Hutchison Research Center

Cambridge, United Kingdom

Hutchison Research Center

Cambridge, United Kingdom
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Faure A.J.,European Bioinformatics Institute | Schmidt D.,Cancer Research UK Research Institute | Schmidt D.,Hutchison Research Center | Watt S.,Cancer Research UK Research Institute | And 14 more authors.
Genome Research | Year: 2012

The cohesin protein complex contributes to transcriptional regulation in a CTCF-independent manner by colocalizing with master regulators at tissue-specific loci. The regulation of transcription involves the concerted action of multiple transcription factors (TFs) and cohesin's role in this context of combinatorial TF binding remains unexplored. To investigate cohesin-non-CTCF (CNC) binding events in vivo we mapped cohesin and CTCF, as well as a collection of tissuespecific and ubiquitous transcriptional regulators using ChIP-seq in primary mouse liver. We observe a positive correlation between the number of distinct TFs bound and the presence of CNC sites. In contrast to regions of the genome where cohesin and CTCF colocalize, CNC sites coincide with the binding of master regulators and enhancer-markers and are significantly associated with liver-specific expressed genes. We also show that cohesin presence partially explains the commonly observed discrepancy between TF motif score and ChIP signal. Evidence from these statistical analyses in wildtype cells, and comparisons to maps of TF binding in Rad21-cohesin haploinsufficient mouse liver, suggests that cohesin helps to stabilize large protein-DNA complexes. Finally, we observe that the presence of mirrored CTCF binding events at promoters and their nearby cohesin-bound enhancers is associated with elevated expression levels. © 2012, Published by Cold Spring Harbor Laboratory Press.

Schmidt D.,Cancer Research UK Research Institute | Schmidt D.,Hutchison Research Center | Schwalie P.C.,European Bioinformatics Institute | Ross-Innes C.S.,Cancer Research UK Research Institute | And 10 more authors.
Genome Research | Year: 2010

The cohesin protein complex holds sister chromatids in dividing cells together and is essential for chromosome segregation. Recently, cohesin has been implicated in mediating transcriptional insulation, via its interactions with CTCF. Here, we show in different cell types that cohesin functionally behaves as a tissue-specific transcriptional regulator, independent of CTCF binding. By performing matched genome-wide binding assays (ChIP-seq) in human breast cancer cells (MCF-7), we discovered thousands of genomic sites that share cohesin and estrogen receptor alpha (ER) yet lack CTCF binding. By use of human hepatocellular carcinoma cells (HepG2), we found that liver-specific transcription factors colocalize with cohesin independently of CTCF at liver-specific targets that are distinct from those found in breast cancer cells. Furthermore, estrogen-regulated genes are preferentially bound by both ER and cohesin, and functionally, the silencing of cohesin caused aberrant re-entry of breast cancer cells into cell cycle after hormone treatment. We combined chromosomal interaction data in MCF-7 cells with our cohesin binding data to show that cohesin is highly enriched at ER-bound regions that capture inter-chromosomal loop anchors. Together, our data show that cohesin cobinds across the genome with transcription factors independently of CTCF, plays a functional role in estrogen-regulated transcription, and may help to mediate tissue-specific transcriptional responses via long-range chromosomal interactions. Copyright © 2010 by Cold Spring Harbor Laboratory Press.

Ip J.Y.,University of Toronto | Schmidt D.,Cancer Research UK Research Institute | Schmidt D.,Hutchison Research Center | Pan Q.,University of Toronto | And 5 more authors.
Genome Research | Year: 2011

The rate of RNA polymerase II (Pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing (AS) is not well understood. We performed experiments to perturb Pol II elongation and then globally compared AS patterns with genome-wide Pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with Pol II elongation inhibition-dependent changes in AS. Under conditions that interfere with Pol II elongation, including cell stress, increased Pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, AS, and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. © 2011 by Cold Spring Harbor Laboratory Press.

