95 results on '"Buckle VJ"'
Search Results
2. Does Transcription of Remote alpha-Globin Regulatory Elements Affect Their Function?
- Author
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Kowalczyk, MS, Hughes, JR, Sharpe, JA, Brown, JM, Buckle, VJ, Wood, WG, and Higgs, DR
- Published
- 2016
3. DNA rearrangements proximal to the EVI1 locus associated with the 3q21q26 syndrome
- Author
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Levy, ER, primary, Parganas, E, additional, Morishita, K, additional, Fichelson, S, additional, James, L, additional, Oscier, D, additional, Gisselbrecht, S, additional, Ihle, JN, additional, and Buckle, VJ, additional
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- 1994
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4. Allelic loss of IRF1 in myelodysplasia and acute myeloid leukemia: retention of IRF1 on the 5q- chromosome in some patients with the 5q- syndrome
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Boultwood, J, primary, Fidler, C, additional, Lewis, S, additional, MacCarthy, A, additional, Sheridan, H, additional, Kelly, S, additional, Oscier, D, additional, Buckle, VJ, additional, and Wainscoat, JS, additional
- Published
- 1993
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5. Alpha-thalassemia caused by a large (62 kb) deletion upstream of the human alpha globin gene cluster
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Hatton, CS, primary, Wilkie, AO, additional, Drysdale, HC, additional, Wood, WG, additional, Vickers, MA, additional, Sharpe, J, additional, Ayyub, H, additional, Pretorius, IM, additional, Buckle, VJ, additional, and Higgs, DR, additional
- Published
- 1990
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6. A new translocation, t(1;3) (p36;q21), in myelodysplastic disorders
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Moir, DJ, primary, Jones, PA, additional, Pearson, J, additional, Duncan, JR, additional, Cook, P, additional, and Buckle, VJ, additional
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- 1984
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7. Intragenic Enhancers Act as Alternative Promoters
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Monika S. Kowalczyk, Douglas Vernimmen, Richard J. Gibbons, Mona Hosseini, David Garrick, Jim R. Hughes, Jacqueline A. Sloane-Stanley, Douglas R. Higgs, Marco De Gobbi, Jacqueline A. Sharpe, William G. Wood, Nicola Gray, Jill M. Brown, Magnus D. Lynch, Licio Collavin, Simon J. McGowan, Thomas A. Milne, Veronica J. Buckle, Stephen S. Taylor, Jonathan Flint, Kowalczyk, M, Hughes, Jr, Garrick, D, Lynch, Md, Sharpe, Ja, Sloane Stanley, Ja, Mcgowan, Sj, De Gobbi, M, Hosseini, M, Vernimmen, D, Brown, Jm, Gray, Ne, Collavin, Licio, Gibbons, Rj, Flint, J, Taylor, S, Buckle, Vj, Milne, Ta, Wood, Wg, and Higgs, Dr
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Gene isoform ,Enhancer RNAs ,Biology ,Genome ,alpha globins ,C16orf35 ,NPRL3 ,Mice ,Erythroid Cells ,Transcription (biology) ,Gene expression ,RNA Isoforms ,Animals ,RNA, Messenger ,Promoter Regions, Genetic ,Enhancer ,Molecular Biology ,Gene ,Cells, Cultured ,Genetics ,alpha globin ,Promoter ,Cell Biology ,Enhancer Elements, Genetic ,Gene Expression Regulation ,RNA ,Poly A ,Transcriptome - Abstract
A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A) + RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole. © 2012 Elsevier Inc..
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- 2016
8. On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.
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Jeziorska DM, Tunnacliffe EAJ, Brown JM, Ayyub H, Sloane-Stanley J, Sharpe JA, Lagerholm BC, Babbs C, Smith AJH, Buckle VJ, and Higgs DR
- Subjects
- Mice, Animals, Cell Differentiation genetics, Chromatin metabolism, alpha-Globins genetics, alpha-Globins metabolism, Transcription, Genetic, Globins genetics, Mammals genetics, Erythroblasts metabolism, Erythropoiesis genetics
- Abstract
Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation., (© 2022. The Author(s).)
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- 2022
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9. RASER-FISH: non-denaturing fluorescence in situ hybridization for preservation of three-dimensional interphase chromatin structure.
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Brown JM, De Ornellas S, Parisi E, Schermelleh L, and Buckle VJ
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- Animals, DNA genetics, DNA metabolism, Exonucleases metabolism, In Situ Hybridization, Fluorescence methods, Interphase, Mammals, Cell Nucleus genetics, Cell Nucleus metabolism, Chromatin metabolism
- Abstract
DNA fluorescence in situ hybridization (FISH) has been a central technique in advancing our understanding of how chromatin is organized within the nucleus. With the increasing resolution offered by super-resolution microscopy, the optimal maintenance of chromatin structure within the nucleus is essential for accuracy in measurements and interpretation of data. However, standard 3D-FISH requires potentially destructive heat denaturation in the presence of chaotropic agents such as formamide to allow access to the DNA strands for labeled FISH probes. To avoid the need to heat-denature, we developed Resolution After Single-strand Exonuclease Resection (RASER)-FISH, which uses exonuclease digestion to generate single-stranded target DNA for efficient probe binding over a 2 d process. Furthermore, RASER-FISH is easily combined with immunostaining of nuclear proteins or the detection of RNAs. Here, we provide detailed procedures for RASER-FISH in mammalian cultured cells to detect single loci, chromatin tracks and topologically associating domains with conventional and super-resolution 3D structured illumination microscopy. Moreover, we provide a validation and characterization of our method, demonstrating excellent preservation of chromatin structure and nuclear integrity, together with improved hybridization efficiency, compared with classic 3D-FISH protocols., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2022
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10. Recapitulation of erythropoiesis in congenital dyserythropoietic anaemia type I (CDA-I) identifies defects in differentiation and nucleolar abnormalities.
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Scott C, Downes DJ, Brown JM, Beagrie R, Olijnik AA, Gosden M, Schwessinger R, Fisher CA, Rose A, Ferguson DJP, Johnson E, Hill QA, Okoli S, Renella R, Ryan K, Brand M, Hughes J, Roy NBA, Higgs DR, Babbs C, and Buckle VJ
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- Erythroid Cells, Erythropoiesis, Glycoproteins genetics, Humans, Nuclear Proteins genetics, Anemia, Dyserythropoietic, Congenital diagnosis, Anemia, Dyserythropoietic, Congenital genetics
- Abstract
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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- 2021
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11. Genetic and functional insights into CDA-I prevalence and pathogenesis.
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Olijnik AA, Roy NBA, Scott C, Marsh JA, Brown J, Lauschke K, Ask K, Roberts N, Downes DJ, Brolih S, Johnson E, Xella B, Proven M, Hipkiss R, Ryan K, Frisk P, Mäkk J, Stattin EM, Sadasivam N, McIlwaine L, Hill QA, Renella R, Hughes JR, Gibbons RJ, Groth A, McHugh PJ, Higgs DR, Buckle VJ, and Babbs C
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- Anemia, Dyserythropoietic, Congenital pathology, Female, Gene Expression Regulation genetics, Genetic Testing, Genetics, Population, Humans, Male, Multiprotein Complexes genetics, Mutation genetics, Anemia, Dyserythropoietic, Congenital genetics, Genetic Predisposition to Disease, Glycoproteins genetics, Nuclear Proteins genetics, Transcription Factors genetics
- Abstract
Background: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41 . 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 CDAN1 . 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 CDAN1 mutations and one novel mutation in C15orf41 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., Competing Interests: Competing interests: JRH is a founder and shareholder of Nucleome Therapeutics., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)
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- 2021
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12. High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale.
