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ABSTRACT: Whereas mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in non-coding regions. Furthermore, recent genome-wide studies have shown that common DNA sequence variants in non-coding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (∼10 fold) changes in expression from a single allele in a tissue-specific manner. We also show how a single nucleotide polymorphism, located at some distance from the recognised TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases.
Human Mutation 04/2013; · 5.69 Impact Factor
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Magnus D Lynch,
Andrew J H Smith,
Marco De Gobbi,
Maria Flenley,
Jim R Hughes,
Douglas Vernimmen, Helena Ayyub,
Jacqueline A Sharpe,
Jacqueline A Sloane-Stanley,
Linda Sutherland,
Stephen Meek,
Tom Burdon,
Richard J Gibbons,
David Garrick,
Douglas R Higgs
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ABSTRACT: The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination-mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase-deficient embryonic stem cells.
The EMBO Journal 11/2011; 31(2):317-29. · 9.20 Impact Factor
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Martin J Law,
Karen M Lower,
Hsiao P J Voon,
Jim R Hughes,
David Garrick,
Vip Viprakasit,
Matthew Mitson,
Marco De Gobbi,
Marco Marra,
Andrew Morris, [......],
Stephen Taylor,
Guilherme M Santos,
Joe Cross, Helena Ayyub,
Steven Jones,
Jiannis Ragoussis,
Daniela Rhodes,
Ian Dunham,
Douglas R Higgs,
Richard J Gibbons
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ABSTRACT: ATRX is an X-linked gene of the SWI/SNF family, mutations in which cause syndromal mental retardation and downregulation of α-globin expression. Here we show that ATRX binds to tandem repeat (TR) sequences in both telomeres and euchromatin. Genes associated with these TRs can be dysregulated when ATRX is mutated, and the change in expression is determined by the size of the TR, producing skewed allelic expression. This reveals the characteristics of the affected genes, explains the variable phenotypes seen with identical ATRX mutations, and illustrates a new mechanism underlying variable penetrance. Many of the TRs are G rich and predicted to form non-B DNA structures (including G-quadruplex) in vivo. We show that ATRX binds G-quadruplex structures in vitro, suggesting a mechanism by which ATRX may play a role in various nuclear processes and how this is perturbed when ATRX is mutated.
Cell 10/2010; 143(3):367-78. · 32.40 Impact Factor
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Karen M Lower,
Jim R Hughes,
Marco De Gobbi,
Shirley Henderson,
Vip Viprakasit,
Chris Fisher,
Anne Goriely, Helena Ayyub,
Jackie Sloane-Stanley,
Douglas Vernimmen,
Cordelia Langford,
David Garrick,
Richard J Gibbons,
Douglas R Higgs
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ABSTRACT: It is well established that all of the cis-acting sequences required for fully regulated human alpha-globin expression are contained within a region of approximately 120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the alpha-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the alpha-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of alpha-globin genes, affects the pattern of NME4 expression by altering the competition for the shared alpha-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.
Proceedings of the National Academy of Sciences 12/2009; 106(51):21771-6. · 9.68 Impact Factor
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ABSTRACT: We have characterized a newly identified 16.6 kb deletion which removes a significant proportion of the human alpha-globin cluster including the psizeta1, alpha(D), psialpha1 and alpha2-globin genes but leaves the duplicated alpha1 gene intact. This complicated rearrangement results from a combination of slippage and strand switching at sites of microhomology during replication. Functional analysis shows that expression of the remaining alpha1 gene is increased, rather than down-regulated by this deletion. This could be related to its proximity to the remote upstream alpha-globin regulatory elements or reduced competition for these elements in the absence of the dominant alpha2-globin gene. The finding of a very mild phenotype associated with such an extensive deletion in the alpha-globin cluster implies that much of the DNA removed by the deletion is likely to be functionally unimportant. These findings suggest that other than the upstream regulatory elements and promoter proximal elements there are unlikely to be additional positive cis-acting sequences in the alpha-globin cluster.
