Publications by authors named "Zoe Webster"

28 Publications

  • Page 1 of 1

Implication of sestrin3 in epilepsy and its comorbidities.

Brain Commun 2021 9;3(1):fcaa130. Epub 2020 Oct 9.

Department of Medical Sciences, Section of Pharmacology, University of Ferrara, Italy.

Epilepsy is a serious neurological disorder affecting about 1% of the population worldwide. Epilepsy may arise as a result of acquired brain injury, or as a consequence of genetic predisposition. To date, genome-wide association studies and exome sequencing approaches have provided limited insights into the mechanisms of acquired brain injury. We have previously reported a pro-epileptic gene network, which is conserved across species, encoding inflammatory processes and positively regulated by sestrin3 (SESN3). In this study, we investigated the phenotype of knock-out rats in terms of susceptibility to seizures and observed a significant delay in status epilepticus onset in knock-out compared to control rats. This finding confirms previous and evidence indicating that SESN3 may favour occurrence and/or severity of seizures. We also analysed the phenotype of knock-out rats for common comorbidities of epilepsy, i.e., anxiety, depression and cognitive impairment. knock-out rats proved less anxious compared to control rats in a selection of behavioural tests. Taken together, the present results suggest that SESN3 may regulate mechanisms involved in the pathogenesis of epilepsy and its comorbidities.
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http://dx.doi.org/10.1093/braincomms/fcaa130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966953PMC
October 2020

Evolution of an Amniote-Specific Mechanism for Modulating Ubiquitin Signaling via Phosphoregulation of the E2 Enzyme UBE2D3.

Mol Biol Evol 2020 07;37(7):1986-2001

Gene Regulation and Chromatin Group, MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom.

Genetic variation in the enzymes that catalyze posttranslational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signaling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as an ubiquitin donor for E3 ligases that catalyze ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologs to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse embryonic stem cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extraembryonic primitive endoderm, levels of the PDGFRα and FGFR1 receptor tyrosine kinases were reduced and primitive endoderm differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the receptor tyrosine kinases. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.
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http://dx.doi.org/10.1093/molbev/msaa060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306689PMC
July 2020

Camk2n1 Is a Negative Regulator of Blood Pressure, Left Ventricular Mass, Insulin Sensitivity, and Promotes Adiposity.

Hypertension 2019 09 22;74(3):687-696. Epub 2019 Jul 22.

From the MRC Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom (N.A., M.B., S.M.d.P., T.J.A., P.M.C.).

Metabolic syndrome is a cause of coronary artery disease and type 2 diabetes mellitus. Camk2n1 resides in genomic loci for blood pressure, left ventricle mass, and type 2 diabetes mellitus, and in the spontaneously hypertensive rat model of metabolic syndrome, Camk2n1 expression is cis-regulated in left ventricle and fat and positively correlates with adiposity. Therefore, we knocked out Camk2n1 in spontaneously hypertensive rat to investigate its role in metabolic syndrome. Compared with spontaneously hypertensive rat, Camk2n1 rats had reduced cardiorenal CaMKII (Ca/calmodulin-dependent kinase II) activity, lower blood pressure, enhanced nitric oxide bioavailability, and reduced left ventricle mass associated with altered hypertrophic networks. Camk2n1 deficiency reduced insulin resistance, visceral fat, and adipogenic capacity through the altered cell cycle and complement pathways, independent of CaMKII. In human visceral fat, CAMK2N1 expression correlated with adiposity and genomic variants that increase CAMK2N1 expression associated with increased risk of coronary artery disease and type 2 diabetes mellitus. Camk2n1 regulates multiple networks that control metabolic syndrome traits and merits further investigation as a therapeutic target in humans.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.118.12409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686962PMC
September 2019

Bioluminescent Reporting of In Vivo IFN-γ Immune Responses during Infection and Autoimmunity.

J Immunol 2019 04 27;202(8):2502-2510. Epub 2019 Feb 27.

Lung Immunology Group, Infectious Diseases and Immunity, Department of Medicine, Imperial College London, London W12 ONN, United Kingdom; and