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Downes DJ, Beagrie RA, Gosden ME, Telenius J, Carpenter SJ, Nussbaum L, De Ornellas S, Sergeant M, Eijsbouts CQ, Schwessinger R, Kerry J, Roberts N, Shivalingam A, El-Sagheer A, Oudelaar AM, Brown T, Buckle VJ, Davies JOJ, and Hughes JR
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- Animals, Cells, Cultured, Chromosome Mapping methods, Computational Biology methods, Gene Expression Regulation, Genomics methods, Humans, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Cell Nucleus genetics, Chromatin genetics, Erythroid Cells metabolism, Genome, Human genetics, Genome-Wide Association Study methods, Regulatory Sequences, Nucleic Acid genetics
- Abstract
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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- 2021
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13. ATR-16 syndrome: mechanisms linking monosomy to phenotype.
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Babbs C, Brown J, Horsley SW, Slater J, Maifoshie E, Kumar S, Ooijevaar P, Kriek M, Dixon-McIver A, Harteveld CL, Traeger-Synodinos J, Wilkie AOM, Higgs DR, and Buckle VJ
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- Chromosome Deletion, Chromosomes, Human, Pair 16 genetics, Female, Gene Deletion, Humans, Intellectual Disability diagnosis, Intellectual Disability pathology, Male, Monosomy diagnosis, Monosomy pathology, Phenotype, alpha-Thalassemia diagnosis, alpha-Thalassemia pathology, Ataxia Telangiectasia Mutated Proteins genetics, DNA Copy Number Variations genetics, Intellectual Disability genetics, Monosomy genetics, alpha-Thalassemia genetics
- Abstract
Background: Deletions removing 100s-1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the contribution of the deletion to an individual's clinical phenotype is challenging., Methods: Here, as an example of this common phenomenon, we analysed 41 patients with simple deletions of ~177 to ~2000 kb affecting one allele of the well-characterised, gene dense, distal region of chromosome 16 (16p13.3), referred to as ATR-16 syndrome. We characterised deletion extents and screened for genetic background effects, telomere position effect and compensatory upregulation of hemizygous genes., Results: We find the risk of developmental and neurological abnormalities arises from much smaller distal chromosome 16 deletions (~400 kb) than previously reported. Beyond this, the severity of ATR-16 syndrome increases with deletion size, but there is no evidence that critical regions determine the developmental abnormalities associated with this disorder. Surprisingly, we find no evidence of telomere position effect or compensatory upregulation of hemizygous genes; however, genetic background effects substantially modify phenotypic abnormalities., Conclusions: Using ATR-16 as a general model of disorders caused by CNVs, we show the degree to which individuals with contiguous gene syndromes are affected is not simply related to the number of genes deleted but depends on their genetic background. We also show there is no critical region defining the degree of phenotypic abnormalities in ATR-16 syndrome and this has important implications for genetic counselling., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)
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- 2020
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14. Dynamics of the 4D genome during in vivo lineage specification and differentiation.
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Oudelaar AM, Beagrie RA, Gosden M, de Ornellas S, Georgiades E, Kerry J, Hidalgo D, Carrelha J, Shivalingam A, El-Sagheer AH, Telenius JM, Brown T, Buckle VJ, Socolovsky M, Higgs DR, and Hughes JR
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- Animals, Cells, Cultured, Chromatin genetics, Chromosomes, Mammalian genetics, Female, Gene Expression Profiling methods, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism, Mice, Mice, Inbred C57BL, Mouse Embryonic Stem Cells cytology, Mouse Embryonic Stem Cells metabolism, Stem Cells cytology, Cell Differentiation genetics, Enhancer Elements, Genetic genetics, Gene Expression Regulation, Developmental, Genome genetics, Promoter Regions, Genetic genetics, Stem Cells metabolism
- Abstract
Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of specific contacts between enhancers and promoters. Our high-resolution data show that these enhancer-promoter interactions are not established prior to gene expression, but formed gradually during differentiation, concomitant with progressive upregulation of gene activity. Together, these results provide new insight into the close, interdependent relationship between chromatin architecture and gene regulation during development.
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- 2020
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15. A Dynamic Folded Hairpin Conformation Is Associated with α-Globin Activation in Erythroid Cells.
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Chiariello AM, Bianco S, Oudelaar AM, Esposito A, Annunziatella C, Fiorillo L, Conte M, Corrado A, Prisco A, Larke MSC, Telenius JM, Sciarretta R, Musella F, Buckle VJ, Higgs DR, Hughes JR, and Nicodemi M
- Subjects
- Animals, Humans, Mice, Erythroid Cells metabolism, Inverted Repeat Sequences genetics, alpha-Globins genetics
- Abstract
We investigate the three-dimensional (3D) conformations of the α-globin locus at the single-allele level in murine embryonic stem cells (ESCs) and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent fluorescence in situ hybridization (FISH) data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ESCs to erythroid cells, associated with the activation of the globin genes. We find that in ESCs, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, whereas in erythroid cells the α-globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites, multiple molecular mechanisms cooperate to form an interacting domain., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2020
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16. Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells.
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Rhodes JDP, Feldmann A, Hernández-Rodríguez B, Díaz N, Brown JM, Fursova NA, Blackledge NP, Prathapan P, Dobrinic P, Huseyin MK, Szczurek A, Kruse K, Nasmyth KA, Buckle VJ, Vaquerizas JM, and Klose RJ
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- Animals, CCCTC-Binding Factor metabolism, Cell Line, Chromatin metabolism, Gene Expression Regulation, Male, Mice, Cohesins, Cell Cycle Proteins metabolism, Chromosomal Proteins, Non-Histone metabolism, Chromosomes metabolism, Embryonic Stem Cells metabolism, Polycomb-Group Proteins metabolism
- Abstract
How chromosome organization is related to genome function remains poorly understood. Cohesin, loop extrusion, and CCCTC-binding factor (CTCF) have been proposed to create topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find, as in other cell types, that cohesin is required to create TADs and regulate A/B compartmentalization. However, in the absence of cohesin, we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system and are dependent on PRC1. Importantly, we discover that cohesin counteracts these polycomb-dependent interactions, but not interactions between super-enhancers. This disruptive activity is independent of CTCF and insulation and appears to modulate gene repression by the polycomb system. Therefore, we discover that cohesin disrupts polycomb-dependent chromosome interactions to modulate gene expression in embryonic stem cells., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2020
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17. Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains.
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Oudelaar AM, Davies JOJ, Hanssen LLP, Telenius JM, Schwessinger R, Liu Y, Brown JM, Downes DJ, Chiariello AM, Bianco S, Nicodemi M, Buckle VJ, Dekker J, Higgs DR, and Hughes JR
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- Animals, Base Sequence, Binding Sites genetics, Cells, Cultured, Enhancer Elements, Genetic, Female, Gene Expression Regulation, Developmental, Globins genetics, Linkage Disequilibrium, Mice, Mice, Inbred C57BL, Promoter Regions, Genetic, Transcription Factors metabolism, Alleles, Chromatin chemistry, Chromatin genetics, Chromatin metabolism, Genetic Loci, Regulatory Sequences, Nucleic Acid
- 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 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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- 2018
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18. A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions.
