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Cambridge, United Kingdom

Grewal S.,University of Oxford | Grewal S.,Gurdon Institute | Carver J.,University of Oxford | Ridley A.J.,Kings College London | Mardon H.J.,University of Oxford
Biology of Reproduction | Year: 2010

Implantation of the embryo into the uterine compartment is a multistep event involving attachment of the embryo to the endometrial epithelia, followed by invasion of the embryo through the endometrial stroma. RHOA, RAC1, and CDC42 are members of the Rho GTPase family of proteins, which control cell functions such as cell migration and cytoskeletal reorganization. Herein, using a heterologous in vitro coculture model, we show that implantation of mouse blastocysts into human endometrial stromal cells (hESCs) is regulated by Rho GTPase activity in hESCs. Whereas iRNA-mediated silencing of RAC1 expression in hESCs led to inhibition of embryo implantation, silencing of either RHOA or CDC42 in hESCs promoted embryo implantation in coculture assays. Analysis of downstream signaling pathways demonstrated that RAC1 silencing was associated with decreased focal adhesion disassembly and resulted in large focal adhesion complexes in hESCs. In contrast, RHOA or CDC42 silencing resulted in perturbed focal adhesion assembly, leading to a decrease in the number of focal adhesions observed. Furthermore, inhibition of Rho signaling using a Rho kinase inhibitor, Y27632, led to decreased activation of protein tyrosine kinase 2 (PTK2, also called focal adhesion kinase) and decreased focal adhesion assembly. Importantly, perturbation of focal adhesion turnover in hESCs, mediated by PTK2 silencing, resulted in inhibition of embryo implantation into hESC monolayers. These findings suggest that Rho GTPase-PTK2-dependent remodeling of the endometrial stromal cell compartment may be critical for successful embryo implantation. © 2010 by the Society for the Study of Reproduction, Inc. Source

Schiavone D.,Medical Research Council | Guilbaud G.,Medical Research Council | Murat P.,University of Cambridge | Papadopoulou C.,Medical Research Council | And 5 more authors.
EMBO Journal | Year: 2014

REV1-deficient chicken DT40 cells are compromised in replicating G quadruplex (G4)-forming DNA. This results in localised, stochastic loss of parental chromatin marks and changes in gene expression. We previously proposed that this epigenetic instability arises from G4-induced replication fork stalls disrupting the accurate propagation of chromatin structure through replication. Here, we test this model by showing that a single G4 motif is responsible for the epigenetic instability of the BU-1 locus in REV1-deficient cells, despite its location 3.5 kb from the transcription start site (TSS). The effect of the G4 is dependent on it residing on the leading strand template, but is independent of its in vitro thermal stability. Moving the motif to more than 4 kb from the TSS stabilises expression of the gene. However, loss of histone modifications (H3K4me3 and H3K9/14ac) around the transcription start site correlates with the position of the G4 motif, expression being lost only when the promoter is affected. This supports the idea that processive replication is required to maintain the histone modification pattern and full transcription of this model locus. © 2014 MRC Laboratory of Molecular Biology. Published under the terms of the CC BY 4.0 license. Source

Pines J.,Gurdon Institute | Hagan I.,Paterson Institute for Cancer Research
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2011

Orson Welles might have been a little unfair on the Swiss, after all cuckoo clocks were developed in the Schwartzwald, but, more importantly, Swiss democracy gives remarkably stable government with considerable decision-making at the local level. The alternative is the battling city-states of Renaissance Italy: culturally rich but chaotic at a higher level of organization. As our understanding of the cell cycle improves, it appears that the cell is organized more along the lines of Switzerland than Renaissance Italy, and one major challenge is to determine how local decisions are made and coordinated to produce the robust cell cycle mechanisms that we observe in the cell as a whole. © 2011 The Royal Society. Source