IFN-γ is a key cytokine of innate and adaptive immunity. It is important to understand temporal changes in IFN-γ production and how these changes relate to the role of IFN-γ in diverse models of infectious and autoimmune disease, making the ability to monitor and track IFN-γ production in vivo of a substantial benefit. IFN-γ ELISPOTs have been a central methodology to measure T cell immunity for many years. In this study, we add the capacity to analyze IFN-γ responses with high sensitivity and specificity, longitudinally, in vitro and in vivo. This allows the refinement of experimental protocols because immunity can be tracked in real-time through a longitudinal approach. We have generated a novel murine IFN-γ reporter transgenic model that allows IFN-γ production to be visualized and quantified in vitro and in vivo as bioluminescence using an imaging system. At baseline, in the absence of an inflammatory stimulus, IFN-γ signal from lymphoid tissue is detectable in vivo. Reporter transgenics are used in this study to track the IFN-γ response to infection in the lung over time in vivo. The longitudinal development of the adaptive T cell immunity following immunization with Ag is identified from day 7 in vivo. Finally, we show that we are able to use this reporter transgenic to follow the onset of autoimmune T cell activation after regulatory T cell depletion in an established model of systemic autoimmunity. This IFN-γ reporter transgenic, termed "Gammaglow," offers a valuable new modality for tracking IFN-γ immunity, noninvasively and longitudinally over time.
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http://dx.doi.org/10.4049/jimmunol.1801453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452029PMC
April 2019

LEFTY2 inhibits endometrial receptivity by downregulating Orai1 expression and store-operated Ca entry.

J Mol Med (Berl) 2018 02 11;96(2):173-182. Epub 2017 Dec 11.

Department of Physiology I, Eberhard-Karls University Tuebingen, Wilhelmstr 56, D-72074, Tuebingen, Germany.

Early embryo development and endometrial differentiation are initially independent processes, and synchronization, imposed by a limited window of implantation, is critical for reproductive success. A putative negative regulator of endometrial receptivity is LEFTY2, a member of the transforming growth factor (TGF)-β family. LEFTY2 is highly expressed in decidualizing human endometrial stromal cells (HESCs) during the late luteal phase of the menstrual cycle, coinciding with the closure of the window of implantation. Here, we show that flushing of the uterine lumen in mice with recombinant LEFTY2 inhibits the expression of key receptivity genes, including Cox2, Bmp2, and Wnt4, and blocks embryo implantation. In Ishikawa cells, a human endometrial epithelial cell line, LEFTY2 downregulated the expression of calcium release-activated calcium channel protein 1, encoded by ORAI1, and inhibited store-operated Ca entry (SOCE). Furthermore, LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, as well as YM-58483, inhibited, whereas the Ca ionophore, ionomycin, strongly upregulated COX2, BMP2 and WNT4 expression in decidualizing HESCs. These findings suggest that LEFTY2 closes the implantation window, at least in part, by downregulating Orai1, which in turn limits SOCE and antagonizes expression of Ca-sensitive receptivity genes.

Key Messages: •Endometrial receptivity is negatively regulated by LEFTY2. •LEFTY2 inhibits the expression of key murine receptivity genes, including Cox2, Bmp2 and Wnt4, and blocks embryo implantation. •LEFTY2 downregulates the expression of Orai1 and inhibits SOCE. •LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, and YM-58483 inhibit COX2, BMP2, and WNT4 expression in endometrial cells. •Targeting LEFTY2 and Orai1 may represent a novel approach for treating unexplained infertility.
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http://dx.doi.org/10.1007/s00109-017-1610-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778154PMC
February 2018

Complement Factor B Is a Determinant of Both Metabolic and Cardiovascular Features of Metabolic Syndrome.

Hypertension 2017 Jul 24. Epub 2017 Jul 24.

From the Centre for Genomic and Experimental Medicine, MRC Institute for Genetics and Molecular Medicine, Edinburgh, United Kingdom (P.M.C., M.B., N.A., S.M.P., X.C.D., D.R., J.M., T.J.A.); British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute (P.M.C., M.B., N.A., R.N.C., A.T., L.H.J.-J., N.M.M., T.J.A.) and Royal (Dick) School of Veterinary Studies (X.C.D.), University of Edinburgh, United Kingdom; Department of Medicine (A.G.D., T.J.A) and Embryonic Stem Cell and Transgenics Facility, MRC Clinical Sciences Centre (B.M., Z.W.), Imperial College London, United Kingdom; and Division of Pathology, Centre for Comparative Pathology, Cancer Research UK Edinburgh Centre, United Kingdom (M.J.A.).