Laudadio I.,Catholic University of Louvain | Manfroid I.,University of Liège | Achouri Y.,Catholic University of Louvain | Schmidt D.,Cancer Research UK Research Institute | And 13 more authors.
Gastroenterology | Year: 2012

Hepatocyte differentiation is controlled by liver-enriched transcription factors (LETFs). We investigated whether LETFs control microRNA expression during development and whether this control is required for hepatocyte differentiation. Using in vivo DNA binding assays, we identified miR-122 as a direct target of the LETF hepatocyte nuclear factor (HNF) 6. The role and mechanisms of the HNF6miR-122 gene cascade in hepatocyte differentiation were studied in vivo and in vitro by gain-of-function and loss-of-function experiments, using developing mice and zebrafish as model organisms. HNF6 and its paralog Onecut2 are strong transcriptional stimulators of miR-122 expression. Specific levels of miR-122 were required for proper progression of hepatocyte differentiation; miR-122 stimulated the expression of hepatocyte-specific genes and most LETFs, including HNF6. This indicates that HNF6 and miR-122 form a positive feedback loop. Stimulation of hepatocyte differentiation by miR-122 was lost in HNF6-null mice, revealing that a transcription factor can mediate microRNA function. All hepatocyte-specific genes whose expression was stimulated by miR-122 bound HNF6 in vivo, confirming their direct regulation by this factor. Hepatocyte differentiation is directed by a positive feedback loop that includes a transcription factor (HNF6) and a microRNA (miR-122) that are specifically expressed in liver. These findings could lead to methods to induce differentiation of hepatocytes in vitro and improve our understanding of liver cell dedifferentiation in pathologic conditions. © 2012 AGA Institute.

Aylon Y.,Weizmann Institute of Science | Ofir-Rosenfeld Y.,Hutchison Research Center | Yabuta N.,Osaka University | Lapi E.,University of Oxford | And 3 more authors.
Genes and Development | Year: 2010

Apoptosis is an important mechanism to eliminate potentially tumorigenic cells. The tumor suppressor p53 plays a pivotal role in this process. Many tumors harbor mutant p53, but others evade its tumor-suppressive effects by altering the expression of proteins that regulate the p53 pathway. ASPP1 (apoptosis-stimulating protein of p53-1) is a key mediator of the nuclear p53 apoptotic response. Under basal conditions, ASPP1 is cytoplasmic. We report that, in response to oncogenic stress, the tumor suppressor Lats2 (large tumor suppressor 2) phosphorylates ASPP1 and drives its translocation into the nucleus. Together, Lats2 and ASPP1 shunt p53 to proapoptotic promoters and promote the death of polyploid cells. These effects are overridden by the Yap1 (Yes-associated protein 1) oncoprotein, which disrupts Lats2-ASPP1 binding and antagonizes the tumor-suppressing function of the Lats2/ASPP1/p53 axis. © 2010 by Cold Spring Harbor Laboratory Press.

Marshall A.,Cancer Research UK Research Institute | Marshall A.,Addenbrookes Hospital | Lukk M.,Cancer Research UK Research Institute | Kutter C.,Cancer Research UK Research Institute | And 4 more authors.
PLoS ONE | Year: 2013

Background: Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets. Methodology and Principal Findings: Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC. Conclusion: HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies. © 2013 Marshall et al.

Murray M.J.,Hutchison Research Center | Murray M.J.,Addenbrookes Hospital | Nicholson J.C.,Hutchison Research Center | Nicholson J.C.,Addenbrookes Hospital
Paediatrics and Child Health | Year: 2010

