Prof Veronica J Buckle

Research Area: Cell and Molecular Biology
Technology Exchange: Chromosome mapping, In situ hybridisation and Microscopy (Confocal)
Scientific Themes: Molecular, Cell & Systems Biology and Genes, Genetics, Epigenetics & Genomics
Keywords: alpha globin, erythropoiesis, chromatin, nuclear organisation and transcription
Web Links:
Erythroblast nuclei showing positioning of alpha globin (red), beta globin (white), SLC4A1 (green) and ERAF (blue) genes. Erythroid genes associate much more frequently when active and the association occurs at nuclear speckles.

Erythroblast nuclei showing positioning of alpha globin (red), beta globin (white), SLC4A1 (green) ...

Nascent RNA transcripts of α globin and RNA polymerase II protein in an erythroblast nucleus captured by super resolution imaging using structured illumination microscopy (3D-SIM) (Micron Advanced BioImaging Unit). Enlarged regions shown for comparison of standard widefield, deconvolved and super-resolved images, with and without DAPI counterstain.

Nascent RNA transcripts of α globin and RNA polymerase II protein in an erythroblast nucleus ...

Electron micrographs of erythroblast nuclei from a normal individual (left) and CDA-I patient (R), showing gross abnormalities of chromatin assembly in the dark-staining areas of the patient nuclei.

Electron micrographs of erythroblast nuclei from a normal individual (left) and CDA-I patient (R), ...

Our studies are directed at understanding the relevance of nuclear organisation to gene expression during erythropoiesis. Our ultimate aim is to gain better insight into transcriptional regulation and to contribute to an understanding of how normal transcription may be restored in disease. Erythroid cells differentiate over the course of a few days from committed blast-forming cells, through the pronormoblast stage where they are highly proliferative and begin to produce large quantities of haemoglobin, to a condensed pyknotic state when nuclei are finally extruded from the cells. During this process, erythroid-specific genes are switched on and transcribe heavily before being shut down, which makes this process an ideal model for following gene regulation.

The nucleus is a highly ordered but plastic body of chromatin and nuclear substructures. Chromatin organisation within the nucleus has a key role to play in the processes of transcription and replication but the mechanisms governing how chromatin domains are established and altered during the course of development and differentiation remain poorly understood. Many genes, particularly those expressed in a tissue-specific manner, are regulated by enhancer elements that can lie at some distance from the target gene. There is a broad consensus that enhancer elements exert their effect on gene promoters by close interaction but many questions remain about what may drive such formations, how long they are stable for and how close is close ie are molecular interactions required or is a diffusible compartment established, enriched in factors required for upregulated transcription?

We are focusing on the organisation of chromatin around the α-globin genes, looking at what changes in decondensation and interactions are necessary for transcription, by imaging the positioning of DNA and RNA probes within single cells, both normal and from a panel of knockouts at regulatory sites across the region. Such analysis is an essential complement to the whole cell population studies of chromatin configurations undertaken within the Unit.To understand the dynamics of spatial organisation at the α-globin gene regulatory region during commitment, differentiation and transcription, we are developing systems in which we can visualise looping and transcription in real time in live cells.

In addition to these fundamental aspects of gene regulation we continue to characterise disorders of red blood cells. Having identified a second gene underlying congenital dyserythropoietic anaemia type 1 (CDA-I) we are undertaking functional studies to investigate the role that the two CDA-I genes, CDAN1 and c15orf41, play in normal erythropoiesis, and why mutations in either of these genes cause such devastating disruption to chromatin structure in differentiating erythroblasts.

Name Department Institution Country
Prof Doug Higgs FRS Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Jim R Hughes Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Christian Eggeling Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Kerstin Bystricky University of Toulouse France
Dr Lothar Schermelleh Biochemistry Department University of Oxford United Kingdom
Prof Tom Brown Dept. of Chemistry University of Oxford United Kingdom
Taylor JC, Martin HC, Lise S, Broxholme J, Cazier J-B, Rimmer A, Kanapin A, Lunter G, Fiddy S, Allan C et al. 2015. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nat Genet, 47 (7), pp. 717-726. | Show Abstract | Read more

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.

Silva AM, Brown JM, Buckle VJ, Wade-Martins R, Lufino MMP. 2015. Expanded GAA repeats impair FXN gene expression and reposition the FXN locus to the nuclear lamina in single cells. Hum Mol Genet, 24 (12), pp. 3457-3471. | Show Abstract | Read more

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.