CFB (complement factor B) is elevated in adipose tissue and serum from patients with type 2 diabetes mellitus and cardiovascular disease, but the causal relationship to disease pathogenesis is unclear. Cfb is also elevated in adipose tissue and serum of the spontaneously hypertensive rat, a well-characterized model of metabolic syndrome. To establish the role of CFB in metabolic syndrome, we knocked out the gene in the spontaneously hypertensive rat. rats showed improved glucose tolerance and insulin sensitivity, redistribution of visceral to subcutaneous fat, increased adipocyte mitochondrial respiration, and marked changes in gene expression. rats also had lower blood pressure, increased ejection fraction and fractional shortening, and reduced left ventricular mass. These changes in metabolism and gene expression, in adipose tissue and left ventricle, suggest new adipose tissue-intrinsic and blood pressure-independent mechanisms for insulin resistance and cardiac hypertrophy in the spontaneously hypertensive rat. In silico analysis of the human locus revealed 2 -regulated expression quantitative trait loci for expression significantly associated with visceral fat, circulating triglycerides and hypertension in genome-wide association studies. Together, these data demonstrate a key role for in the development of spontaneously hypertensive rat metabolic syndrome phenotypes and of related traits in humans and indicate the potential for CFB as a novel target for treatment of cardiometabolic disease.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.117.09242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548512PMC
July 2017

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults.

Cell Rep 2017 01;18(5):1090-1099

MRC London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.
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http://dx.doi.org/10.1016/j.celrep.2017.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300902PMC
January 2017

Activation of SGK1 in Endometrial Epithelial Cells in Response to PI3K/AKT Inhibition Impairs Embryo Implantation.

Cell Physiol Biochem 2016 31;39(5):2077-2087. Epub 2016 Oct 31.

Departments of Cardiology, Vascular Medicine and Physiology, Institute for Ophthalmic Research and Experimental Retinal Prosthetics Group, Eberhard-Karls Universitaet Tuebingen, Tuebingen, Germany.

Background: Serum & Glucocorticoid Regulated Kinase 1 (SGK1) plays a fundamental role in ion and solute transport processes in epithelia. In the endometrium, down-regulation of SGK1 during the window of receptivity facilitates embryo implantation whereas expression of a constitutively active mutant in the murine uterus blocks implantation.

Methods/results: Here, we report that treatment of endometrial epithelial cells with specific inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT activity pathway results in reciprocal activation of SGK1. Flushing of the uterine lumen of mice with a cell permeable, substrate competitive phosphatidylinositol analogue that inhibits AKT activation (AKT inhibitor III) resulted in Sgk1 phosphorylation, down-regulation of the E3 ubiquitin-protein ligase Nedd4-2, and increased expression of epithelial Na+ channels (ENaC). Furthermore, exposure of the uterine lumen to AKT inhibitor III prior to embryo transfer induced a spectrum of early pregnancy defects, ranging from implantation failure to aberrant spacing of implantation sites.

Conclusion: Taken together, our data indicate that the balanced activities of two related serine/threonine kinases, AKT and SGK1, critically govern the implantation process.
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http://dx.doi.org/10.1159/000447903DOI Listing
February 2017

LEFTYA Activates the Epithelial Na+ Channel (ENaC) in Endometrial Cells via Serum and Glucocorticoid Inducible Kinase SGK1.

Cell Physiol Biochem 2016 8;39(4):1295-306. Epub 2016 Sep 8.

Department of Cardiology, Vascular Medicine and Physiology I, University of Tuebingen, Tuebigen, Germany.

Background: Serum & glucocorticoid inducible kinase (SGK1) regulates several ion channels, including amiloride sensitive epithelial Na+ channel (ENaC). SGK1 and ENaC in the luminal endometrium epithelium, are critically involved in embryo implantation, although little is known about their regulation. The present study explored whether SGK1 and ENaC are modulated by LEFTYA, a negative regulator of uterine receptivity.

Methods: Expression levels were determined by qRT-PCR and Western blotting, ENaC channel activity by whole cell patch clamp and transepithelial current by Ussing chamber experiments.

Results: Treatment of Ishikawa cells, an endometrial adenocarcinoma model cell line of endometrial epithelial cells, with LEFTYA rapidly up-regulated SGK1 and ENaC transcript and protein levels. Induction of ENaC in response to LEFTYA was blunted upon co-treatment with the SGK1 inhibitor EMD638683. ENaC levels also significantly upregulated upon expression of a constitutively active, but not a kinase dead, SGK1 mutant in Ishikawa cells. LEFTYA increased amiloride sensitive Na+-currents in Ishikawa cells and amiloride sensitive transepithelial current across the murine endometrium. Furthermore, LEFTYA induced the expression of ENaC in the endometrium of wild-type but not of Sgk1-deficient mice.

Conclusions: LEFTYA regulates the expression and activity of ENaC in endometrial epithelial cells via SGK1. Aberrant regulation of SGK1 and ENaC by LEFTYA could contribute to the pathogenesis of unexplained infertility.
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http://dx.doi.org/10.1159/000447834DOI Listing
January 2017

The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia.

Nature 2015 Aug 10;524(7565):356-60. Epub 2015 Aug 10.

Centre for Pharmacology and Therapeutics, Division of Experimental Medicine, Imperial College London, Hammersmith Hospital, London W12 0NN, UK.