News Article
Site: http://www.biosciencetechnology.com/rss-feeds/all/rss.xml/all

The world of epigenetics – where molecular ‘switches’ attached to DNA turn genes on and off – has just got bigger with the discovery by a team of scientists from the University of Cambridge of a new type of epigenetic modification. Published in the journal Nature Structural and Molecular Biology, the discovery suggests that many more DNA modifications than previously thought may exist in human, mouse and other vertebrates. DNA is made up of four ‘bases’: molecules known as adenine, cytosine, guanine and thymine – the A, C, G and T letters. Strings of these letters form genes, which provide the code for essential proteins, and other regions of DNA, some of which can regulate these genes. Epigenetics (epi - the Greek prefix meaning ‘on top of’) is the study of how genes are switched on or off. It is thought to be one explanation for how our environment and behavior, such as our diet or smoking habit, can affect our DNA and how these changes may even be passed down to our children and grandchildren. Epigenetics has so far focused mainly on studying proteins called histones that bind to DNA. Such histones can be modified, which can result in genes being switched on or of. In addition to histone modifications, genes are also known to be regulated by a form of epigenetic modification that directly affects one base of the DNA, namely the base C. More than 60 years ago, scientists discovered that C can be modified directly through a process known as methylation, whereby small molecules of carbon and hydrogen attach to this base and act like switches to turn genes on and off, or to ‘dim’ their activity. Around 75 million (one in ten) of the Cs in the human genome are methylated. Now, researchers at the Wellcome Trust-Cancer Research UK Gurdon Institute and the Medical Research Council Cancer Unit at the University of Cambridge have identified and characterized a new form of direct modification – methylation of the base A – in several species, including frogs, mouse and humans. Methylation of A appears to be far less common that C methylation, occurring on around 1,700 As in the genome, but is spread across the entire genome. However, it does not appear to occur on sections of our genes known as exons, which provide the code for proteins. “These newly-discovered modifiers only seem to appear in low abundance across the genome, but that does not necessarily mean they are unimportant,” said Dr. Magdalena Koziol from the Gurdon Institute. “At the moment, we don’t know exactly what they actually do, but it could be that even in small numbers they have a big impact on our DNA, gene regulation and ultimately human health.” More than two years ago, Dr. Koziol made the discovery while studying modifications of RNA. There are 66 known RNA modifications in the cells of complex organisms. Using an antibody that identifies a specific RNA modification, Dr. Koziol looked to see if the analogous modification was also present on DNA, and discovered that this was indeed the case. Researchers at the MRC Cancer Unit then confirmed that this modification was to DNA, rather than from any RNA contaminating the sample. “It’s possible that we struck lucky with this modifier,” said Dr. Koziol, “but we believe it is more likely that there are many more modifications that directly regulate our DNA. This could open up the field of epigenetics.” The research was funded by the Biotechnology and Biological Sciences Research Council, Human Frontier Science Program, Isaac Newton Trust, Wellcome Trust, Cancer Research UK and the Medical Research Council.

Conduit P.T.,University of Oxford | Brunk K.,Gurdon Institute | Dobbelaere J.,Gurdon Institute | Dobbelaere J.,Research Institute of Molecular Pathology | And 5 more authors.
Current Biology | Year: 2010

Background: Centrosomes are major microtubule organizing centers in animal cells, and they comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Centrosome size is tightly regulated during the cell cycle, and it has recently been shown that the two centrosomes in certain stem cells are often asymmetric in size. There is compelling evidence that centrioles influence centrosome size, but how centrosome size is set remains mysterious. Results: We show that the conserved Drosophila PCM protein Cnn exhibits an unusual dynamic behavior, because Cnn molecules only incorporate into the PCM closest to the centrioles and then spread outward through the rest of the PCM. Cnn incorporation into the PCM is driven by an interaction with the conserved centriolar proteins Asl (Cep152 in humans) and DSpd-2 (Cep192 in humans). The rate of Cnn incorporation into the PCM is tightly regulated during the cell cycle, and this rate influences the amount of Cnn in the PCM, which in turn is an important determinant of overall centrosome size. Intriguingly, daughter centrioles in syncytial embryos only start to incorporate Cnn as they disengage from their mothers; this generates a centrosome size asymmetry, with mother centrioles always initially organizing more Cnn than their daughters. Conclusions: Centrioles can control the amount of PCM they organize by regulating the rate of Cnn incorporation into the PCM. This mechanism can explain how centrosome size is regulated during the cell cycle and also allows mother and daughter centrioles to set centrosome size independently of one another. © 2010 Elsevier Ltd. All rights reserved. Source

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