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Brown JM, Roberts NA, Graham B, Waithe D, Lagerholm C, Telenius JM, De Ornellas S, Oudelaar AM, Scott C, Szczerbal I, Babbs C, Kassouf MT, Hughes JR, Higgs DR, and Buckle VJ
- Subjects
- Animals, Erythroid Cells metabolism, In Situ Hybridization, Fluorescence, Mice, Primary Cell Culture, Protein Domains, alpha-Globins genetics, Chromatin metabolism, Regulatory Sequences, Nucleic Acid
- Abstract
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.
- Published
- 2018
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19. Robust CRISPR/Cas9 Genome Editing of the HUDEP-2 Erythroid Precursor Line Using Plasmids and Single-Stranded Oligonucleotide Donors.
- Author
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Moir-Meyer G, Cheong PL, Olijnik AA, Brown J, Knight S, King A, Kurita R, Nakamura Y, Gibbons RJ, Higgs DR, Buckle VJ, and Babbs C
- Abstract
The study of cellular processes and gene regulation in terminal erythroid development has been greatly facilitated by the generation of an immortalised erythroid cell line derived from Human Umbilical Derived Erythroid Precursors, termed HUDEP-2 cells. The ability to efficiently genome edit HUDEP-2 cells and make clonal lines hugely expands their utility as the insertion of clinically relevant mutations allows study of potentially every genetic disease affecting red blood cell development. Additionally, insertion of sequences encoding short protein tags such as Strep, FLAG and Myc permits study of protein behaviour in the normal and disease state. This approach is useful to augment the analysis of patient cells as large cell numbers are obtainable with the additional benefit that the need for specific antibodies may be circumvented. This approach is likely to lead to insights into disease mechanisms and provide reagents to allow drug discovery. HUDEP-2 cells provide a favourable alternative to the existing immortalised erythroleukemia lines as their karyotype is much less abnormal. These cells also provide sufficient material for a broad range of analyses as it is possible to generate in vitro-differentiated erythroblasts in numbers 4-7 fold higher than starting cell numbers within 9-12 days of culture. Here we describe an efficient, robust and reproducible plasmid-based methodology to introduce short (<20 bp) DNA sequences into the genome of HUDEP-2 cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 Cas9 system combined with single-stranded oligodeoxynucleotide (ssODN) donors. This protocol produces genetically modified lines in ~30 days and could also be used to generate knock-out and knock-in mutations.
- Published
- 2018
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20. Predicting the three-dimensional folding of cis-regulatory regions in mammalian genomes using bioinformatic data and polymer models.
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Brackley CA, Brown JM, Waithe D, Babbs C, Davies J, Hughes JR, Buckle VJ, and Marenduzzo D
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- Animals, Chromosomes, Mammalian genetics, Chromosomes, Mammalian metabolism, Humans, In Situ Hybridization, Fluorescence, Mice, Models, Biological, Models, Molecular, Nucleic Acid Conformation, Polymers, Chromosomes, Mammalian chemistry, Computational Biology methods, Regulatory Sequences, Nucleic Acid
- Abstract
The three-dimensional (3D) organization of chromosomes can be probed using methods like Capture-C. However, it is unclear how such population-level data relate to the organization within a single cell, and the mechanisms leading to the observed interactions are still largely obscure. We present a polymer modeling scheme based on the assumption that chromosome architecture is maintained by protein bridges, which form chromatin loops. To test the model, we perform FISH experiments and compare with Capture-C data. Starting merely from the locations of protein binding sites, our model accurately predicts the experimentally observed chromatin interactions, revealing a population of 3D conformations.
- Published
- 2016
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21. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders.
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Taylor JC, Martin HC, Lise S, Broxholme J, Cazier JB, Rimmer A, Kanapin A, Lunter G, Fiddy S, Allan C, Aricescu AR, Attar M, Babbs C, Becq J, Beeson D, Bento C, Bignell P, Blair E, Buckle VJ, Bull K, Cais O, Cario H, Chapel H, Copley RR, Cornall R, Craft J, Dahan K, Davenport EE, Dendrou C, Devuyst O, Fenwick AL, Flint J, Fugger L, Gilbert RD, Goriely A, Green A, Greger IH, Grocock R, Gruszczyk AV, Hastings R, Hatton E, Higgs D, Hill A, Holmes C, Howard M, Hughes L, Humburg P, Johnson D, Karpe F, Kingsbury Z, Kini U, Knight JC, Krohn J, Lamble S, Langman C, Lonie L, Luck J, McCarthy D, McGowan SJ, McMullin MF, Miller KA, Murray L, Németh AH, Nesbit MA, Nutt D, Ormondroyd E, Oturai AB, Pagnamenta A, Patel SY, Percy M, Petousi N, Piazza P, Piret SE, Polanco-Echeverry G, Popitsch N, Powrie F, Pugh C, Quek L, Robbins PA, Robson K, Russo A, Sahgal N, van Schouwenburg PA, Schuh A, Silverman E, Simmons A, Sørensen PS, Sweeney E, Taylor J, Thakker RV, Tomlinson I, Trebes A, Twigg SR, Uhlig HH, Vyas P, Vyse T, Wall SA, Watkins H, Whyte MP, Witty L, Wright B, Yau C, Buck D, Humphray S, Ratcliffe PJ, Bell JI, Wilkie AO, Bentley D, Donnelly P, and McVean G
- Subjects
- Base Sequence, DNA Mutational Analysis, Genetic Diseases, Inborn genetics, Genome, Human, Humans, Molecular Sequence Annotation, Polymorphism, Single Nucleotide, Sensitivity and Specificity, Genetic Diseases, Inborn diagnosis, High-Throughput Nucleotide Sequencing, Molecular Diagnostic Techniques
- Abstract
To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.
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- 2015
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22. Expanded GAA repeats impair FXN gene expression and reposition the FXN locus to the nuclear lamina in single cells.
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Silva AM, Brown JM, Buckle VJ, Wade-Martins R, and Lufino MM
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- Alleles, Cell Line, Gene Order, Gene Silencing, Humans, Protein Transport, RNA, Messenger genetics, Single-Cell Analysis, Transcription Initiation Site, Transcription Initiation, Genetic, Transcription, Genetic, Frataxin, Gene Expression, Genetic Loci, Iron-Binding Proteins genetics, Iron-Binding Proteins metabolism, Nuclear Lamina metabolism, Trinucleotide Repeat Expansion
- Abstract
Abnormally expanded DNA repeats are associated with several neurodegenerative diseases. In Friedreich's ataxia (FRDA), expanded GAA repeats in intron 1 of the frataxin gene (FXN) reduce FXN mRNA levels in averaged cell samples through a poorly understood mechanism. By visualizing FXN expression and nuclear localization in single cells, we show that GAA-expanded repeats decrease the number of FXN mRNA molecules, slow transcription, and increase FXN localization at the nuclear lamina (NL). Restoring histone acetylation reverses NL positioning. Expanded GAA-FXN loci in FRDA patient cells show increased NL localization with increased silencing of alleles and reduced transcription from alleles positioned peripherally. We also demonstrate inefficiencies in transcription initiation and elongation from the expanded GAA-FXN locus at single-cell resolution. We suggest that repressive epigenetic modifications at the expanded GAA-FXN locus may lead to NL relocation, where further repression may occur., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2015
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23. De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder.