The typical response of the adult mammalian pulmonary circulation to a low oxygen environment is vasoconstriction and structural remodelling of pulmonary arterioles, leading to chronic elevation of pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. Some mammals, however, exhibit genetic resistance to hypoxia-induced pulmonary hypertension. We used a congenic breeding program and comparative genomics to exploit this variation in the rat and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodelling. Slc39a12 encodes the zinc transporter ZIP12. Here we report that ZIP12 expression is increased in many cell types, including endothelial, smooth muscle and interstitial cells, in the remodelled pulmonary arterioles of rats, cows and humans susceptible to hypoxia-induced pulmonary hypertension. We show that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia dependent and that targeted inhibition of ZIP12 inhibits the rise in intracellular labile zinc in hypoxia-exposed pulmonary vascular smooth muscle cells and their proliferation in culture. We demonstrate that genetic disruption of ZIP12 expression attenuates the development of pulmonary hypertension in rats housed in a hypoxic atmosphere. This new and unexpected insight into the fundamental role of a zinc transporter in mammalian pulmonary vascular homeostasis suggests a new drug target for the pharmacological management of pulmonary hypertension.
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http://dx.doi.org/10.1038/nature14620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091855PMC
August 2015

Jarid2 Coordinates Nanog Expression and PCP/Wnt Signaling Required for Efficient ESC Differentiation and Early Embryo Development.

Cell Rep 2015 Jul 16;12(4):573-86. Epub 2015 Jul 16.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

Jarid2 is part of the Polycomb Repressor complex 2 (PRC2) responsible for genome-wide H3K27me3 deposition. Unlike other PRC2-deficient embryonic stem cells (ESCs), however, Jarid2-deficient ESCs show a severe differentiation block, altered colony morphology, and distinctive patterns of deregulated gene expression. Here, we show that Jarid2(-/-) ESCs express constitutively high levels of Nanog but reduced PCP signaling components Wnt9a, Prickle1, and Fzd2 and lowered β-catenin activity. Depletion of Wnt9a/Prickle1/Fzd2 from wild-type ESCs or overexpression of Nanog largely phenocopies these cellular defects. Co-culture of Jarid2(-/-) with wild-type ESCs restores variable Nanog expression and β-catenin activity and can partially rescue the differentiation block of mutant cells. In addition, we show that ESCs lacking Jarid2 or Wnt9a/Prickle1/Fzd2 or overexpressing Nanog induce multiple ICM formation when injected into normal E3.5 blastocysts. These data describe a previously unrecognized role for Jarid2 in regulating a core pluripotency and Wnt/PCP signaling circuit that is important for ESC differentiation and for pre-implantation development.
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http://dx.doi.org/10.1016/j.celrep.2015.06.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534826PMC
July 2015

Macrophage epoxygenase determines a profibrotic transcriptome signature.

J Immunol 2015 May 3;194(10):4705-4716. Epub 2015 Apr 3.

Physiological Genomics and Medicine, MRC Clinical Sciences Centre, Imperial College London, W12 0NN, UK.

Epoxygenases belong to the cytochrome P450 family. They generate epoxyeicosatrienoic acids, which are known to have anti-inflammatory effects, but little is known about their role in macrophage function. By high-throughput sequencing of RNA in primary macrophages derived from rodents and humans, we establish the relative expression of epoxygenases in these cells. Zinc-finger nuclease-mediated targeted gene deletion of the major rat macrophage epoxygenase Cyp2j4 (ortholog of human CYP2J2) resulted in reduced epoxyeicosatrienoic acid synthesis. Cyp2j4(-/-) macrophages have relatively increased peroxisome proliferator-activated receptor-γ levels and show a profibrotic transcriptome, displaying overexpression of a specific subset of genes (260 transcripts) primarily involved in extracellular matrix, with fibronectin being the most abundantly expressed transcript. Fibronectin expression is under the control of epoxygenase activity in human and rat primary macrophages. In keeping with the in vitro findings, Cyp2j4(-/-) rats show upregulation of type I collagen following unilateral ureter obstruction of the kidney, and quantitative proteomics analysis (liquid chromatography-tandem mass spectrometry) showed increased renal type I collagen and fibronectin protein abundance resulting from experimentally induced crescentic glomerulonephritis in these rats. Taken together, these results identify the rat epoxygenase Cyp2j4 as a determinant of a profibrotic macrophage transcriptome that could have implications in various inflammatory conditions, depending on macrophage function.
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http://dx.doi.org/10.4049/jimmunol.1402979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417646PMC
May 2015

Targeted glomerular angiopoietin-1 therapy for early diabetic kidney disease.