Human Molecular Genetics 10/2008; 17(19):3084-93. · 7.64 Impact Factor
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David Garrick,
Marco De Gobbi,
Vasiliki Samara,
Michelle Rugless,
Michelle Holland, Helena Ayyub,
Karen Lower,
Jackie Sloane-Stanley,
Nicki Gray,
Christoph Koch,
Ian Dunham,
Douglas R Higgs
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ABSTRACT: Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human alpha- and beta-globin genes are silenced by fundamentally different mechanisms. The alpha-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the beta-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the alpha-cluster by sequences within the CpG islands associated with their promoters; the beta-globin promoters do not lie within such islands. Chromatin associated with the alpha-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The alpha- and beta-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.
Blood 09/2008; 112(9):3889-99. · 9.90 Impact Factor
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Blood 06/2006; 107(9):3811-2. · 9.90 Impact Factor
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Marco De Gobbi,
Vip Viprakasit,
Jim R Hughes,
Chris Fisher,
Veronica J Buckle, Helena Ayyub,
Richard J Gibbons,
Douglas Vernimmen,
Yuko Yoshinaga,
Pieter de Jong,
Jan-Fang Cheng,
Edward M Rubin,
William G Wood,
Don Bowden,
Douglas R Higgs
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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.
Science 06/2006; 312(5777):1215-7. · 31.20 Impact Factor
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Thomas S Price,
Regina Regan,
Richard Mott,
Asa Hedman,
Ben Honey,
Rachael J Daniels,
Lee Smith,
Andy Greenfield,
Ana Tiganescu,
Veronica Buckle,
Nicki Ventress, Helena Ayyub,
Anita Salhan,
Susana Pedraza-Diaz,
John Broxholme,
Jiannis Ragoussis,
Douglas R Higgs,
Jonathan Flint,
Samantha J L Knight
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ABSTRACT: 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.
Nucleic Acids Research 02/2005; 33(11):3455-64. · 8.03 Impact Factor
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ABSTRACT: We describe a family of Italian origin in which the father and his two children had hypochromia and microcytosis with normal iron status. All individuals underwent an uneventful clinical course and required no treatment. To investigate the molecular basis of this phenotype, which is a prerequisite for further genetic counselling, we revealed that all affected family members are carriers of a common form of alpha+ thalassaemia resulting from the deletion of 3.7 kb of the alpha-globin cluster (alphaalpha/-alpha3.7). However, this genotype alone could not account for the phenotype presenting in this family. Further characterization of the alpha-globin genes demonstrated an additional AC deletion in the vicinity of the initiation codon of the -alpha3.7 allele. This secondary mutation causes an additional impaired translation of the affected allele producing increased globin chain imbalance. This leads to a more severe phenotype, as heterozygotes for such mutation (alphaalpha/-alphaT) have hypochromic microcytosis and abnormal globin chain synthesis that mimic alpha0 thalassaemia trait (--/alphaalpha). Accurate genotyping of alpha globin determinant is absolutely required as there is a possibility that an interaction of this unusual double mutation with other common alpha0 thalassaemias (--/-alphaT) can give rise to a very severe, probably fatal, alpha thalassaemia.
European Journal Of Haematology 09/2003; 71(2):133-6. · 2.61 Impact Factor
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ABSTRACT: Inherited mutations of specific genes have elucidated the normal roles of the proteins they encode by relating specific mutations to particular phenotypes. But many potentially informative mutations in such genes are lethal early in development. Consequently, inherited mutations may not reflect all the functional roles of such proteins. Acquired, somatic defects should reflect a wider spectrum of mutations because they are not prone to negative selection in development. It has been difficult to identify such mutations so far, but microarray analysis provides a new opportunity to do so. Using this approach, we have shown that in individuals with myelodysplasia associated with alpha-thalassemia (ATMDS), somatic mutations of the gene encoding the chromatin remodeling factor ATRX cause an unexpectedly severe hematological phenotype compared with the wide spectrum of inherited mutations affecting this gene. These findings cast new light on this pleiotropic cofactor, which appears to be an essential component rather than a mere facilitator of globin gene expression.
