Publications by authors named "Nigel Roberts"

13 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295333PMC
July 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

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

A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions.

Nat Commun 2018 09 21;9(1):3849. Epub 2018 Sep 21.

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

Self-interacting chromatin domains encompass genes and their cis-regulatory elements; however, the three-dimensional form a domain takes, whether this relies on enhancer-promoter interactions, and the processes necessary to mediate the formation and maintenance of such domains, remain unclear. To examine these questions, here we use a combination of high-resolution chromosome conformation capture, a non-denaturing form of fluorescence in situ hybridisation and super-resolution imaging to study a 70 kb domain encompassing the mouse α-globin regulatory locus. We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. Formation of this domain does not rely on interactions between the α-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. However, absence of the major enhancers does alter internal domain interactions. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within.
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http://dx.doi.org/10.1038/s41467-018-06248-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155075PMC
September 2018

Fungal burden exposure assessment in podiatry clinics from Ireland.

Int J Environ Health Res 2018 Apr 26;28(2):167-177. Epub 2018 Mar 26.

j School of Natural Sciences , National University of Ireland , Galway , Ireland.

Fungi are amongst the bioaerosols of most importance, as indicated by the growing interest in this field of research. The aim was to characterize the exposure to fungal burden in podiatry clinics using culture-based and molecular methods.

Methods: Airborne fungi were collected using an impaction air sampler and surface samples were also performed. Fourteen air samples were collected for direct detection of fungal DNA from filamentous fungi and dermatophytes. Overall, 63.6 % of the evening samples and 46 % of the morning samples surpassed the threshold values (150 CFU/m). Molecular detection, by real time PCR, of the target fungal species/strains (Aspergillus and Stachybotrys species) was negative for all samples collected. Trichophyton rubrum was detected by PCR analysis in one DNA sample collected on day six. Results suggest the use of both culture-based and molecular methodologies are desirable for a complete evaluation of fungal burden in this particular health care setting.
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http://dx.doi.org/10.1080/09603123.2018.1453053DOI Listing
April 2018

Health and Safety in Podiatric Medicine .

J Am Podiatr Med Assoc 2017 Nov;107(6):522-530

Background: Much of the research into health and safety in podiatric medicine to date has focused on measuring particular hazards. This study examines legislative awareness and compliance in Irish podiatric medical practices and aspects of health and safety practice.

Methods: Podiatric physicians practicing in Ireland completed a cross-sectional questionnaire survey that included measures of health and safety knowledge and awareness, compliance with legislative requirements, perceived risks, and health status.

Results: Of 250 podiatric physicians who were contacted, 101 completed the survey (response rate, 40%). Legislative knowledge and compliance were low among respondents. A Student t test revealed that the use of safety control measures was more frequent among podiatric physicians in practice for less than 20 years ( P < .05). Musculoskeletal disorders and back injuries were the most frequently reported health concerns.

Conclusions: This study demonstrates the need for interventions to increase awareness of legislative requirements among podiatric physicians as a first step to increase levels of regulatory compliance.
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http://dx.doi.org/10.7547/15-141DOI Listing
November 2017

Multiplexed analysis of chromosome conformation at vastly improved sensitivity.

Nat Methods 2016 Jan 23;13(1):74-80. Epub 2015 Nov 23.

Medical Research Council, Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford, UK.

Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.
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http://dx.doi.org/10.1038/nmeth.3664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724891PMC
January 2016

Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment.

Nat Genet 2014 Feb 12;46(2):205-12. Epub 2014 Jan 12.

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

Gene expression during development and differentiation is regulated in a cell- and stage-specific manner by complex networks of intergenic and intragenic cis-regulatory elements whose numbers and representation in the genome far exceed those of structural genes. Using chromosome conformation capture, it is now possible to analyze in detail the interaction between enhancers, silencers, boundary elements and promoters at individual loci, but these techniques are not readily scalable. Here we present a high-throughput approach (Capture-C) to analyze cis interactions, interrogating hundreds of specific interactions at high resolution in a single experiment. We show how this approach will facilitate detailed, genome-wide analysis to elucidate the general principles by which cis-acting sequences control gene expression. In addition, we show how Capture-C will expedite identification of the target genes and functional effects of SNPs that are associated with complex diseases, which most frequently lie in intergenic cis-acting regulatory elements.
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http://dx.doi.org/10.1038/ng.2871DOI Listing
February 2014

Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I.

Haematologica 2013 Sep 28;98(9):1383-7. Epub 2013 May 28.