J Am Soc Nephrol 2014 Jan 5;25(1):33-42. Epub 2013 Sep 5.

Cardiovascular Division, King's College London, London, United Kingdom;

Vascular growth factors play an important role in maintaining the structure and integrity of the glomerular filtration barrier. In healthy adult glomeruli, the proendothelial survival factors vascular endothelial growth factor-A (VEGF-A) and angiopoietin-1 are constitutively expressed in glomerular podocyte epithelia. We demonstrate that this milieu of vascular growth factors is altered in streptozotocin-induced type 1 diabetic mice, with decreased angiopoietin-1 levels, VEGF-A upregulation, decreased soluble VEGF receptor-1 (VEGFR1), and increased VEGFR2 phosphorylation. This was accompanied by marked albuminuria, nephromegaly, hyperfiltration, glomerular ultrastructural alterations, and aberrant angiogenesis. We subsequently hypothesized that restoration of angiopoietin-1 expression within glomeruli might ameliorate manifestations of early diabetic glomerulopathy. Podocyte-specific inducible repletion of angiopoietin-1 in diabetic mice caused a 70% reduction of albuminuria and prevented diabetes-induced glomerular endothelial cell proliferation; hyperfiltration and renal morphology were unchanged. Furthermore, angiopoietin-1 repletion in diabetic mice increased Tie-2 phosphorylation, elevated soluble VEGFR1, and was paralleled by a decrease in VEGFR2 phosphorylation and increased endothelial nitric oxide synthase Ser(1177) phosphorylation. Diabetes-induced nephrin phosphorylation was also reduced in mice with angiopoietin-1 repletion. In conclusion, targeted angiopoietin-1 therapy shows promise as a renoprotective tool in the early stages of diabetic kidney disease.
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http://dx.doi.org/10.1681/ASN.2012121218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871771PMC
January 2014

Disordered IL-33/ST2 activation in decidualizing stromal cells prolongs uterine receptivity in women with recurrent pregnancy loss.

PLoS One 2012 27;7(12):e52252. Epub 2012 Dec 27.

Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry, United Kingdom.

Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. Here we show that human endometrial stromal cells (HESCs) rapidly release IL-33, a key regulator of innate immune responses, upon decidualization. In parallel, differentiating HESCs upregulate the IL-33 transmembrane receptor ST2L and other pro-inflammatory mediators before mounting a profound anti-inflammatory response that includes downregulation of ST2L and increased expression of the soluble decoy receptor sST2. We demonstrate that HESCs secrete factors permissive of embryo implantation in mice only during the pro-inflammatory phase of the decidual process. IL-33 knockdown in undifferentiated HESCs was sufficient to abrogate this pro-inflammatory decidual response. Further, sequential activation of the IL-33/ST2L/sST2 axis was disordered in decidualizing HESCs from women with recurrent pregnancy loss. Signals from these cultures prolonged the implantation window but also caused subsequent pregnancy failure in mice. Thus, Il-33/ST2 activation in HESCS drives an autoinflammatory response that controls the temporal expression of receptivity genes. Failure to constrain this response predisposes to miscarriage by allowing out-of-phase implantation in an unsupportive uterine environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052252PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531406PMC
July 2013

Developmental stalling and organ-autonomous regulation of morphogenesis.

Proc Natl Acad Sci U S A 2011 Nov 14;108(48):19270-5. Epub 2011 Nov 14.

Department of Craniofacial Development, Dental Institute, Kings College London, Guys Hospital, London SE1 6RT, United Kingdom.

Timing of organ development during embryogenesis is coordinated such that at birth, organ and fetal size and maturity are appropriately proportioned. The extent to which local developmental timers are integrated with each other and with the signaling interactions that regulate morphogenesis to achieve this end is not understood. Using the absolute requirement for a signaling pathway activity (bone morphogenetic protein, BMP) during a critical stage of tooth development, we show that suboptimal levels of BMP signaling do not lead to abnormal morphogenesis, as suggested by mutants affecting BMP signaling, but to a 24-h stalling of the intrinsic developmental clock of the tooth. During this time, BMP levels accumulate to reach critical levels whereupon tooth development restarts, accelerates to catch up with development of the rest of the embryo and completes normal morphogenesis. This suggests that individual organs can autonomously control their developmental timing to adjust their stage of development to that of other organs. We also find that although BMP signaling is critical for the bud-to-cap transition in all teeth, levels of BMP signaling are regulated differently in multicusped teeth. We identify an interaction between two homeodomain transcription factors, Barx1 and Msx1, which is responsible for setting critical levels of BMP activity in multicusped teeth and provides evidence that correlates the levels of Barx1 transcriptional activity with cuspal complexity. This study highlights the importance of absolute levels of signaling activity for development and illustrates remarkable self-regulation in organogenesis that ensures coordination of developmental processes such that timing is subordinate to developmental structure.
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http://dx.doi.org/10.1073/pnas.1112801108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228462PMC
November 2011

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure.