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Babbs C, Lloyd D, Pagnamenta AT, Twigg SR, Green J, McGowan SJ, Mirza G, Naples R, Sharma VP, Volpi EV, Buckle VJ, Wall SA, Knight SJ, Parr JR, and Wilkie AO
- Subjects
- Child, Chromosome Breakpoints, Humans, In Situ Hybridization, Fluorescence, Infant, Male, Sequence Analysis, DNA, Child Development Disorders, Pervasive genetics, Chromosomes, Human, Pair 22 genetics, Gene Rearrangement genetics, Mutation genetics, Transcription Factors genetics
- Abstract
Background: Autism spectrum disorders (ASDs) are common and have a strong genetic basis, yet the cause of ∼70-80% ASDs remains unknown. By clinical cytogenetic testing, we identified a family in which two brothers had ASD, mild intellectual disability and a chromosome 22 pericentric inversion, not detected in either parent, indicating de novo mutation with parental germinal mosaicism. We hypothesised that the rearrangement was causative of their ASD and localised the chromosome 22 breakpoints., Methods: The rearrangement was characterised using fluorescence in situ hybridisation, Southern blotting, inverse PCR and dideoxy-sequencing. Open reading frames and intron/exon boundaries of the two physically disrupted genes identified, TCF20 and TNRC6B, were sequenced in 342 families (260 multiplex and 82 simplex) ascertained by the International Molecular Genetic Study of Autism Consortium (IMGSAC)., Results: IMGSAC family screening identified a de novo missense mutation of TCF20 in a single case and significant association of a different missense mutation of TCF20 with ASD in three further families. Through exome sequencing in another project, we independently identified a de novo frameshifting mutation of TCF20 in a woman with ASD and moderate intellectual disability. We did not identify a significant association of TNRC6B mutations with ASD., Conclusions: TCF20 encodes a transcriptional coregulator (also termed SPBP) that is structurally and functionally related to RAI1, the critical dosage-sensitive protein implicated in the behavioural phenotypes of the Smith-Magenis and Potocki-Lupski 17p11.2 deletion/duplication syndromes, in which ASD is frequently diagnosed. This study provides the first evidence that mutations in TCF20 are also associated with ASD., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)
- Published
- 2014
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24. Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I.
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Babbs C, Roberts NA, Sanchez-Pulido L, McGowan SJ, Ahmed MR, Brown JM, Sabry MA, Bentley DR, McVean GA, Donnelly P, Gileadi O, Ponting CP, Higgs DR, and Buckle VJ
- Subjects
- Endonucleases chemistry, Endonucleases genetics, Female, Glycoproteins chemistry, Humans, Male, Nuclear Proteins, Pedigree, Predictive Value of Tests, Protein Structure, Secondary, Protein Structure, Tertiary, Anemia, Dyserythropoietic, Congenital diagnosis, Anemia, Dyserythropoietic, Congenital genetics, Glycoproteins genetics, Homozygote, Mutation, Missense genetics
- Abstract
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.
- Published
- 2013
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25. A biophysical model for transcription factories.
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Canals-Hamann AZ, das Neves RP, Reittie JE, Iñiguez C, Soneji S, Enver T, Buckle VJ, and Iborra FJ
- Abstract
Summary: Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene. Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active and inactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in the chromatin fibre. The result of this change in flexibility is that chromatin could behave like a multi-block copolymer with repetitions of stiff-flexible (active-inactive chromatin) components. Copolymers with such structure self-organize through spontaneous phase separation into microdomains. Consistent with such model H4K16ac chromatin form foci that associates with nascent transcripts. We propose that transcription factories are the result of the spontaneous concentration of H4K16ac chromatin that are in proximity, mainly in cis.
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- 2013
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26. Intragenic enhancers act as alternative promoters.
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Kowalczyk MS, Hughes JR, Garrick D, Lynch MD, Sharpe JA, Sloane-Stanley JA, McGowan SJ, De Gobbi M, Hosseini M, Vernimmen D, Brown JM, Gray NE, Collavin L, Gibbons RJ, Flint J, Taylor S, Buckle VJ, Milne TA, Wood WG, and Higgs DR
- Subjects
- Animals, Cells, Cultured, Erythroid Cells, Mice, Poly A, RNA chemistry, RNA physiology, RNA Isoforms chemistry, RNA, Messenger chemistry, RNA, Messenger physiology, Transcriptome, Enhancer Elements, Genetic physiology, Gene Expression Regulation, Promoter Regions, Genetic physiology
- Abstract
A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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27. Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1{alpha} localization in erythroblasts.
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Renella R, Roberts NA, Brown JM, De Gobbi M, Bird LE, Hassanali T, Sharpe JA, Sloane-Stanley J, Ferguson DJ, Cordell J, Buckle VJ, Higgs DR, and Wood WG
- Subjects
- Animals, Carrier Proteins genetics, Cell Line, Tumor, Cells, Cultured, Chromatin pathology, Chromobox Protein Homolog 5, Erythroblasts metabolism, Female, Glycoproteins analysis, Humans, Male, Mice, Mice, Inbred C57BL, Nuclear Proteins, Vesicular Transport Proteins analysis, Anemia, Dyserythropoietic, Congenital genetics, Anemia, Dyserythropoietic, Congenital pathology, Chromosomal Proteins, Non-Histone analysis, Erythroblasts pathology, Glycoproteins genetics, Mutation
- Abstract
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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- 2011
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28. Global gene expression analysis of human erythroid progenitors.
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Merryweather-Clarke AT, Atzberger A, Soneji S, Gray N, Clark K, Waugh C, McGowan SJ, Taylor S, Nandi AK, Wood WG, Roberts DJ, Higgs DR, Buckle VJ, and Robson KJ
- Subjects
- Cell Differentiation genetics, Cells, Cultured, Cluster Analysis, Erythroblasts metabolism, Erythroblasts physiology, Erythroid Precursor Cells chemistry, Erythropoiesis physiology, Flow Cytometry, Gene Expression Regulation, Developmental, Humans, Polymerase Chain Reaction, Erythroid Precursor Cells metabolism, Erythroid Precursor Cells physiology, Erythropoiesis genetics, Gene Expression Profiling methods, Microarray Analysis methods
- Abstract
Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html. [corrected].
- Published
- 2011
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29. Detection of nascent RNA transcripts by fluorescence in situ hybridization.
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Brown JM and Buckle VJ
- Subjects
- Animals, Blood Cells cytology, Blood Cells metabolism, Cell Culture Techniques, Cell Separation, DNA metabolism, Humans, Oligonucleotide Probes metabolism, RNA, Messenger metabolism, In Situ Hybridization, Fluorescence methods
- Abstract
The development of cellular diversity within any organism depends on the timely and correct expression of differing subsets of genes within each tissue type. Many techniques exist which allow a global, average analysis of RNA expression; however, RNA-FISH permits the sensitive detection of specific transcripts within individual cells while preserving the cellular morphology. The technique can provide insight into the spatial and temporal organization of gene transcription as well the relationship of gene expression and mature RNA distribution to nuclear and cellular compartments. It can also reveal the intercellular variation of gene expression within a given tissue. Here, we describe RNA-FISH methodologies that allow the detection of nascent transcripts within the cell nucleus as well as protocols that allow the detection of RNA alongside DNA or proteins. Such techniques allow the placing of gene transcription within a functional context of the whole cell.
