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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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ABSTRACT: Remote distal enhancers may be located tens or thousands of kilobases away from their promoters. How they control gene expression is still poorly understood. Here, we analyze the influence of a remote enhancer on the balance between repression (Polycomb-PcG) and activation (Trithorax-TrxG) of a developmentally regulated gene associated with a CpG island. We reveal its essential, nonredundant role in clearing the PcG complex and H3K27me3 from the CpG island. In the absence of the enhancer, the H3K27me3 demethylase (JMJD3) is not recruited to the CpG island. We propose a new role of long-range regulatory elements in removing repressive PcG complexes.
Genes & development 08/2011; 25(15):1583-8. · 12.08 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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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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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: ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation.
PLoS Genetics 05/2006; 2(4):e58. · 8.69 Impact Factor
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Diane L Sherman,
Steven Tait,
Shona Melrose,
Richard Johnson,
Barbara Zonta,
Felipe A Court,
Wendy B Macklin,
Stephen Meek, Andrew J H Smith,
David F Cottrell,
Peter J Brophy
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ABSTRACT: Voltage-gated sodium channels are concentrated in myelinated nerves at the nodes of Ranvier flanked by paranodal axoglial junctions. Establishment of these essential nodal and paranodal domains is determined by myelin-forming glia, but the mechanisms are not clear. Here, we show that two isoforms of Neurofascin, Nfasc155 in glia and Nfasc186 in neurons, are required for the assembly of these specialized domains. In Neurofascin-null mice, neither paranodal adhesion junctions nor nodal complexes are formed. Transgenic expression of Nfasc155 in the myelinating glia of Nfasc-/- nerves rescues the axoglial adhesion complex by recruiting the axonal proteins Caspr and Contactin to the paranodes. However, in the absence of Nfasc186, sodium channels remain diffusely distributed along the axon. Our study shows that the two major Neurofascins play essential roles in assembling the nodal and paranodal domains of myelinated axons; therefore, they are essential for the transition to saltatory conduction in developing vertebrate nerves.
Neuron 01/2006; 48(5):737-42. · 14.74 Impact Factor
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ABSTRACT: Butyrophilin 1a1 (Btn1a1), which is a member of the Ig superfamily, is highly expressed in the lactating mammary gland and is secreted into milk in association with lipid droplets. To determine the potential function of Btn1a1 in milk secretion, we ablated Btn1a1 in mice and analyzed the lactation phenotype of homozygous (Btn1a1(-/-)) animals. Two mutant mouse lines were generated in which expression of Btn1a1 was either disrupted or eliminated, respectively. The regulated secretion of milk-lipid droplets was severely compromised in both mutant mouse lines in comparison to wild-type animals. Large pools of triacylglycerol accumulated in the cytoplasm of secretory cells, and lipid droplets escaped from the apical surface with disrupted outer membranes. Luminal spaces became engorged with unstable lipid droplets, which coalesced to form large aggregates. The amount of lipid (wt/vol) was elevated, on average by 50%, during the first 10 days of lactation, and the diameter of the droplets was up to seven times larger than the normal diameter. In contrast, there was no significant difference between wild-type and null animals in the relative amounts of skim-milk proteins secreted from Golgi-derived secretory vesicles. Approximately half the pups suckling Btn1a1(-/-) animals died within the first 20 days, and weaning weights for the surviving pups were 60-80% of those suckling wild-type mice. Thus, expression of Btn1a1 is essential for the regulated secretion of milk-lipid droplets. We speculate that Btn1a1 functions either as a structural protein or as a signaling receptor by binding to xanthine dehydrogenase/oxidase.
Proceedings of the National Academy of Sciences 08/2004; 101(27):10084-9. · 9.68 Impact Factor
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ABSTRACT: Mutations in the ATRX gene cause a severe X-linked mental retardation syndrome that is frequently associated with alpha thalassemia (ATR-X syndrome). The previously characterized ATRX protein (approximately 280 kDa) contains both a Plant homeodomain (PHD)-like zinc finger motif as well as an ATPase domain of the SNF2 family. These motifs suggest that ATRX may function as a regulator of gene expression, probably by exerting an effect on chromatin structure, although the exact cellular role of ATRX has not yet been fully elucidated. Here we characterize a truncated (approximately 200 kDa) isoform of ATRX (called here ATRXt) that has been highly conserved between mouse and human. In both species, ATRXt arises due to the failure to splice intron 11 from the primary transcript, and the use of a proximal intronic poly(A) signal. We show that the relative expression of the full length and ATRXt isoforms is subject to tissue-specific regulation. The ATRXt isoform contains the PHD-like domain but not the SWI/SNF-like motifs and is therefore unlikely to be functionally equivalent to the full length protein. We used indirect immunofluorescence to demonstrate that the full length and ATRXt isoforms are colocalized at blocks of pericentromeric heterochromatin but unlike full length ATRX, the truncated isoform does not associate with promyelocytic leukemia (PML) nuclear bodies. The high degree of conservation of ATRXt and the tight regulation of its expression relative to the full length protein suggest that this truncated isoform fulfills an important biological function.