Nat Med 2011 Oct 16;17(11):1509-13. Epub 2011 Oct 16.

Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, London, UK.

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.
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http://dx.doi.org/10.1038/nm.2498DOI Listing
October 2011

Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome.

Dev Biol 2009 Jul 24;331(1):38-49. Epub 2009 Apr 24.

Department of Craniofacial Development and Orthodontics, Dental Institute, King's College London, Floor 27, Guy's Hospital, London SE1 9RT, UK.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant or spontaneous disorder characterized by multiple cutaneous basal cell carcinomas, odontogenic keratocysts, skeletal anomalies and facial dysmorphology, including cleft lip and palate. Causative mutations for NBCCS occur in the PTCH1 gene on chromosome 9q22.3-q31, which encodes the principle receptor for the Hedgehog signalling pathway. We have investigated the molecular basis of craniofacial defects seen in NBCCS using a transgenic mouse model expressing Shh in basal epithelium under a Keratin-14 promoter. These mice have an absence of flat bones within the skull vault, hypertelorism, open-bite malocclusion, cleft palate and arrested tooth development. Significantly, increased Hedgehog signal transduction in these mice can influence cell fate within the craniofacial region. In medial edge epithelium of the palate, Shh activity prevents apoptosis and subsequent palatal shelf fusion. In contrast, high levels of Shh in odontogenic epithelium arrests tooth development at the bud stage, secondary to a lack of cell proliferation in this region. These findings illustrate the importance of appropriately regulated Hedgehog signalling during early craniofacial development and demonstrate that oro-facial clefting and hypodontia seen in NBCCS can occur as a direct consequence of increased Shh signal activity within embryonic epithelial tissues.
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http://dx.doi.org/10.1016/j.ydbio.2009.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2696601PMC
July 2009

A reappraisal of evidence for probabilistic models of allelic exclusion.

Proc Natl Acad Sci U S A 2009 Jan 30;106(2):516-21. Epub 2008 Dec 30.

Lymphocyte Development Group and ES Cell and Transgenics Facility, Medical Research Council Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom.

B cell development requires the coordinated rearrangement of Ig heavy (IgH) and light chain loci (IgL). Most mature B cells express a single B cell receptor of unique specificity, and a central question in immunology concerns the mechanisms that prevent the productive rearrangement of >1 IgH and IgL allele per cell. Probabilistic models of allelic exclusion maintain that simultaneous rearrangement of both alleles is rare, because the likelihood of undergoing rearrangement is low for a given Ig allele. Strong support for this idea came from studies in which a GFP marker was inserted into the Igk locus. In this system, the probability of high-level germ-line transcription and subsequent locus rearrangement appeared to be low in pre-B cells. Readdressing the validity of GFP expression as a reporter for the level of germ-line transcription, we found a striking discordance between GFP transcript and protein levels at the pre-B cell stage, which is explained at least in part by the developmentally regulated usage of 2 alternative Igk-J germ-line promoters. These results question the validity of the kappa-GFP system as evidence for probabilistic models of allelic exclusion.
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http://dx.doi.org/10.1073/pnas.0808764105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626735PMC
January 2009

Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice.

Diabetes 2008 Oct 22;57(10):2824-33. Epub 2008 Jul 22.

Cardiovascular Division, King's College London School of Medicine, Guy's Hospital, King's College London, London, UK.

Objective: Podocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.

Research Design And Methods: We studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques.

Results: Nondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an approximately 70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69-199) vs. 43 (26.8-69) mug/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.

Conclusions: Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.
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http://dx.doi.org/10.2337/db08-0647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2551695PMC
October 2008

Cohesins functionally associate with CTCF on mammalian chromosome arms.

Cell 2008 Feb 31;132(3):422-33. Epub 2008 Jan 31.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.

Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.
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http://dx.doi.org/10.1016/j.cell.2008.01.011DOI Listing
February 2008

GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology.

Genomics 2006 Nov 17;88(5):580-90. Epub 2006 Aug 17.

Biosciences, School of Health Sciences & Social Care, Brunel University, Uxbridge UB8 3PH, UK.