Babbs C, Lloyd D, Pagnamenta AT, Twigg SRF, Green J, McGowan SJ, Mirza G, Naples R, Sharma VP, Volpi EV et al. 2014. De novo and rare inherited mutations implicate the transcriptional coregulator TCF20/SPBP in autism spectrum disorder. J Med Genet, 51 (11), pp. 737-747. | Show Abstract | Read more

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.

Babbs C, Roberts NA, Sanchez-Pulido L, McGowan SJ, Ahmed MR, Brown JM, Sabry MA, WGS500 Consortium, Bentley DR, McVean GA et al. 2013. Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I. Haematologica, 98 (9), pp. 1383-1387. | Show Abstract | Read more

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.

Canals-Hamann AZ, das Neves R, Reittie JE, Iñiguez C, Soneji S, Enver T, Buckle VJ, Iborra FJ. 2013. A biophysical model for transcription factories BMC Biophysics, 6 (1), pp. 2-2. | Read more

Lutz C, Woll PS, Hall G, Castor A, Dreau H, Cazzaniga G, Zuna J, Jensen C, Clark SA, Biondi A et al. 2013. Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia. Leukemia, 27 (5), pp. 1204-1207. | Read more

Kowalczyk MS, Hughes JR, Garrick D, Lynch MD, Sharpe JA, Sloane-Stanley JA, McGowan SJ, De Gobbi M, Hosseini M, Vernimmen D et al. 2012. Intragenic enhancers act as alternative promoters. Mol Cell, 45 (4), pp. 447-458. | Show Abstract | Read more

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.

Cited:

71

Scopus

Schödel J, Bardella C, Sciesielski LK, Brown JM, Pugh CW, Buckle V, Tomlinson IP, Ratcliffe PJ, Mole DR. 2012. Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1 expression Nature Genetics, 44 (4), pp. 420-425. | Show Abstract | Read more

Although genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here, we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma, modulates the binding and function of hypoxia-inducible factor (HIF) at a previously unrec ognized transcriptional enhancer of CCND1 (encoding cyclin D1) that is specific for renal cancers characterized by inactivation of the von Hippelg-Lindau tumor suppressor (pVHL). The protective haplotype impairs binding of HIF-2, resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control. © 2012 Nature America, Inc. All rights reserved.

Renella R, Roberts NA, Brown JM, De Gobbi M, Bird LE, Hassanali T, Sharpe JA, Sloane-Stanley J, Ferguson DJP, Cordell J et al. 2011. Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1 alpha localization in erythroblasts BLOOD, 117 (25), pp. 6928-6938. | Show Abstract | Read more

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 byWestern blot.Adetailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genomewide 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. © 2011 by The American Society of Hematology.

Merryweather-Clarke AT, Atzberger A, Soneji S, Gray N, Clark K, Waugh C, McGowan SJ, Taylor S, Nandi AK, Wood WG et al. 2011. Global gene expression analysis of human erythroid progenitors. Blood, 117 (13), pp. e96-108. | Show Abstract | Read more

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].

Brown JM, Buckle VJ. 2010. Detection of nascent RNA transcripts by fluorescence in situ hybridization. Methods Mol Biol, 659 pp. 33-50. | Show Abstract | Read more

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.

Brown JM, Green J, das Neves RP, Wallace HAC, Smith AJH, Hughes J, Gray N, Taylor S, Wood WG, Higgs DR et al. 2008. Association between active genes occurs at nuclear speckles and is modulated by chromatin environment. J Cell Biol, 182 (6), pp. 1083-1097. | Show Abstract | Read more

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.

Hong D, Gupta R, Ancliff P, Atzberger A, Brown J, Soneji S, Green J, Colman S, Piacibello W, Buckle V et al. 2008. Initiating and cancer-propagating cells in TEL-AML1-associated childhood leukemia. Science, 319 (5861), pp. 336-339. | Show Abstract | Read more

Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice. By studying a monochorionic twin pair, one preleukemic and one with frank leukemia, we establish the lineal relationship between these "cancer-propagating" cells and the preleukemic cell in which the TEL-AML1 fusion first arises or has functional impact. Analysis of TEL-AML1-transduced cord blood cells suggests that TEL-AML1 functions as a first-hit mutation by endowing this preleukemic cell with altered self-renewal and survival properties.

Iborra FJ, Buckle V. 2008. Wide confocal cytometry: a new approach to study proteomic and structural changes in the cell nucleus during the cell cycle. Histochem Cell Biol, 129 (1), pp. 45-53. | Show Abstract | Read more

Wide-confocal-cytometry (WCC) is a new method developed in this paper that uses a standard confocal system to gather quantitative information on contents and fine structural details of cells. The system is operated under conditions of non-confocality, in order to capture the maximum amount of light emitted by the specimen (comparable to LSC). After analysis of macromolecule content (DNA, RNA, specific proteins, lipids, etc.), cells can be sampled using conventional confocal microscopy. We analyzed the illumination and acquiring capabilities of WCC. The quantitative power of WCC was validated by analysis of cell cycle stage in Hela cells, looking at DNA content and markers for S phase and mitosis. As an example of the potential of this methodology we have documented changes in cell nucleus during the cell cycle. After mitosis the cell nucleus changes its shape from elongated to ellipsoid and remains constant until G2. This change is associated with nuclear volume increase. As nuclear volume increases, chromatin becomes decondensed in an isometric manner, probably due to the increase in gene expression and factors necessary for RNA metabolism.

Merryweather-Clarke AT, Atzberger A, Shah W, Woffendin HB, Soneji S, Waugh C, Clark K, Brown J, Sloane-Stanley J, Wood WG et al. 2007. Expression profile of healthy erythroid progenitors AMERICAN JOURNAL OF HEMATOLOGY, 82 (6), pp. 571-571.

De Gobbi M, Viprakasit V, Hughes JR, Fisher C, Buckle VJ, Ayyub H, Gibbons RJ, Vernimmen D, Yoshinaga Y, de Jong P et al. 2006. A regulatory SNP causes a human genetic disease by creating a new transcriptional promoter. Science, 312 (5777), pp. 1215-1217. | Show Abstract | Read more

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.

Brown JM, Leach J, Reittie JE, Atzberger A, Lee-Prudhoe J, Wood WG, Higgs DR, Iborra FJ, Buckle VJ. 2006. Coregulated human globin genes are frequently in spatial proximity when active. J Cell Biol, 172 (2), pp. 177-187. | Show Abstract | Read more

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.

Higgs DR, Vernimmen D, De Gobbi M, Anguita E, Hughes J, Buckle V, Iborra F, Garrick D, Wood WG. 2006. How transcriptional and epigenetic programmes are played out on an individual mammalian gene cluster during lineage commitment and differentiation. Biochem Soc Symp, 73 (73), pp. 11-22. | Show Abstract | Read more

In the post-genomic era, a great deal of work has focused on understanding how DNA sequence is used to programme complex nuclear, cellular and tissue functions throughout differentiation and development. There are many approaches to these issues, but we have concentrated on understanding how a single mammalian gene cluster is activated or silenced as stem cells undergo lineage commitment, differentiation and maturation. In particular we have analysed the alpha globin cluster, which is expressed in a cell-type- and developmental stage-specific manner in the haemopoietic system. Our studies include analysis of the transcriptional programme that accompanies globin gene activation, focusing on the expression of relevant transcription factors and cofactors. Binding of these factors to the chromosomal domain containing the alpha globin cluster has been characterized by ChIP (chromatin immunoprecipitation). In addition, we have monitored the epigenetic modifications (e.g. nuclear position, timing of replication, chromatin modification, DNA methylation) that occur as the genes are activated (in erythroid cells) or silenced (e.g. in granulocytes) as haemopoiesis proceeds. Together, these observations provide a uniquely well-characterized model illustrating the mechanisms that regulate and memorize patterns of mammalian gene expression as stem cells undergo lineage specification, differentiation and terminal maturation.

Price TS, Regan R, Mott R, Hedman A, Honey B, Daniels RJ, Smith L, Greenfield A, Tiganescu A, Buckle V et al. 2005. SW-ARRAY: a dynamic programming solution for the identification of copy-number changes in genomic DNA using array comparative genome hybridization data. Nucleic Acids Res, 33 (11), pp. 3455-3464. | Show Abstract | Read more

Comparative genome hybridization (CGH) to DNA microarrays (array CGH) is a technique capable of detecting deletions and duplications in genomes at high resolution. However, array CGH studies of the human genome noting false negative and false positive results using large insert clones as probes have raised important concerns regarding the suitability of this approach for clinical diagnostic applications. Here, we adapt the Smith-Waterman dynamic-programming algorithm to provide a sensitive and robust analytic approach (SW-ARRAY) for detecting copy-number changes in array CGH data. In a blind series of hybridizations to arrays consisting of the entire tiling path for the terminal 2 Mb of human chromosome 16p, the method identified all monosomies between 267 and 1567 kb with a high degree of statistical significance and accurately located the boundaries of deletions in the range 267-1052 kb. The approach is unique in offering both a nonparametric segmentation procedure and a nonparametric test of significance. It is scalable and well-suited to high resolution whole genome array CGH studies that use array probes derived from large insert clones as well as PCR products and oligonucleotides.

Horsley SW, Daniels RJ, Anguita E, Raynham HA, Peden JF, Villegas A, Vickers MA, Green S, Waye JS, Chui DH et al. 2001. Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects. Eur J Hum Genet, 9 (3), pp. 217-225. | Show Abstract | Read more

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.

McDonell N, Ramser J, Francis F, Vinet MC, Rider S, Sudbrak R, Riesselman L, Yaspo ML, Reinhardt R, Monaco AP et al. 2000. Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia. Genomics, 64 (3), pp. 221-229. | Show Abstract | Read more

The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region.

Fakis G, Boukouvala S, Buckle V, Payton M, Denning C, Sim E. 2000. Chromosome mapping of the genes for murine arylamine N-acetyltransferases (NATs), enzymes involved in the metabolism of carcinogens: identification of a novel upstream noncoding exon for murine Nat2. Cytogenet Cell Genet, 90 (1-2), pp. 134-138. | Show Abstract | Read more

Arylamine N-acetyltransferases (NATs) catalyse acetylation reactions which can result in either detoxification or activation of arylamine carcinogens. The human NAT loci (NAT1, NAT2, and a pseudogene, NATP) have been mapped to human chromosome 8p22, a region frequently deleted in tumours. There are three functional genes in mice (Nat1, Nat2, and Nat3) encoding for three NAT isoenzymes. Different alleles at the Nat2 locus are responsible for the acetylation polymorphism identified in different mouse strains. We show that Nat3 is close to Nat1 and Nat2, by screening of a P1 artificial chromosome (PAC) library and provide cytogenetic evidence for co-localisation of the three genes in chromosome region 8 B3.1-B3.3. The Nat region of mouse and human is homologous. We also provide sequence information and a restriction map in the vicinity of Nat1 and Nat2 and describe a noncoding exon located 6 kb upstream of the Nat2 coding region.

Pritchard L, Sloane-Stanley JA, Sharpe JA, Aspinwall R, Lu W, Buckle V, Strmecki L, Walker D, Ward CJ, Alpers CE et al. 2000. A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype. Hum Mol Genet, 9 (18), pp. 2617-2627. | Show Abstract | Read more

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease.

Brackley CA, Brown JM, Waithe D, Babbs C, Davies J, Hughes JR, Buckle VJ, Marenduzzo D. 2016. Predicting the three-dimensional folding of cis-regulatory regions in mammalian genomes using bioinformatic data and polymer models. Genome Biol, 17 (1), pp. 59. | Show Abstract | Read more

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.

Lutz C, Woll PS, Hall G, Castor A, Dreau H, Cazzaniga G, Zuna J, Jensen C, Clark SA, Biondi A et al. 2013. Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia Leukemia, 27 (5), pp. 1204-1207. | Read more

Azuara V, Brown KE, Williams RRE, Webb N, Dillon N, Festenstein R, Buckle V, Merkenschlager M, Fisher AG. 2003. Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication. Nat Cell Biol, 5 (7), pp. 668-674. | Show Abstract | Read more

Temporal control of DNA replication has been implicated in epigenetic regulation of gene expression on the basis of observations that certain tissue-specific genes replicate earlier in expressing than non-expressing cells. Here, we show evidence that several leukocyte-specific genes replicate early in lymphocytes regardless of their transcription and also in fibroblasts, where these genes are never normally expressed. Instead, the heritable silencing of some genes (Rag-1, TdT, CD8alpha and lambda5) and their spatial recruitment to heterochromatin domains within the nucleus of lymphocytes resulted in a markedly delayed resolution of sister chromatids into doublet signals discernable by 3D fluorescence in situ hybridization (FISH). Integration of transgenes within heterochromatin (in cis) did, however, confer late replication and this was reversed after variegated transgene expression. These findings emphasise that chromosomal location is important for defining the replication timing of genes and show that retarded sister-chromatid resolution is a novel feature of inactive chromatin.

Taylor JC, Martin HC, Lise S, Broxholme J, Cazier J-B, Rimmer A, Kanapin A, Lunter G, Fiddy S, Allan C et al. 2015. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nat Genet, 47 (7), pp. 717-726. | Show Abstract | Read more

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.

Silva AM, Brown JM, Buckle VJ, Wade-Martins R, Lufino MMP. 2015. Expanded GAA repeats impair FXN gene expression and reposition the FXN locus to the nuclear lamina in single cells. Hum Mol Genet, 24 (12), pp. 3457-3471. | Show Abstract | Read more

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.

Babbs C, Roberts NA, Sanchez-Pulido L, McGowan SJ, Ahmed MR, Brown JM, Sabry MA, WGS500 Consortium, Bentley DR, McVean GA et al. 2013. Homozygous mutations in a predicted endonuclease are a novel cause of congenital dyserythropoietic anemia type I. Haematologica, 98 (9), pp. 1383-1387. | Show Abstract | Read more

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.

Kowalczyk MS, Hughes JR, Garrick D, Lynch MD, Sharpe JA, Sloane-Stanley JA, McGowan SJ, De Gobbi M, Hosseini M, Vernimmen D et al. 2012. Intragenic enhancers act as alternative promoters. Mol Cell, 45 (4), pp. 447-458. | Show Abstract | Read more

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.

Cited:

71

Scopus

Schödel J, Bardella C, Sciesielski LK, Brown JM, Pugh CW, Buckle V, Tomlinson IP, Ratcliffe PJ, Mole DR. 2012. Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1 expression Nature Genetics, 44 (4), pp. 420-425. | Show Abstract | Read more

Although genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here, we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma, modulates the binding and function of hypoxia-inducible factor (HIF) at a previously unrec ognized transcriptional enhancer of CCND1 (encoding cyclin D1) that is specific for renal cancers characterized by inactivation of the von Hippelg-Lindau tumor suppressor (pVHL). The protective haplotype impairs binding of HIF-2, resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control. © 2012 Nature America, Inc. All rights reserved.

Renella R, Roberts NA, Brown JM, De Gobbi M, Bird LE, Hassanali T, Sharpe JA, Sloane-Stanley J, Ferguson DJP, Cordell J et al. 2011. Codanin-1 mutations in congenital dyserythropoietic anemia type 1 affect HP1 alpha localization in erythroblasts BLOOD, 117 (25), pp. 6928-6938. | Show Abstract | Read more

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 byWestern blot.Adetailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genomewide 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. © 2011 by The American Society of Hematology.

Merryweather-Clarke AT, Atzberger A, Soneji S, Gray N, Clark K, Waugh C, McGowan SJ, Taylor S, Nandi AK, Wood WG et al. 2011. Global gene expression analysis of human erythroid progenitors. Blood, 117 (13), pp. e96-108. | Show Abstract | Read more

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].

Brown JM, Buckle VJ. 2010. Detection of nascent RNA transcripts by fluorescence in situ hybridization. Methods Mol Biol, 659 pp. 33-50. | Show Abstract | Read more

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.

Brown JM, Green J, das Neves RP, Wallace HAC, Smith AJH, Hughes J, Gray N, Taylor S, Wood WG, Higgs DR et al. 2008. Association between active genes occurs at nuclear speckles and is modulated by chromatin environment. J Cell Biol, 182 (6), pp. 1083-1097. | Show Abstract | Read more

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.

Hong D, Gupta R, Ancliff P, Atzberger A, Brown J, Soneji S, Green J, Colman S, Piacibello W, Buckle V et al. 2008. Initiating and cancer-propagating cells in TEL-AML1-associated childhood leukemia. Science, 319 (5861), pp. 336-339. | Show Abstract | Read more

Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice. By studying a monochorionic twin pair, one preleukemic and one with frank leukemia, we establish the lineal relationship between these "cancer-propagating" cells and the preleukemic cell in which the TEL-AML1 fusion first arises or has functional impact. Analysis of TEL-AML1-transduced cord blood cells suggests that TEL-AML1 functions as a first-hit mutation by endowing this preleukemic cell with altered self-renewal and survival properties.

Brown JM, Leach J, Reittie JE, Atzberger A, Lee-Prudhoe J, Wood WG, Higgs DR, Iborra FJ, Buckle VJ. 2006. Coregulated human globin genes are frequently in spatial proximity when active. J Cell Biol, 172 (2), pp. 177-187. | Show Abstract | Read more

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.

Brackley CA, Brown JM, Waithe D, Babbs C, Davies J, Hughes JR, Buckle VJ, Marenduzzo D. 2016. Predicting the three-dimensional folding of cis-regulatory regions in mammalian genomes using bioinformatic data and polymer models. Genome Biol, 17 (1), pp. 59. | Show Abstract | Read more

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.

1519