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Monika S Kowalczyk,
Jim R Hughes,
Christian Babbs,
Luis Sanchez-Pulido,
Dorota Szumska,
Jacqueline A Sharpe,
Jacqueline A Sloane-Stanley,
Gillian M Morriss-Kay,
Leslie B Smoot,
Amy E Roberts,
Hugh Watkins,
Shoumo Bhattacharya,
Richard J Gibbons,
Chris P Ponting, William G Wood,
Douglas R Higgs
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ABSTRACT: C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system.
Mammalian Genome 04/2012; 23(7-8):404-15. · 2.89 Impact Factor
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Monika S Kowalczyk,
Jim R Hughes,
David Garrick,
Magnus D Lynch,
Jacqueline A Sharpe,
Jacqueline A Sloane-Stanley,
Simon J McGowan,
Marco De Gobbi,
Mona Hosseini,
Douglas Vernimmen,
Jill M Brown,
Nicola E Gray,
Licio Collavin,
Richard J Gibbons,
Jonathan Flint,
Stephen Taylor,
Veronica J Buckle,
Thomas A Milne, William G Wood,
Douglas R Higgs
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ABSTRACT: A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)(+) RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.
Molecular cell 02/2012; 45(4):447-58. · 14.61 Impact Factor
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Raffaele Renella,
Nigel A Roberts,
Jill M Brown,
Marco De Gobbi,
Louise E Bird,
Tasneem Hassanali,
Jacqueline A Sharpe,
Jacqueline Sloane-Stanley,
David J P Ferguson,
Jacqueline Cordell,
Veronica J Buckle,
Douglas R Higgs, William G Wood
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ABSTRACT: Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1(gt/gt) homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.
Blood 03/2011; 117(25):6928-38. · 9.90 Impact Factor
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Alison T Merryweather-Clarke,
Ann Atzberger,
Shamit Soneji,
Nicki Gray,
Kevin Clark,
Craig Waugh,
Simon J McGowan,
Stephen Taylor,
Asoke K Nandi, William G Wood,
David J Roberts,
Douglas R Higgs,
Veronica J Buckle,
Kathryn J H Robson
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ABSTRACT: Understanding the pattern of gene expression during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here, we isolated 4 distinct populations at successive erythropoietin-dependent stages of erythropoiesis, including the terminal, pyknotic stage. The transcriptome was determined using Affymetrix arrays. First, we demonstrated the importance of using defined cell populations to identify lineage and temporally specific patterns of gene expression. Cells sorted by surface expression profile not only express significantly fewer genes than unsorted cells but also demonstrate significantly greater differences in the expression levels of particular genes between stages than unsorted cells. Second, using standard software, we identified more than 1000 transcripts not previously observed to be differentially expressed during erythroid maturation, 13 of which are highly significantly terminally regulated, including RFXAP and SMARCA4. Third, using matched filtering, we identified 12 transcripts not previously reported to be continuously up-regulated in maturing human primary erythroblasts. Finally, using transcription factor binding site analysis, we identified potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts containing many genes with undiscovered functions in erythroblasts are a resource for future functional studies of erythropoiesis. Our Human Erythroid Maturation database is available at https://cellline.molbiol.ox.ac.uk/eryth/index.html. [corrected].
Blood 01/2011; 117(13):e96-108. · 9.90 Impact Factor
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ABSTRACT: Previous studies in the mouse have shown that high levels of alpha-globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human alpha cluster and their interactions with the alpha-globin promoter. All of these elements interact with the alpha-globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human alpha globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS -40) results in loss of all the interactions and alpha-globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human alpha-globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.
Blood 09/2009; 114(19):4253-60. · 9.90 Impact Factor
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ABSTRACT: Severe intrauterine anemia of unknown cause presents a diagnostic challenge. We describe a Norwegian case, managed successfully by intrauterine transfusions, that further investigations demonstrated to be due to a rare type of thalassemia. A deletion of the 5' end of the beta globin gene cluster was characterized, the breakpoints sequenced and a new type of epsilongammagammadeltabeta thalassemia identified. This case highlights the need to consider diagnoses of rare conditions not normally associated with a particular population.
Haematologica 07/2009; 94(8):1157-9. · 6.42 Impact Factor
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ABSTRACT: The congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of hereditary disorders that seem to be restricted to the erythroid lineage. They are characterized by morphologic abnormalities of erythroid precursors in the bone marrow, resulting in ineffective erythropoiesis and a suboptimal reticulocyte response. As with many rare disorders, cases of CDA are often misdiagnosed, which may lead to inappropriate management. In this review, the authors highlight the relevant clinical data together with recent molecular advances that should aid decision making in diagnosis and patient management.