- Published
- 2010
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30. Association between active genes occurs at nuclear speckles and is modulated by chromatin environment.
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Brown JM, Green J, das Neves RP, Wallace HA, Smith AJ, Hughes J, Gray N, Taylor S, Wood WG, Higgs DR, Iborra FJ, and Buckle VJ
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- Animals, Anion Exchange Protein 1, Erythrocyte genetics, Anion Exchange Protein 1, Erythrocyte metabolism, Cell Nucleus metabolism, Gene Expression Regulation, Globins genetics, Humans, In Situ Hybridization, Fluorescence, Mice, Multigene Family, Blood Cells physiology, Chromatin metabolism, Chromosomes metabolism, Erythropoiesis genetics, Intranuclear Inclusion Bodies metabolism, Transcription, Genetic
- Abstract
Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse alpha-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human alpha-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.
- Published
- 2008
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31. A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter.
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De Gobbi M, Viprakasit V, Hughes JR, Fisher C, Buckle VJ, Ayyub H, Gibbons RJ, Vernimmen D, Yoshinaga Y, de Jong P, Cheng JF, Rubin EM, Wood WG, Bowden D, and Higgs DR
- Subjects
- Binding Sites, Cells, Cultured, Chromatin Immunoprecipitation, Erythroblasts, GATA1 Transcription Factor metabolism, Gene Expression, Gene Expression Profiling, Haplotypes, Humans, Melanesia, Minisatellite Repeats, Multigene Family, Oligonucleotide Array Sequence Analysis, Regulatory Elements, Transcriptional, Transcription, Genetic, Chromosomes, Human, Pair 16 genetics, Globins genetics, Polymorphism, Single Nucleotide, Promoter Regions, Genetic, alpha-Thalassemia genetics
- Abstract
We describe a pathogenetic mechanism underlying a variant form of the inherited blood disorder alpha thalassemia. Association studies of affected individuals from Melanesia localized the disease trait to the telomeric region of human chromosome 16, which includes the alpha-globin gene cluster, but no molecular defects were detected by conventional approaches. After resequencing and using a combination of chromatin immunoprecipitation and expression analysis on a tiled oligonucleotide array, we identified a gain-of-function regulatory single-nucleotide polymorphism (rSNP) in a nongenic region between the alpha-globin genes and their upstream regulatory elements. The rSNP creates a new promoterlike element that interferes with normal activation of all downstream alpha-like globin genes. Thus, our work illustrates a strategy for distinguishing between neutral and functionally important rSNPs, and it also identifies a pathogenetic mechanism that could potentially underlie other genetic diseases.
- Published
- 2006
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32. Coregulated human globin genes are frequently in spatial proximity when active.
- Author
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Brown JM, Leach J, Reittie JE, Atzberger A, Lee-Prudhoe J, Wood WG, Higgs DR, Iborra FJ, and Buckle VJ
- Subjects
- Animals, Cell Nucleus metabolism, Cell Separation, Cells, Cultured, Chromosomes, Erythroblasts cytology, Erythroblasts physiology, Globins metabolism, Humans, In Situ Hybridization, Fluorescence, Mice, Transcription, Genetic, Gene Expression Regulation, Globins genetics
- Abstract
The organization of genes within the nucleus may influence transcription. We have analyzed the nuclear positioning of the coordinately regulated alpha- and beta-globin genes and show that the gene-dense chromatin surrounding the human alpha-globin genes is frequently decondensed, independent of transcription. Against this background, we show the frequent juxtaposition of active alpha- and beta-globin genes and of homologous alpha-globin loci that occurs at nuclear speckles and correlates with transcription. However, we did not see increased colocalization of signals, which would be expected with direct physical interaction. The same degree of proximity does not occur between human beta-globin genes or between murine globin genes, which are more constrained to their chromosome territories. Our findings suggest that the distribution of globin genes within erythroblast nuclei is the result of a self-organizing process, involving transcriptional status, diffusional ability of chromatin, and physical interactions with nuclear proteins, rather than a directed form of higher-order control.
- Published
- 2006
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33. Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells.
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Brown KE, Amoils S, Horn JM, Buckle VJ, Higgs DR, Merkenschlager M, and Fisher AG
- Subjects
- Cells, Cultured, Gene Expression, Humans, Lymphocytes physiology, Cell Nucleus physiology, Globins genetics, Hematopoietic Stem Cells physiology
- Abstract
The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.
- Published
- 2001
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34. Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.
- Author
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Horsley SW, Daniels RJ, Anguita E, Raynham HA, Peden JF, Villegas A, Vickers MA, Green S, Waye JS, Chui DH, Ayyub H, MacCarthy AB, Buckle VJ, Gibbons RJ, Kearney L, and Higgs DR
- Subjects
- Adolescent, Adult, Base Sequence, Child, Child, Preschool, Female, Humans, In Situ Hybridization, Fluorescence, Male, Middle Aged, Molecular Sequence Data, Phenotype, Sequence Deletion, Sequence Homology, Nucleic Acid, Chromosomes, Human, Pair 16, Monosomy, Telomere
- Abstract
We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.
- Published
- 2001
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35. Sequence, structure and pathology of the fully annotated terminal 2 Mb of the short arm of human chromosome 16.
- Author
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Daniels RJ, Peden JF, Lloyd C, Horsley SW, Clark K, Tufarelli C, Kearney L, Buckle VJ, Doggett NA, Flint J, and Higgs DR
- Subjects
- Adolescent, Animals, Asthma genetics, Base Composition, Bipolar Disorder genetics, Child, Child, Preschool, CpG Islands genetics, Epilepsy genetics, Female, Genetic Linkage genetics, Humans, Intellectual Disability genetics, Male, Mice, Monosomy, Phenotype, Polycystic Kidney, Autosomal Dominant genetics, Recombination, Genetic, Sequence Analysis, DNA, Syndrome, Telomere chemistry, Telomere genetics, Tuberous Sclerosis genetics, alpha-Thalassemia genetics, Chromosomes, Human, Pair 16 chemistry, Chromosomes, Human, Pair 16 genetics, Physical Chromosome Mapping
- Abstract
We have sequenced 1949 kb from the terminal Giemsa light band of human chromosome 16p, enabling us to fully annotate the region extending from the telomeric repeats to the previously published tuberous sclerosis disease 2 (TSC2) and polycystic kidney disease 1 (PKD1) genes. This region can be subdivided into two GC-rich, Alu-rich domains and one GC-rich, Alu-poor domain. The entire region is extremely gene rich, containing 100 confirmed genes and 20 predicted genes. Many of the genes encode widely expressed proteins orchestrating basic cellular processes (e.g. DNA recombination, repair, transcription, RNA processing, signal transduction, intracellular signalling and mRNA translation). Others, such as the alpha globin genes (HBA1 and HBA2), PDIP and BAIAP3, are specialized tissue-restricted genes. Some of the genes have been previously implicated in the pathophysiology of important human genetic diseases (e.g. asthma, cataracts and the ATR-16 syndrome). Others are known disease genes for alpha thalassaemia, adult polycystic kidney disease and tuberous sclerosis. There is also linkage evidence for bipolar affective disorder, epilepsy and autism in this region. Sixty-three chromosomal deletions reported here and elsewhere allow us to interpret the results of removing progressively larger numbers of genes from this well defined human telomeric region.