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

The congenital dyserythropoietic anemias are a heterogeneous group of rare disorders primarily affecting erythropoiesis with characteristic morphological abnormalities and a block in erythroid maturation. Mutations in the CDAN1 gene, which encodes Codanin-1, underlie the majority of congenital dyserythropoietic anemia type I cases. However, no likely pathogenic CDAN1 mutation has been detected in approximately 20% of cases, suggesting the presence of at least one other locus. We used whole genome sequencing and segregation analysis to identify a homozygous T to A transversion (c.533T>A), predicted to lead to a p.L178Q missense substitution in C15ORF41, a gene of unknown function, in a consanguineous pedigree of Middle-Eastern origin. Sequencing C15ORF41 in other CDAN1 mutation-negative congenital dyserythropoietic anemia type I pedigrees identified a homozygous transition (c.281A>G), predicted to lead to a p.Y94C substitution, in two further pedigrees of SouthEast Asian origin. The haplotype surrounding the c.281A>G change suggests a founder effect for this mutation in Pakistan. Detailed sequence similarity searches indicate that C15ORF41 encodes a novel restriction endonuclease that is a member of the Holliday junction resolvase family of proteins.
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http://dx.doi.org/10.3324/haematol.2013.089490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762094PMC
September 2013

Workplace exposure to bioaerosols in podiatry clinics.

Ann Occup Hyg 2012 Jul 30;56(6):746-53. Epub 2012 Jan 30.

School of Physics, National University of Ireland, Galway, Ireland.

Objectives: The aim of this study was to design and execute a pilot study to collect information on the personal exposure levels of podiatrists to microbial hazards in podiatry clinics and also to assess health and safety knowledge within the sector using a questionnaire survey.

Methods: A self-report quantitative questionnaire dealing with health and safety/health issues was issued to 250 podiatrist clinics. Fifteen podiatry clinics were randomly recruited to participate in the exposure study. Concentrations of airborne bacteria, fungi, yeasts, and moulds were assessed using a six-stage viable microbial cascade impactor. Personal samples of total inhalable dust and endotoxin were measured in the breathing zone of the podiatrist.

Results: A questionnaire response rate of 42% (N = 101) was achieved. Thirty-two per cent of respondents indicated that they had a respiratory condition; asthma was the most prevalent condition reported. The most frequently employed control measures reported were use of disposable gloves during patient treatments (73.3%), use of respiratory protective equipment (34.6%), use of protective aprons (16.8%), and eye protection (15.8%). A total of 15.8% of respondents used mechanical room ventilation, 47.5% used nail drills with local exhaust ventilation systems, and 11% used nail drills with water spray dust suppression. The geometric mean concentrations of bacteria, Staphylococci, fungi, and yeasts/moulds were 590, 190, 422, and 59 CFU m(-3), respectively. The geometric mean endotoxin exposure was 9.6 EU m(-3). A significant percentage of all the bioaerosols that were in the respirable fraction was representative of yeasts and moulds (65%) and Fungi (87%).

Conclusions: Even if statistical analysis of data is limited by low sample numbers, this study showed that the frequency of cleaning and use of RPE varied between clinics sampled, and it is likely that refresher health and safety training focusing on health and safety hazards inherent in podiatry work and practical control measures is warranted.
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http://dx.doi.org/10.1093/annhyg/mer124DOI Listing
July 2012

Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1{alpha} localization in erythroblasts.

Blood 2011 Jun 1;117(25):6928-38. Epub 2011 Mar 1.

Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1(gt/gt) homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.
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http://dx.doi.org/10.1182/blood-2010-09-308478DOI Listing
June 2011

Severe intrauterine anemia: a new form of epsilongammagammadeltabeta thalassemia presenting in utero in a Norwegian family.

Haematologica 2009 Aug 22;94(8):1157-9. Epub 2009 Jun 22.

National Center for Fetal Medicine, Department of Obstetrics and Gynecology, St Olavs Hospital, Trondheim University Hospital, Norwegian University of Science and Technology, Trondheim, Norway.

Severe intrauterine anemia of unknown cause presents a diagnostic challenge. We describe a Norwegian case, managed successfully by intrauterine transfusions, that further investigations demonstrated to be due to a rare type of thalassemia. A deletion of the 5' end of the beta globin gene cluster was characterized, the breakpoints sequenced and a new type of epsilongammagammadeltabeta thalassemia identified. This case highlights the need to consider diagnoses of rare conditions not normally associated with a particular population.
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http://dx.doi.org/10.3324/haematol.2009.007534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719038PMC
August 2009

Globin gene expression in Hb Lepore-BAC transgenic mice.

Br J Haematol 2006 Dec;135(5):735-7

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

We generated five lines of transgenic mice carrying 1-3 copies of the Hb Lepore deltabeta fusion gene, in the context of a Bacterial Artificial Chromosome containing the whole human beta globin gene cluster. Normal developmental regulation of human genes occurred at levels approximating to those of endogenous genes. Deltabeta transgene expression became detectable during fetal life and rose to a mean level of 13.0% in adults, similar to that of humans. Low levels of human gamma chains were detectable as F cells in adult mice, but numbers did not increase after treatment with drugs that raise F cells in human subjects, even on a thalassaemic background.
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http://dx.doi.org/10.1111/j.1365-2141.2006.06354.xDOI Listing
December 2006
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