Gene 03/2004; 326:23-34. · 2.34 Impact Factor
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ABSTRACT: Disruption of the PPP1R3A gene encoding the glycogen targeting subunit (G(M)/R(GL)) of protein phosphatase 1 (PP1) causes substantial lowering of the glycogen synthase activity and a 10-fold decrease in the glycogen levels in skeletal muscle. Homozygous G(M)(-/-) mice show increased weight gain after 3 months of age and become obese, weighing approximately 20% more than their wild-type (WT) littermates after 12 months of age. Glucose tolerance is impaired in 11-month-old G(M)(-/-) mice, and their skeletal muscle is insulin-resistant at > or =12 months of age. The massive abdominal and other fat depositions observed at this age are likely to be a consequence of impaired blood glucose utilization in skeletal muscle. PP1-G(M) activity, assayed after specific immunoadsorption, was absent from G(M)(-/-) mice and stimulated in the hind limb muscles of WT mice by intravenous infusion of insulin. PP1-R5/PTG, another glycogen targeted form of PP1, was not significantly stimulated by insulin in the skeletal muscle of WT mice but showed compensatory stimulation by insulin in G(M)(-/-) mice. Our results suggest that dysfunction of PP1-G(M) may contribute to the pathophysiology of human type 2 diabetes.
Diabetes 03/2003; 52(3):596-604. · 8.29 Impact Factor
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ABSTRACT: Chromosomal deletions are a common feature of epithelial tumours and when further defined by homozygous deletions, are often the location of tumour suppressor genes. Deletions within the short arm of chromosome 3 occur very frequently in human carcinomas: a minimal region of loss at 3p21.3 (the Luca) region has been defined by overlapping homozygous deletions in lung and breast cancer cell lines. Using a rapid strategy for Cre-loxP chromosome engineering, a deletion of approximately 370 kb was created in the mouse germline corresponding to the deleted region at 3p21.3. The deletion when homozygous is embryonic lethal. Heterozygotes develop normally despite being haplo-insufficient for twelve genes including the candidate tumour suppressor gene Rassf1. Because damage to 3p21.3 often occurs very early in the sequence of genetic changes that lead to malignancy, particularly in lung and breast cancer, further genetic damage to these mice will provide the opportunity to model multi-step tumorigenesis of these tumours.
Oncogene 08/2002; 21(29):4521-9. · 6.37 Impact Factor
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ABSTRACT: NOTE: THE SPECIAL CHARACTERS IN THIS ABSTRACT CANNOT BE DISPLAYED CORRECTLY ON THIS PAGE. PLEASE REFER TO THE ABSTRACT IN THE PUBLISHER'S WEBSITE FOR AN ACCURATE DISPLAY. Disruption of the PPP1R3A gene encoding the glycogen targeting subunit (GM/RGL) of protein phosphatase 1 (PP1) causes substantial lowering of the glycogen synthase activity and a 10-fold decrease in the glycogen levels in skeletal muscle. Homozygous GM ⁻/⁻ mice show increased weight gain after 3 months of age and become obese, weighing ~20% more than their wild-type (WT) littermates after 12 months of age. Glucose tolerance is impaired in 11-month-old GM ⁻/⁻ mice, and their skeletal muscle is insulin-resistant at ≥12 months of age. The massive abdominal and other fat depositions observed at this age are likely to be a consequence of impaired blood glucose utilization in skeletal muscle. PP1-GM activity, assayed after specific immunoadsorption, was absent from GM ⁻/⁻ mice and stimulated in the hind limb muscles of WT mice by intravenous infusion of insulin. PP1-R5/PTG, another glycogen targeted form of PP1, was not significantly stimulated by insulin in the skeletal muscle of WT mice but showed compensatory stimulation by insulin in GM ⁻/⁻ mice. Our results suggest that dysfunction of PP1-GM may contribute to the pathophysiology of human type 2 diabetes. Diabetes 52: 596–604, 2003 U.K. Medical Research Council AstraZeneca Boehringer Ingelheim GlaxoSmithKline Novo Nordisk Pfizer
