CRUK Cambridge Research Institute

Cambridge, United Kingdom

CRUK Cambridge Research Institute

Cambridge, United Kingdom

Time filter

Source Type

Massie C.E.,CRUK Cambridge Research Institute | Massie C.E.,Cambridge Institute for Medical Research | Mills I.G.,CRUK Cambridge Research Institute | Mills I.G.,University of Oslo
Methods in Molecular Biology | Year: 2012

Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method. Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.


Farrar D.,University of Essex | Rai S.,University of Essex | Rai S.,Anglia Ruskin University | Chernukhin I.,University of Essex | And 7 more authors.
Molecular and Cellular Biology | Year: 2010

Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADPribosyl) ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Baker C.M.,CRUK Cambridge Research Institute | Verstuyf A.,Catholic University of Leuven | Jensen K.B.,Wellcome Trust Center for Stem Cell Research | Watt F.M.,CRUK Cambridge Research Institute | Watt F.M.,Wellcome Trust Center for Stem Cell Research
Developmental Biology | Year: 2010

Wnt signalling is required for hair follicle development and for the growth phase (anagen) of postnatal follicles. When the pathway is activated at high levels in adult mouse epidermis, ectopic follicles form from existing follicles, interfollicular epidermis (IFE) and sebaceous glands, revealing a remarkable ability of the tissue to be reprogrammed. To compare the competence of different epidermal cell populations to form ectopic follicles, we expressed a 4-hydroxy-tamoxifen (4OHT) inducible, stabilised β-catenin transgene (δNβ-cateninER) under the control of two different promoters. We targeted the reservoir of stem cells in the hair follicle bulge via the keratin 15 (K15) promoter and targeted the sebaceous glands and base of the follicle (bulb) with a truncated K5 promoter (δK5). No ectopic follicles formed in the IFE in either model, establishing the autonomy of the IFE stem cell compartment in undamaged epidermis. Activation of β-catenin in the bulge stimulated proliferation and bulge expansion. