Veronica J Buckle

University of Oxford | OX · Weatherall Institute of Molecular Medicine

Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin’s stoichiometry in vivo. Using quantitative super-resolu...

Extensive genetic and epigenetic variegation has been demonstrated in many malignancies. Importantly, their interplay has the potential to contribute to disease progression and treatment resistance. To shed light on the complex relationships between these different sources of intra-tumour heterogeneity, we explored their relative contributions to t...

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cell...

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...

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 confo...

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at up to several hundred loci. All 3C methods are affected to varying degrees by inefficiency, bias and noi...

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...

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 phenomen...

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 t...

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes¹. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers...

Genome-wide association studies (GWAS) have identified over 150,000 links between common genetic variants and human traits or complex diseases. Over 80% of these associations map to polymorphisms in non-coding DNA. Therefore, the challenge is to identify disease-causing variants, the genes they affect, and the cells in which these effects occur. We...

Background: Sporadic 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 clinical phenotype is challenging. Methods: Here, as an example of this common phenomenon, we...

Precise gene expression patterns during mammalian development are controlled by regulatory elements in the non-coding genome. Active enhancer elements interact with gene promoters within Topologically Associating Domains (TADs). However, the precise relationships between chromatin accessibility, nuclear architecture and gene activation are not comp...

We employ and extensively characterise an ex vivo culture system to study terminal erythroid maturation of CD34+ progenitors from the peripheral blood of normal individuals and patients with Congenital Dyserythropoietic Anaemia type 1 (CDA-I). Using morphological analysis, FACS analysis and the proteomic approach CyTOF, we analysed patient-derived...

The Application of SEM-Based EDS Microanalysis to the Study of Congenital Dyserythropoietic Anaemia Type-1 (CDA-I) - Volume 25 Supplement - Errin Johnson, Patrick Trimby, Caroline Scott, Jill Brown, Christian Babbs, Aude-Anais Olijnik, Veronica Buckle

How chromosome organisation is related to genome function remains poorly understood. Cohesin, loop-extrusion, and CTCF have been proposed to create structures called 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 t...

Three-dimensional (3D) chromatin organisation plays a key role in regulating genome function in higher eukaryotes. Despite recognition that the genome partitions into ~1Mb-sized topological associated domains (TADs) based on ensemble Hi-C measurements, many features of the physical organisation at the single cell level remain underexplored. Using 3...

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 c...

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 o...

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 u...

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact within single cells is not understood. To address this we developed Tri-C, a new Chromosome Conformation Capture (3C) approach t...

The three-dimensional organisation of chromosomes can be probed using methods such as Capture-C. However it is unclear how such population level data relates to the organisation within a single cell, and the mechanisms leading to the observed interactions are still largely obscure. We present a polymer modelling scheme based on the assumption that...

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 s...

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 visualising FXN expression and nuclear localisation in single cells we show that...

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 mutati...

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 like...

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 mod...

Relapses after therapy-induced remissions remain a major challenge in cancer.1, 2 Molecular detection of minimal residual disease (MRD) is valuable in relapse prediction but its cellular basis remains largely unknown.3, 4, 5 Delineating therapy-resistant cells (Figure 1a) might inform new therapeutic strategies. ‘Cancer stem cells’, defined by xeno...

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...

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...

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 demons...

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 b...

1003 Introduction The Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of rare inborn disorders mainly affecting erythropoiesis. Distinct from other inherited bone marrow failure syndromes, they are marked by morphological abnormalities of the erythroblasts leading to ineffective erythropoiesis/dyserythropoiesis, while the oth...

1585 Recent studies utilising surrogate leukaemic stem cell (LSC) assays have suggested that LSCs in acute lymphoblastic leukaemias (ALLs) might be neither rare, nor phenotypically or functionally distinct. However, studies of candidate LSCs in surrogate assays might not recapitulate the full leukaemic potential of candidate LSCs in patients, and i...

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 preservi...

4060 Poster Board III-995 Expression of many tissue and developmental stage specific genes is controlled by remote regulatory elements. The mammalian a-globin gene cluster includes four previously characterised remote regulatory elements (MCS-R1 to R4) upstream of the a-globin genes. Naturally occurring deletions and experiments in transgenic mice,...

Introduction: In this chapter we describe three relatively rare, clinically complex syndromes in which the occurrence of α thalassemia provided the clue to understanding the molecular basis of each condition. These conditions exemplify the important interplay between clinical observation and human molecular genetics. Two of these syndromes (ATR-16...

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 ot...

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...

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...

We describe a pathogenetic mechanism underlying a variant form of the inherited blood disorder α thalassemia. Association studies of affected individuals from Melanesia localized the disease trait to the telomeric region of human chromosome 16, which includes the α-globin gene cluster, but no molecular defects were detected by conventional approach...

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...

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 fr...

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 suitabili...

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draf...

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draf...

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 a...

The alpha- and beta -globin gene clusters have been extensively studied(1-3). 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 i...

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 haem...

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...

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+CD38lo/− cell proliferation. We have identified three novel human CD164 variants derived by alternative splicing of bona fide exons from a single gen...

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...

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 g...

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 Na...

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 i...

The probe StB12.3 has been used to screen the FMR-1 gene in 42 pedigrees with a distal Xq fragile site for expansion of the CCG repeat and aberrant methylation of the FRAXA locus. Four families did not have a FRAXA mutation and were investigated further. Fluorescent in situ hybridisation (FISH) and molecular analyses showed that three of these fami...

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...

The hematological and clinical features of 26 patients with myelodysplasia and a chromosome 5q deletion in the bone marrow are presented. We have examined the relationship of French-American-British Co-operative Group (FAB) 1982 classification and bone marrow karyotype at diagnosis with patient outcome and the presence or absence of the classical f...

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 abn...

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...

A young adult female with multiple exostoses, short stature, autism, mental retardation and 46,X,t(X;8)(p22.13;q22.1) is described. Although the clinical features and translocation breakpoints raise the possibility of a number of specific conditions, the constellation of problems is not consistent with any previously reported genetic syndrome. It i...

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 pre...

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...

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 duplicatio...

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...