Friedreich ataxia (FRDA) is a neurodegenerative disorder caused by an unstable GAA repeat expansion mutation within intron 1 of the FXN gene. However, the origins of the GAA repeat expansion, its unstable dynamics within different cells and tissues, and its effects on frataxin expression are not yet completely understood. Therefore, we have chosen to generate representative FRDA mouse models by using the human FXN GAA repeat expansion itself as the genetically modified mutation. We have previously reported the establishment of two lines of human FXN YAC transgenic mice that contain unstable GAA repeat expansions within the appropriate genomic context. We now describe the generation of FRDA mouse models by crossbreeding of both lines of human FXN YAC transgenic mice with heterozygous Fxn knockout mice. The resultant FRDA mice that express only human-derived frataxin show comparatively reduced levels of frataxin mRNA and protein expression, decreased aconitase activity, and oxidative stress, leading to progressive neurodegenerative and cardiac pathological phenotypes. Coordination deficits are present, as measured by accelerating rotarod analysis, together with a progressive decrease in locomotor activity and increase in weight. Large vacuoles are detected within neurons of the dorsal root ganglia (DRG), predominantly within the lumbar regions in 6-month-old mice, but spreading to the cervical regions after 1 year of age. Secondary demyelination of large axons is also detected within the lumbar roots of older mice. Lipofuscin deposition is increased in both DRG neurons and cardiomyocytes, and iron deposition is detected in cardiomyocytes after 1 year of age. These mice represent the first GAA repeat expansion-based FRDA mouse models that exhibit progressive FRDA-like pathology and thus will be of use in testing potential therapeutic strategies, particularly GAA repeat-based strategies.
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http://dx.doi.org/10.1016/j.ygeno.2006.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842930PMC
November 2006

Post-embryonic ablation of AgRP neurons in mice leads to a lean, hypophagic phenotype.

FASEB J 2005 Oct 11;19(12):1680-2. Epub 2005 Aug 11.

Department of Metabolic Medicine, Faculty of Medicine, Imperial College London, London, UK.

Agouti-related protein (AgRP) and neuropeptide Y (NPY) are colocalized in arcuate nucleus (arcuate) neurons implicated in the regulation of energy balance. Both AgRP and NPY stimulate food intake when administered into the third ventricle and are up-regulated in states of negative energy balance. However, mice with targeted deletion of either NPY or AgRP or both do not have major alterations in energy homeostasis. Using bacterial artificial chromosome (BAC) transgenesis we have targeted expression of a neurotoxic CAG expanded form of ataxin-3 to AgRP-expressing neurons in the arcuate. This resulted in a 47% loss of AgRP neurons by 16 weeks of age, a significantly reduced body weight, (wild-type mice (WT) 34.7+/-0.7 g vs. transgenic mice (Tg) 28.6+/-0.6 g, P<0.001), and reduced food intake (WT 5.0+/-0.2 vs. Tg 3.6+/-0.1 g per day, P<0.001). Transgenic mice had significantly reduced total body fat, plasma insulin, and increased brown adipose tissue UCP1 expression. Transgenic mice failed to respond to peripherally administered ghrelin but retained sensitivity to PYY 3-36. These data suggest that postembryonic partial loss of AgRP/NPY neurons leads to a lean, hypophagic phenotype.
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http://dx.doi.org/10.1096/fj.04-3434fjeDOI Listing
October 2005

Mapping and functional analysis of regulatory sequences in the mouse lambda5-VpreB1 domain.

Mol Immunol 2005 Jul 26;42(11):1283-92. Epub 2005 Feb 26.

Gene regulation and Chromatin Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London W12 ONN, UK.

The lambda5 and VpreB genes encode the components of the surrogate light-chain which forms part of the pre-B cell receptor and plays a key role in B cell development. In the mouse, the lambda5 and VpreB1 genes are closely linked and are co-regulated by a multi-component locus control region. To identify the sequences that regulate lambda5 and VpreB1 expression during B cell development, we have comprehensively mapped the DNaseI hypersensitive sites (HS) in the lambda5-VpreB1 functional domain. The active domain contains 12 HS that are distributed at high density across the 18.3 kb region that forms the lambda5 and VpreB1 functional unit. Analysis of a reporter gene driven by the VpreB1 promoter in transgenic mice identified a novel enhancer associated with two HS located upstream of lambda5. The lambda5-VpreB1 locus was also found to be closely linked to the ubiquitously expressed Topoisomerase-3beta (Topo3beta) gene. The VpreB1 and Topo3beta genes have entirely different expression patterns despite the fact that the two promoters are separated by a distance of only 1.5 kb.
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http://dx.doi.org/10.1016/j.molimm.2005.01.003DOI Listing
July 2005

The regulated long-term delivery of therapeutic proteins by using antigen-specific B lymphocytes.

Proc Natl Acad Sci U S A 2004 Nov 1;101(46):16298-303. Epub 2004 Nov 1.