Germ cell tumours (GCTs) are a heterogeneous group of predominantly midline tumours uniquely occurring from birth to late adulthood. All are believed to arise from the totipotent primordial germ cell. In childhood, ∼50% are gonadal and ∼50% extragonadal (∼20% intracranial and ∼30% extracranial) and clinical presentation depends on tumour site. Teratomas account for ∼50% of all paediatric GCTs and are considered benign in childhood. Malignant GCTs often secrete the tumour markers alpha-fetoprotein (AFP) and human chorionic gonadotrophin (ß-HCG), which may help in diagnosis and follow-up. Management involves complete surgical resection for teratomas and non-metastatic gonadal tumours. In the UK, chemotherapy is reserved for stage 2-4 extracranial malignant GCTs, resulting in >90% five-year overall survival (OS). Radiotherapy is rarely indicated for extracranial disease. Intracranial tumours typically occur in the pineal or suprasellar region. Intracranial germinomas are cured in >90% cases with radiotherapy or combined chemo-radiotherapy. About two-thirds of non-germinomatous intracranial tumours are cured with combined chemo-radiotherapy. Current issues relating to the management of teenagers and young adults (TYAs) with GCTs and implications of tumour biology are highlighted in this review. © 2009 Elsevier Ltd. All rights reserved.

Sledz P.,University of Cambridge | Stubbs C.J.,University of Cambridge | Lang S.,University of Cambridge | Yang Y.-Q.,University of Cambridge | And 4 more authors.
Angewandte Chemie - International Edition | Year: 2011

Picking the pocket: Crystal-packing interactions are present in every macromolecular structure solved with X-ray methods; however, they have not been extensively used to gain insight into molecular recognition. A novel approach is described to utilize these packing interactions for the discovery of a flexible binding site and ligands recognized by it (see picture). © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Esposito A.,University of Cambridge | Esposito A.,Hutchison Research Center | Choimet J.-B.,University of Cambridge | Skepper J.N.,University of Cambridge | And 4 more authors.
Biophysical Journal | Year: 2010

During its 48 h asexual reproduction cycle, the malaria parasite Plasmodium falciparum ingests and digests hemoglobin in excess of its metabolic requirements and causes major changes in the homeostasis of the host red blood cell (RBC). A numerical model suggested that this puzzling excess consumption of hemoglobin is necessary for the parasite to reduce the colloidosmotic pressure within the host RBC, thus preventing lysis before completion of its reproduction cycle. However, the validity of the colloidosmotic hypothesis appeared to be compromised by initial conflicts between model volume predictions and experimental observations. Here, we investigated volume and membrane area changes in infected RBCs (IRBCs) using fluorescence confocal microscopy on calcein-loaded RBCs. Substantial effort was devoted to developing and testing a new threshold-independent algorithm for the precise estimation of cell volumes and surface areas to overcome the shortfalls of traditional methods. We confirm that the volume of IRBCs remains almost constant during parasite maturation, suggesting that the reported increase in IRBCs' osmotic fragility results from a reduction in surface area and increased lytic propensity on volume expansion. These results support the general validity of the colloidosmotic hypothesis, settle the IRBC volume debate, and help to constrain the range of parameter values in the numerical model. © 2010 by the Biophysical Society.

Barbera M.,Hutchison Research Center | Fitzgerald R.C.,Hutchison Research Center
Biochemical Society Transactions | Year: 2010

Barrett's oesophagus is a metaplastic pre-malignant disorder and the only established precursor lesion for oesophageal adenocarcinoma. Barrett's oesophagus develops when the normal stratified squamous epithelium of the lower oesophagus is replaced by a columnar lined mucosa with intestinal differentiation, usually in the context of chronic gastro-oesophageal reflux disease. The cellular and molecular mechanisms by which this metaplastic transformation occurs are poorly understood. Abnormal differentiation of multipotent stem cells in the squamous oesophagus, triggered by exposure to refluxate, is one potential mechanism. These stem cells could be located in the basal layer of the squamous oesophageal epithelium and/or in the neck region of the oesophageal submucosal gland ducts; however, their exact location and identification are still matter of discussion. Three-dimensional models combined with state-of-the-art imaging techniques are now applied to characterize the squamous epithelium in human oesophageal samples, and this could unveil essential information to identify these progenitor cells. Locating stem cells in human squamous oesophagus could have important implications for our understanding of Barrett's oesophagus and remarkably improve our future strategies for its prevention. ©The Authors.

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