- Published
- 2001
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36. Relationship between novel isoforms, functionally important domains, and subcellular distribution of CD164/endolyn.
- Author
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Chan JY, Lee-Prudhoe JE, Jorgensen B, Ihrke G, Doyonnas R, Zannettino AC, Buckle VJ, Ward CJ, Simmons PJ, and Watt SM
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, CD146 Antigen, Cell Line, Chromosome Mapping, DNA, Complementary, Endolyn, Humans, Molecular Sequence Data, Polymerase Chain Reaction, RNA Splicing, RNA, Messenger genetics, Receptors, Cell Surface chemistry, Receptors, Cell Surface genetics, Structure-Activity Relationship, Antigens, CD, Membrane Glycoproteins, Neural Cell Adhesion Molecules, Protein Isoforms metabolism, Receptors, Cell Surface metabolism, Subcellular Fractions metabolism
- Abstract
Functional analyses have indicated that the human CD164 sialomucin may play a key role in hematopoiesis by facilitating the adhesion of human CD34(+) cells to the stroma and by negatively regulating CD34(+)CD38(lo/-) cell proliferation. We have identified three novel human CD164 variants derived by alternative splicing of bona fide exons from a single genomic transcription unit. The predominant CD164(E1-6) isoform, encoded by six exons, is a type I transmembrane protein containing two extracellular mucin domains (I and II) interrupted by a cysteine-rich non-mucin domain. The 103B2/9E10 and 105A5 epitopes, which specify ligand binding characteristics, are located on the exon 1-encoded mucin domain I. Three human CD164(E1-6) mRNA species, exhibiting differential polyadenylation site usage, are differentially expressed in hematopoietic and non-hematopoietic tissues. This study provides additional evidence that human CD164(E1-6) represents the ortholog of murine MGC-24v and rat endolyn. Comparative analysis of murine MGC-24v/CD164(E1-6) with human CD164(E1-6) revealed two potential splice variants and a similar genomic structure. Whereas the human CD164 gene is located on chromosome 6q21, the mouse gene occurs in a syntenic region on chromosome 10B1-B2. By confocal microscopy, human CD164 in CD34(+)CD38(+) hematopoietic progenitor (KG1B) and epithelial cell lines appears to be localized primarily in endosomes and lysosomes, with low concentrations at the cell surface. However, in a minority of KG1B cells, CD164 is more prominently expressed at the plasma membrane and in the recycling endosomes, suggesting that its distribution is regulated in cells of hematopoietic origin.
- Published
- 2001
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37. Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes.
- Author
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McDowell TL, Gibbons RJ, Sutherland H, O'Rourke DM, Bickmore WA, Pombo A, Turley H, Gatter K, Picketts DJ, Buckle VJ, Chapman L, Rhodes D, and Higgs DR
- Subjects
- Animals, Antibodies, Monoclonal immunology, COS Cells, Cell Fractionation, Cell Line, Transformed, Chromobox Protein Homolog 5, Chromosomal Proteins, Non-Histone metabolism, Chromosomes chemistry, DNA-Binding Proteins genetics, DNA-Binding Proteins immunology, HeLa Cells, Humans, Mice, Mice, Inbred BALB C, Nuclear Proteins genetics, Nuclear Proteins immunology, Sheep, Transcription Factors genetics, Transcription Factors immunology, X-linked Nuclear Protein, Centromere chemistry, DNA Helicases, DNA-Binding Proteins analysis, Heterochromatin chemistry, Nuclear Proteins analysis, Transcription Factors analysis
- Abstract
ATRX is a member of the SNF2 family of helicase/ATPases that is thought to regulate gene expression via an effect on chromatin structure and/or function. Mutations in the hATRX gene cause severe syndromal mental retardation associated with alpha-thalassemia. Using indirect immunofluorescence and confocal microscopy we have shown that ATRX protein is associated with pericentromeric heterochromatin during interphase and mitosis. By coimmunofluorescence, ATRX localizes with a mouse homologue of the Drosophila heterochromatic protein HP1 in vivo, consistent with a previous two-hybrid screen identifying this interaction. From the analysis of a trap assay for nuclear proteins, we have shown that the localization of ATRX to heterochromatin is encoded by its N-terminal region, which contains a conserved plant homeodomain-like finger and a coiled-coil domain. In addition to its association with heterochromatin, at metaphase ATRX clearly binds to the short arms of human acrocentric chromosomes, where the arrays of ribosomal DNA are located. The unexpected association of a putative transcriptional regulator with highly repetitive DNA provides a potential explanation for the variability in phenotype of patients with identical mutations in the ATRX gene.
- Published
- 1999
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38. Chromosomal stabilisation by a subtelomeric rearrangement involving two closely related Alu elements.
- Author
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Flint J, Rochette J, Craddock CF, Dodé C, Vignes B, Horsley SW, Kearney L, Buckle VJ, Ayyub H, and Higgs DR
- Subjects
- Adolescent, Adult, Alleles, Base Sequence, Chromosome Mapping, Chromosomes, Human, Pair 16 ultrastructure, DNA genetics, DNA Primers genetics, Female, Gene Rearrangement, Genotype, Globins genetics, Haplotypes, Humans, In Situ Hybridization, Fluorescence, Male, Middle Aged, Molecular Sequence Data, Multigene Family, Polymerase Chain Reaction, Recombination, Genetic, Sequence Homology, Nucleic Acid, Telomere genetics, Telomere ultrastructure, Chromosomes, Human, Pair 16 genetics, Repetitive Sequences, Nucleic Acid, alpha-Thalassemia genetics
- Abstract
We have characterised a subtelomeric rearrangement involving the short arm of chromosome 16 that gives rise to alpha-thalassaemia by deleting the major, remote regulatory element controlling alpha-globin expression. The chromosomal breakpoint lies in an Alu family repeat located only approximately 105 kb from the 16p subtelomeric region. The broken chromosome has been stabilised with a newly positioned telomere acquired by recombination between this 16p Alu element and a closely related subtelomeric Alu element of the Sx subfamily. It seems most likely that this abnormal chromosome has been rescued by the mechanism of telomere capture which may reflect a more general process by which subtelomeric sequences are normally dispersed between chromosomal ends.
- Published
- 1996
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39. Radiation-induced genomic instability: delayed cytogenetic aberrations and apoptosis in primary human bone marrow cells.
- Author
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Kadhim MA, Lorimore SA, Townsend KM, Goodhead DT, Buckle VJ, and Wright EG
- Subjects
- Alpha Particles, Bone Marrow Cells, Cells, Cultured, Chromatids radiation effects, Clone Cells, Colony-Forming Units Assay, Hematopoietic Stem Cells radiation effects, Hematopoietic Stem Cells ultrastructure, Humans, X-Rays, Apoptosis radiation effects, Bone Marrow radiation effects, Chromosome Aberrations
- Abstract
Transmissible chromosomal instability, characterized by non-clonal cytogenetic aberrations with a high frequency of chromatid-type aberrations together with a lower frequency of chromosome-type aberrations, has been demonstrated in the clonal descendants of human haemopoietic stem cells after alpha- but not X-irradiation. Comparable cytogenetic abnormalities have also been demonstrated in non-clonal cultures of alpha-irradiated primary human bone marrow, but a different pattern of delayed aberrations, mainly of chromosome-type, was found after X-irradiation in non-clonal cultures. In clonal analyses, delayed apoptotic cell death was evident after both X- and alpha-irradiation. It is suggested that the type of radiation exposure, the type of cell and its genetically determined susceptibility are factors that may influence the expression of delayed effects of radiation.
- Published
- 1995
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40. The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation.
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Flint J, Wilkie AO, Buckle VJ, Winter RM, Holland AJ, and McDermid HE
- Subjects
- Adult, Child, Child, Preschool, Chromosome Aberrations diagnosis, Chromosome Aberrations epidemiology, Chromosome Disorders, Chromosomes, Human, Pair 13, Chromosomes, Human, Pair 22, Female, Gene Deletion, Gene Rearrangement, Humans, Intellectual Disability diagnosis, Karyotyping, Male, Prevalence, Telomere physiology, Intellectual Disability etiology, Intellectual Disability genetics, Telomere genetics
- Abstract
A major challenge for human genetics is to identify new causes of mental retardation, which, although present in about 3% of individuals, is unexplained in more than half of all cases. We have developed a strategy to screen for the abnormal inheritance of subtelomeric DNA polymorphisms in individuals with mental retardation and have detected three abnormalities in 99 patients with normal routine karyotypes. Pulsed-field gel electrophoresis and reverse chromosome painting showed that one case arose from an interstitial or terminal deletion and two from the de novo inheritance of derivative translocation chromosomes. At least 6% of unexplained mental retardation is accounted for by these relatively small chromosomal abnormalities, which will be an important resource in the characterization of the genetic basis of neurodevelopment.
- Published
- 1995
- Full Text
- View/download PDF
41. Clinical and hematologic aspects of the X-linked alpha-thalassemia/mental retardation syndrome (ATR-X).
- Author
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Gibbons RJ, Brueton L, Buckle VJ, Burn J, Clayton-Smith J, Davison BC, Gardner RJ, Homfray T, Kearney L, and Kingston HM
- Subjects
- Abnormalities, Multiple genetics, Adolescent, Child, Child, Preschool, Erythrocyte Volume, Female, Genetic Linkage, Hemoglobin H analysis, Heterozygote, Humans, Infant, Male, Pedigree, Phenotype, Syndrome, alpha-Thalassemia genetics, alpha-Thalassemia pathology, Intellectual Disability genetics, X Chromosome, alpha-Thalassemia blood
- Abstract
The hallmarks of the X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome are severe psychomotor retardation, minor facial anomalies, genital abnormalities, and an unusual form of alpha-thalassemia. The demonstration of HbH inclusions in red blood cells after incubation with brilliant cresyl blue confirms the diagnosis. We describe 15 previously unreported cases and analyse the phenotypic and hematologic findings in these subjects and compare them with previously published cases. This study demonstrates the consistency of the main characteristics of this syndrome and extends the phenotype. Developmental changes in phenotype, in particular the coarsening of the facial appearance, are illustrated. The hematologic findings are shown to vary widely; in some cases the manifestation of alpha-thalassemia may be subtle and missed without repeated examination.
- Published
- 1995
- Full Text
- View/download PDF
42. Alpha-particle-induced chromosomal instability in human bone marrow cells.
- Author
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Kadhim MA, Lorimore SA, Hepburn MD, Goodhead DT, Buckle VJ, and Wright EG
- Subjects
- Animals, Bone Marrow ultrastructure, Hematopoietic Stem Cells radiation effects, Hematopoietic Stem Cells ultrastructure, Humans, In Vitro Techniques, Leukemia, Radiation-Induced etiology, Leukemia, Radiation-Induced genetics, Mice, Alpha Particles, Bone Marrow radiation effects, Chromosome Aberrations, Linear Energy Transfer
- Abstract
alpha-particles, which are ionising radiation of high linear-energy-transfer emitted, for example, from radon or plutonium, pass through tissue as highly structured tracks. Single target cells in the path of the tracks might be damaged by even low-dose alpha-irradiation. We found non-clonal cytogenetic aberrations, characterised by a high frequency of chromatid aberrations with chromosome aberrations, in clonal descendants of haemopoietic stem cells after exposure to alpha-particles of bone marrow cells from two of four haematologically normal individuals (up to 25% abnormal metaphases). The data are consistent with a transmissible genetic instability induced in a stem cell resulting in a diversity of aberrations in its clonal progeny many cell divisions later.
- Published
- 1994
- Full Text
- View/download PDF
43. A large duplicated area in the polycystic kidney disease 1 (PKD1) region of chromosome 16 is prone to rearrangement.
- Author
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Harris PC, Thomas S, MacCarthy AB, Stallings RL, Breuning MH, Jenne DE, Fink TM, Buckle VJ, Ratcliffe PJ, and Ward CJ
- Subjects
- Chromosome Mapping, Cloning, Molecular, Cosmids, DNA genetics, Electrophoresis, Gel, Pulsed-Field, Female, Genetic Markers, Humans, In Situ Hybridization, Fluorescence, Male, Pedigree, Polymorphism, Genetic, Recombination, Genetic, Chromosomes, Human, Pair 16, Gene Rearrangement, Multigene Family, Polycystic Kidney, Autosomal Dominant genetics
- Abstract
An area of 500 kb at the proximal end of the polycystic kidney disease 1 (PKD1) region has been mapped in detail, with 260 kb cloned in cosmids. The area cloned from normal individuals contains two homologous but divergent regions each of 75 kb, including the previously described marker 26-6. Pulsed-field gel electrophoresis identified a duplication of 75 kb of this region, referred to as the OX duplication (OXdup), in three patients with PKD1. The OXdup probably arose by an unequal exchange promoted by misalignment of partially homologous areas. Study of the OXdup in a large PKD1 family showed that it segregated with PKD1 in just one-half of the family, indicating that a recent crossover had occurred between the OXdup and PKD1 and showing that it was not a PKD1 mutation. Further analysis identified an OXdup breakpoint fragment: the OXdup was subsequently identified in 2 normal individuals of 110 assayed. The finding of the OXdup and in other individuals an 11-kb deletion (OXdel) at a similar point within this duplicated area indicates that this is an unusually unstable genomic region.
- Published
- 1994
- Full Text
- View/download PDF
44. Dissecting the centromere of the human Y chromosome with cloned telomeric DNA.
- Author
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Brown KE, Barnett MA, Burgtorf C, Shaw P, Buckle VJ, and Brown WR
- Subjects
- Cells, Cultured, Humans, Restriction Mapping, Telomere genetics, Centromere genetics, Cloning, Molecular, DNA genetics, Y Chromosome
- Abstract
We have used telomeric DNA to break the human Y chromosome within the centromeric array of alphoid satellite DNA and have created two derivative chromosomes; one consists of the short arm and 140 kb of alphoid DNA, the other consists of the long arm and 480 kb of alphoid DNA. Both segregate accurately at mitosis. It is known that there is no large scale sequence duplication around the alphoid DNA and so the simplest interpretation of our results is that the sequence responsible for accurate segregation is the alphoid DNA itself. Although the long arm acrocentric derivative segregates accurately it lags with respect to the other chromosomes in about 10% of anaphase cells and thus additional sequences may be required for orderly segregation. The short arm acrocentric chromosome is probably no larger than 12 Mb in size and thus our results also demonstrate that chromosomes of this size are capable of accurate segregation.
- Published
- 1994
- Full Text
- View/download PDF
45. New methods in cytogenetics.
- Author
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Buckle VJ and Kearney L
- Subjects
- Animals, Chromosome Aberrations, DNA Primers, DNA Replication, Female, Genome, Humans, In Situ Hybridization, Fluorescence, Interphase genetics, Meiosis genetics, Molecular Probes, Nucleic Acid Hybridization, Pregnancy, Prenatal Diagnosis, Cytogenetics methods
- Abstract
Developments in the technique of fluorescence in situ hybridization (FISH) now permit hybridization of sequences ranging from 1 kb to whole genomes. The technique can be used in applications from coarse mapping of whole chromosomes to high-resolution analysis of extended strands of DNA. The complexity, and hence the coverage, of 'paints' prepared by amplification is being improved to the extent that such methods are used in cloning strategies for the generation of region-specific probes. Interphase analysis and comparative genomic hybridization are becoming important tools in cancer cytogenetics, and the potential for routine analysis of fetal cells obtained from maternal blood may provide a fresh approach to prenatal cytogenetic screening. Functional studies of gene activity and nuclear organization are now also possible.
- Published
- 1994
- Full Text
- View/download PDF
46. Molecular mapping of uncharacteristically small 5q deletions in two patients with the 5q- syndrome: delineation of the critical region on 5q and identification of a 5q- breakpoint.
- Author
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Boultwood J, Fidler C, Lewis S, Kelly S, Sheridan H, Littlewood TJ, Buckle VJ, and Wainscoat JS
- Subjects
- Adult, Alleles, Chromosome Mapping, Electrophoresis, Gel, Pulsed-Field, Female, Genes, Tumor Suppressor, Genetic Markers, Granulocytes ultrastructure, Humans, Lymphocytes ultrastructure, Middle Aged, Syndrome, Anemia, Refractory genetics, Chromosome Deletion, Chromosomes, Human, Pair 5 ultrastructure
- Abstract
Molecular mapping techniques have defined the region of gene loss in two patients with the 5q- syndrome and uncharacteristically small 5q deletions (5q31-q33). The allelic loss of 10 genes localized to 5q23-qter (centromere-CSF2-EGR1-FGFA-GRL-ADRB2-CS F1R-SPARC-GLUH1-NKSF1-FLT4-telomere) was investigated in peripheral blood cell fractions. Gene dosage experiments demonstrated that CSF2, EGR1, NKSF1, and FLT4 were retained on the 5q- chromosome in both patients and that FGFA was retained in one patient, thus placing these genes outside the critical region. GRL, ADRB2, CSF1R, SPARC, and GLUH1 were shown to be deleted in both patients. The proximal breakpoint is localized between EGR1 and FGFA in one patient and between FGFA and ADRB2 in the other, and the distal breakpoint is localized between GLUH1 and NKSF1 in both patients. Pulsed-field gel electrophoresis was used to map the 5q deletion breakpoints, and breakpoint-specific fragments were detected with FGFA in the granulocyte but not the lymphocyte fraction of one patient. This study has established the critical region of gene loss of the 5q- chromosome in the 5q- syndrome, giving the location for a putative tumor-suppressor gene in the 5.6-Mb region between FGFA and NKSF1.
- Published
- 1994
- Full Text
- View/download PDF
47. Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA).
- Author
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Thomas NS, Chelly J, Zonana J, Davies KJ, Morgan S, Gault J, Rack KA, Buckle VJ, Brockdorff N, and Clarke A
- Subjects
- Cell Line, Chromosome Mapping, Chromosomes, Artificial, Yeast, Chromosomes, Human, Pair 9 ultrastructure, Cosmids, DNA Mutational Analysis, Female, Humans, Hybrid Cells, In Situ Hybridization, Fluorescence, Male, Sequence Deletion, Translocation, Genetic, Ectodermal Dysplasia genetics, X Chromosome ultrastructure
- Abstract
A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region. One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
- Full Text
- View/download PDF
48. Untwirling dirvish.
- Author
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Buckle VJ and Kearney L
- Subjects
- Animals, Chromatin ultrastructure, Chromosome Mapping, Chromosomes ultrastructure, Interphase, Rhodamines, Sensitivity and Specificity, In Situ Hybridization, Fluorescence methods
- Published
- 1993
- Full Text
- View/download PDF
49. The identification of a third fragile site, FRAXF, in Xq27--q28 distal to both FRAXA and FRAXE.
- Author
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Hirst MC, Barnicoat A, Flynn G, Wang Q, Daker M, Buckle VJ, Davies KE, and Bobrow M
- Subjects
- Adolescent, Adult, Chromatin ultrastructure, Chromosome Fragile Sites, Female, Genetic Markers, Humans, In Situ Hybridization, Fluorescence, Infant, Male, Pedigree, Chromosome Fragility, Fragile X Syndrome genetics, X Chromosome ultrastructure
- Abstract
FRAXA is unique amongst fragile sites in that it is intimately involved with a specific clinical phenotype, the fragile X syndrome. Whilst the majority of fragile X individuals have been found to have a characteristic mutation in the FMR1 gene, a small proportion of individuals exhibiting fragility have no such mutation. Investigation of the site of chromosome fragility in these FMR1 mutation negative, fragile X site positive individuals, has identified a second site of fragility, FRAXE. However, the presence of FRAXE has not explained all such cases. Here we describe a fragile X site positive, FMR1 mutation negative family, in which chromosome fragility is not due to the FRAXA or FRAXE but is due to a third site designated FRAXF. Using fluorescent in situ hybridisation (FISH) this site is shown to lie over 1Mb distal to FRAXA. The identification of a third fragile site in this small region of the X chromosome provides an opportunity to extend our studies of the molecular nature of chromosome fragility.
- Published
- 1993
- Full Text
- View/download PDF
50. Comparison of cytogenetic and restriction fragment length polymorphism analyses for the detection of loss of chromosome material in clonal hemopoietic disorders.
- Author
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Abrahamson GM, Rack K, Oscier DG, Fitchett M, Buckle VJ, and Wainscoat JS
- Subjects
- Aged, Chromosomes, Human, Pair 7, DNA genetics, Female, Humans, Male, Middle Aged, Chromosome Deletion, Cytogenetics methods, Leukemia, Myeloid, Acute genetics, Myelodysplastic Syndromes genetics, Polymorphism, Restriction Fragment Length
- Abstract
Loss of chromosome material, as manifested by monosomy or partial deletion, is commonly found in neoplastic cells. We have undertaken a systematic comparison of standard cytogenetic analysis and molecular analysis for the detection of such loss, using as a model loss of chromosome 7 in 72 patients with a clonal myeloid malignancy. A large number of probes was used to screen three regions of chromosome 7 for loss by restriction fragment length polymorphism (RFLP) analysis. There were nine cases in which loss of chromosome 7 was detected by both techniques, but seven in which loss was detected by only one of the methods, demonstrating the complementary nature of these two techniques.
- Published
- 1993
- Full Text
- View/download PDF
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