utilization.

Sci Rep 2015 Aug 28;5:13592. Epub 2015 Aug 28.

Fungal Physiology, CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1038/srep13592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552001PMC
August 2015

Characterization of the mutagenic spectrum of 4-nitroquinoline 1-oxide (4-NQO) in Aspergillus nidulans by whole genome sequencing.

G3 (Bethesda) 2014 Oct 27;4(12):2483-92. Epub 2014 Oct 27.

Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506

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 diverse 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.
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http://dx.doi.org/10.1534/g3.114.014712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267943PMC
October 2014

Dual DNA binding and coactivator functions of Aspergillus nidulans TamA, a Zn(II)2Cys6 transcription factor.

Mol Microbiol 2014 Jun 7;92(6):1198-211. Epub 2014 May 7.

Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS, 66506, USA; Department of Genetics, The University of Melbourne, Parkville, Vic., 3010, Australia.

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.
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http://dx.doi.org/10.1111/mmi.12620DOI Listing
June 2014

Multiple nuclear localization signals mediate nuclear localization of the GATA transcription factor AreA.

Eukaryot Cell 2014 Apr 21;13(4):527-38. Epub 2014 Feb 21.

Department of Plant Pathology, Kansas State University, Manhattan, Kansas, USA.

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 RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually 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.
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http://dx.doi.org/10.1128/EC.00040-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000105PMC
April 2014

Regulation of the NADP-glutamate dehydrogenase gene gdhA in Aspergillus nidulans by the Zn(II)2Cys6 transcription factor LeuB.

Microbiology (Reading) 2013 Dec 11;159(Pt 12):2467-2480. Epub 2013 Sep 11.

Department of Genetics, University of Melbourne, Parkville, VIC 3010, Australia.

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.
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http://dx.doi.org/10.1099/mic.0.071514-0DOI Listing
December 2013
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