Hematology/oncology clinics of North America 05/2009; 23(2):283-306. · 2.05 Impact Factor
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ABSTRACT: Muenke syndrome, defined by heterozygosity for a Pro250Arg substitution in fibroblast growth factor receptor 3 (FGFR3), is the most common genetic cause of craniosynostosis in humans. We have used gene targeting to introduce the Muenke syndrome mutation (equivalent to P244R) into the murine Fgfr3 gene. A rounded skull and shortened snout (often skewed) with dental malocclusion was observed in a minority of heterozygotes and many homozygotes. Development of this incompletely penetrant skull phenotype was dependent on genetic background and sex, with males more often affected. However, these cranial abnormalities were rarely attributable to craniosynostosis, which was only present in 2/364 mutants; more commonly, we found fusion of the premaxillary and/or zygomatic sutures. We also found decreased cortical thickness and bone mineral densities in long bones. We conclude that although both cranial and long bone development is variably affected by the murine Fgfr3(P244R) mutation, coronal craniosynostosis is not reliably reproduced.
Developmental Dynamics 01/2009; 238(2):331-42. · 2.54 Impact Factor
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ABSTRACT: Muenke syndrome, defined by heterozygosity for a Pro250Arg substitution in fibroblast growth factor receptor 3 (FGFR3), is the most common genetic cause of craniosynostosis in humans. We have used gene targeting to introduce the Muenke syndrome mutation (equivalent to P244R) into the murine Fgfr3 gene. A rounded skull and shortened snout (often skewed) with dental malocclusion was observed in a minority of heterozygotes and many homozygotes. Development of this incompletely penetrant skull phenotype was dependent on genetic background and sex, with males more often affected. However, these cranial abnormalities were rarely attributable to craniosynostosis, which was only present in 2/364 mutants; more commonly, we found fusion of the premaxillary and/or zygomatic sutures. We also found decreased cortical thickness and bone mineral densities in long bones. We conclude that although both cranial and long bone development is variably affected by the murine Fgfr3P244R mutation, coronal craniosynostosis is not reliably reproduced. Developmental Dynamics 238:331–342, 2009. © 2008 Wiley-Liss, Inc.
Developmental Dynamics 12/2008; 238(2):331 - 342. · 2.54 Impact Factor
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Jill M Brown,
Joanne Green,
Ricardo Pires das Neves,
Helen A C Wallace,
Andrew J H Smith,
Jim Hughes,
Nicki Gray,
Steve Taylor, William G Wood,
Douglas R Higgs,
Francisco J Iborra,
Veronica J Buckle
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ABSTRACT: Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse alpha-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human alpha-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.
The Journal of Cell Biology 10/2008; 182(6):1083-97. · 10.26 Impact Factor
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Jill M. Brown,
Joanne Green,
Ricardo Pires das Neves,
Helen A.C. Wallace,
Andrew J.H. Smith,
Jim Hughes,
Nicki Gray,
Steve Taylor, William G. Wood,
Douglas R. Higgs,
Francisco J. Iborra,
Veronica J. Buckle
[show abstract]
[hide abstract]
ABSTRACT: Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations;
however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show
that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but
variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the
mouse α-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 α-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.
The Journal of Cell Biology 09/2008; 182(6):1083-1097. · 10.26 Impact Factor
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Proceedings of the National Academy of Sciences 09/2008; 105(33):11595-6. · 9.68 Impact Factor
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ABSTRACT: The analysis of globin gene expression during erythropoiesis has established many principles underlying normal mammalian gene expression. New aspects of gene regulation have been revealed by natural mutations that downregulate globin gene expression and cause thalassemia. Deletions involving sequences upstream of the alpha and beta clusters suggested that the globin genes might be controlled by remote regulatory elements. This was demonstrated experimentally and suggested that many mammalian genes may be controlled in a similar manner.
Completion of the Human Genome Project and the associated encyclopaedia of DNA elements (ENCODE) project confirmed that human gene expression is commonly controlled by long-range, cis-acting elements. The development of chromatin immunoprecipitation has allowed us to identify binding of transcription factors and chromatin modifications at the key cis-acting sequences in vivo. In addition, chromosome conformation capture has enabled us to address the topological models proposed to mediate long-range interactions. Together, these methods have given us some insight into how long-range elements may influence gene expression and how this process may be subverted in thalassemia.
The review asks how remote elements regulate alpha globin expression and how natural mutations interfere with this mechanism to cause alpha thalassemia. We also speculate as to why long-range control of gene expression may have evolved in higher organisms.
Current Opinion in Hematology 06/2008; 15(3):176-83. · 4.52 Impact Factor
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ABSTRACT: To address the mechanism by which the human globin genes are activated during erythropoiesis, we have used a tiled microarray to analyze the pattern of transcription factor binding and associated histone modifications across the telomeric region of human chromosome 16 in primary erythroid and nonerythroid cells. This 220-kb region includes the alpha globin genes and 9 widely expressed genes flanking the alpha globin locus. This un-biased, comprehensive analysis of transcription factor binding and histone modifications (acetylation and methylation) described here not only identified all known cis-acting regulatory elements in the human alpha globin cluster but also demonstrated that there are no additional erythroid-specific regulatory elements in the 220-kb region tested. In addition, the pattern of histone modification distinguished promoter elements from potential enhancer elements across this region. Finally, comparison of the human and mouse orthologous regions in a unique mouse model, with both regions coexpressed in the same animal, showed significant differences that may explain how these 2 clusters are regulated differently in vivo.
Blood 01/2008; 110(13):4503-10. · 9.90 Impact Factor
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ABSTRACT: Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate.
The American Journal of Human Genetics 07/2007; 80(6):1138-49. · 10.60 Impact Factor
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ABSTRACT: To understand how mammalian genes are regulated from their natural chromosomal environment, we have analysed the molecular events occurring throughout a 150 kb chromatin segment containing the alpha globin gene locus as it changes from a poised, silent state in erythroid progenitors, to the fully activated state in late, erythroid cells. Active transcription requires the late recruitment of general transcription factors, mediator and Pol II not only to the promoter but also to its remote regulatory elements. Natural mutants of the alpha cluster show that whereas recruitment of the pre-initiation complex to the upstream elements occurs independently, recruitment to the promoter is largely dependent on the regulatory elements. An improved, quantitative chromosome conformation capture analysis demonstrates that this recruitment is associated with a conformational change, in vivo, apposing the promoter with its remote regulators, consistent with a chromosome looping mechanism. These findings point to a general mechanism by which a gene can be held in a poised state until the appropriate stage for expression, coordinating the level and timing of gene expression during terminal differentiation.
The EMBO Journal 05/2007; 26(8):2041-51. · 9.20 Impact Factor
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ABSTRACT: We have devised a strategy (called recombinase-mediated genomic replacement, RMGR) to allow the replacement of large segments (>100 kb) of the mouse genome with the equivalent human syntenic region. The technique involves modifying a mouse ES cell chromosome and a human BAC by inserting heterotypic lox sites to flank the proposed exchange interval and then using Cre recombinase to achieve segmental exchange. We have demonstrated the feasibility of this approach by replacing the mouse alpha globin regulatory domain with the human syntenic region and generating homozygous mice that produce only human alpha globin chains. Furthermore, modified ES cells can be used iteratively for functional studies, and here, as an example, we have used RMGR to produce an accurate mouse model of human alpha thalassemia. RMGR has general applicability and will overcome limitations inherent in current transgenic technology when studying the expression of human genes and modeling human genetic diseases.
Cell 01/2007; 128(1):197-209. · 32.40 Impact Factor
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ABSTRACT: We generated five lines of transgenic mice carrying 1-3 copies of the Hb Lepore deltabeta fusion gene, in the context of a Bacterial Artificial Chromosome containing the whole human beta globin gene cluster. Normal developmental regulation of human genes occurred at levels approximating to those of endogenous genes. Deltabeta transgene expression became detectable during fetal life and rose to a mean level of 13.0% in adults, similar to that of humans. Low levels of human gamma chains were detectable as F cells in adult mice, but numbers did not increase after treatment with drugs that raise F cells in human subjects, even on a thalassaemic background.
British Journal of Haematology 01/2007; 135(5):735-7. · 4.94 Impact Factor
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Momin R Ahmed,
Aref Chehal,
Laila Zahed,
Ali Taher,
Joud Haidar,
Ali Shamseddine,
Anne-Marie O'Hea,
Nicola Bienz,
Orly Dgany,
Nili Avidan,
Jacqui S Beckmann,
Hannah Tamary,
Douglas Higgs,
Paresh Vyas, William G Wood,
Sunitha N Wickramasinghe
Blood 07/2006; 107(12):4968-9. · 9.90 Impact Factor
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Blood 06/2006; 107(9):3811-2. · 9.90 Impact Factor