Lymphocyte Development Group, Gene Regulation and Chromatin Group, Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College of Science, Technology, Du Cane Road, London W12 ONN, United Kingdom.

Memory lymphocytes are important mediators of the immune response. These cells are long-lived and undergo clonal expansion upon reexposure to specific antigen, differentiating into effector cells that secrete Ig or cytokines while maintaining a residual pool of memory T and B lymphocytes. Here, the ability of antigen-specific lymphocytes to undergo repeated cycles of antigen-driven clonal expansion and contraction is exploited in a therapeutic protocol aimed at regulating protein delivery. The principle of this strategy is to introduce genes encoding proteins of therapeutic interest into a small number of antigen-specific B lymphocytes. Output of therapeutic protein can then be regulated in vivo by manipulating the size of the responder population by antigen challenge. To evaluate whether such an approach is feasible, we developed a mouse model system in which Emu- and Iglambda-based vectors were used to express human erythropoietin (hEPO) gene in B lymphocytes. These mice were then immunized with the model antigen phycoerythrin (PE), and immune splenocytes (or purified PE-specific B lymphocytes) were adoptively transferred to normal or mutant (EPO-deficient) hosts. High levels of hEPO were detected in the serum of adoptively transferred normal mice after PE administration, and this responsiveness was maintained for several months. Similarly, in EPO-deficient anemic recipients, antigen-driven hEPO expression was shown to restore hematocrit levels to normal. These results show that antigen-mediated regulation of memory lymphocytes can be used as a strategy for delivering therapeutic proteins in vivo.
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http://dx.doi.org/10.1073/pnas.0405271101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC528951PMC
November 2004

GAA repeat instability in Friedreich ataxia YAC transgenic mice.

Genomics 2004 Aug;84(2):301-10

Department of Medical Genetics, Division of Medicine, Imperial College London, North West Thames Regional Genetics Service, Northwick Park Hospital, Watford Road, Harrow, HA1 3UJ, UK.

Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.
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http://dx.doi.org/10.1016/j.ygeno.2004.04.003DOI Listing
August 2004

Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium.

Blood 2004 Sep 4;104(5):1344-9. Epub 2004 May 4.

Department of Immunology, Imperial College London, Hammersmith Hospital, Du Cane Rd, London W12 0NN.

We have generated transgenic mice expressing the leech anticoagulant hirudin and human tissue factor pathway inhibitor tethered to the cell surface by fusion with fragments of human CD4 and P-selectin. Expression of the transgenes is under the control of the CD31 (platelet endothelial cell adhesion molecule [PECAM]) promoter, limiting expression to endothelial cells, monocytes, and platelets. In addition, the P-selectin sequence directs expression to secretory granules. Functional cell surface expression only occurs when the cells are activated. In a mouse model of systemic lipopolysaccharide (LPS)-induced endotoxemia, we show that expression of either anticoagulant on activated endothelium inhibits the widespread intravascular thrombosis, thrombocytopenia, and consumptive coagulopathy associated with endotoxemia. Importantly, non- LPS-treated transgenic mice had normal baseline bleeding times. We speculate that targeted delivery of anticoagulants to the endothelium may be a strategy worth pursuing in clinical sepsis to improve efficacy of systemic anticoagulation while minimizing potential hemorrhagic side effects.
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http://dx.doi.org/10.1182/blood-2003-12-4365DOI Listing
September 2004

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing.

Nature 2003 Apr;422(6934):909-13

CSC Gene Control Mechanisms and Disease Group, Experimental Genetics, Faculty of Medicine, Imperial College School of Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.
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http://dx.doi.org/10.1038/nature01596DOI Listing
April 2003

Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes.

Dev Cell 2003 Apr;4(4):481-95

X Inactivation Group, MRC Clinical Sciences Centre, ICSM, Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom.

Previous studies have implicated the Eed-Enx1 Polycomb group complex in the maintenance of imprinted X inactivation in the trophectoderm lineage in mouse. Here we show that recruitment of Eed-Enx1 to the inactive X chromosome (Xi) also occurs in random X inactivation in the embryo proper. Localization of Eed-Enx1 complexes to Xi occurs very early, at the onset of Xist expression, but then disappears as differentiation and development progress. This transient localization correlates with the presence of high levels of the complex in totipotent cells and during early differentiation stages. Functional analysis demonstrates that Eed-Enx1 is required to establish methylation of histone H3 at lysine 9 and/or lysine 27 on Xi and that this, in turn, is required to stabilize the Xi chromatin structure.
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http://dx.doi.org/10.1016/s1534-5807(03)00068-6DOI Listing
April 2003