Nature Genetics 09/2003; 34(4):446-9. · 35.53 Impact Factor
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ABSTRACT: We describe a family of Italian origin in which the father and his two children had hypochromia and microcytosis with normal iron status. All individuals underwent an uneventful clinical course and required no treatment. To investigate the molecular basis of this phenotype, which is a prerequisite for further genetic counselling, we revealed that all affected family members are carriers of a common form of α+ thalassaemia resulting from the deletion of 3.7 kb of the α-globin cluster (αα/−α3.7). However, this genotype alone could not account for the phenotype presenting in this family. Further characterization of the α-globin genes demonstrated an additional AC deletion in the vicinity of the initiation codon of the −α3.7 allele. This secondary mutation causes an additional impaired translation of the affected allele producing increased globin chain imbalance. This leads to a more severe phenotype, as heterozygotes for such mutation (αα/−αT) have hypochromic microcytosis and abnormal globin chain synthesis that mimic α0 thalassaemia trait (−−/αα). Accurate genotyping of α globin determinant is absolutely required as there is a possibility that an interaction of this unusual double mutation with other common α0 thalassaemias (−−/−αT) can give rise to a very severe, probably fatal, α thalassaemia.
European Journal Of Haematology 07/2003; 71(2):133 - 136. · 2.61 Impact Factor
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ABSTRACT: Nearly all human genetic disorders result from a limited repertoire of mutations in an associated gene or its regulatory elements. We recently described an individual with an inherited form of anemia (alpha-thalassemia) who has a deletion that results in a truncated, widely expressed gene (LUC7L) becoming juxtaposed to a structurally normal alpha-globin gene (HBA2). Although it retains all of its local and remote cis-regulatory elements, expression of HBA2 is silenced and its CpG island becomes completely methylated early during development. Here we show that in the affected individual, in a transgenic model and in differentiating embryonic stem cells, transcription of antisense RNA mediates silencing and methylation of the associated CpG island. These findings identify a new mechanism underlying human genetic disease.
Nature Genetics 07/2003; 34(2):157-65. · 35.53 Impact Factor
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ABSTRACT: We have identified and characterized a Scottish individual with alpha thalassaemia, resulting from a de novo 48 kilobase (kb) deletion from the telomeric flanking region of the alpha globin cluster which occurred as a result of recombination between two misaligned repetitive elements that normally lie approximately 83 kb and 131 kb from the 16p telomere. The deletion removes two previously described putative regulatory elements (HS-40 and HS-33) but leaves two other elements (HS-10 and HS-8) intact. Analysis of this deletion, together with eight other published deletions of the telomeric region, showed that they all severely downregulated alpha globin expression. Together they defined a 20.4-kb region of the human alpha cluster, which contains all of the positive cis-acting elements required to regulate alpha globin expression. Comparative analysis of this region with the corresponding segment of the mouse alpha globin cluster demonstrated conserved non-coding sequences corresponding to the putative regulatory elements HS-40 and HS-33. Although the role of HS-40 as an enhancer of alpha globin expression is fully established, these observations suggest that the role of HS-33 and other sequences in this region should be more fully investigated in the context of the natural human and mouse alpha globin loci.
British Journal of Haematology 04/2003; 120(5):867-75. · 4.94 Impact Factor
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ABSTRACT: To date, more than 35 single or oligonucleotide mutations of the alpha genes that cause alpha thalassaemia have been described. Their interactions give rise to widely variable clinical manifestations, from a mild hypochromic, microcytic anaemia to a lethal intrauterine anaemia associated with hydrops fetalis. Understanding the molecular genetics enables accurate genotyping, genetic counselling and prenatal testing for the most severe forms of alpha thalassaemia. Here we show for the first time that the interaction between two relatively common forms of alpha thalassaemia (--MED/(alpha)TSaudi(alpha)) may be associated with a clinically severe form of alpha thalassaemia, Hb H hydrops fetalis.
British Journal of Haematology 07/2002; 117(3):759-62. · 4.94 Impact Factor
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ABSTRACT: To date, more than 35 single or oligonucleotide mutations of the genes that cause thalassaemia have been described. Their interactions give rise to widely variable clinical manifestations, from a mild hypochromic, microcytic anaemia to a lethal intrauterine anaemia associated with hydrops fetalis. Understanding the molecular genetics enables accurate genotyping, genetic counselling and prenatal testing for the most severe forms of thalassaemia. Here we show for the first time that the interaction between two relatively common forms of thalassaemia (--MED/TSaudi) may be associated with a clinically severe form of thalassaemia, Hb H hydrops fetalis.
British Journal of Haematology 05/2002; 117(3):759 - 762. · 4.94 Impact Factor
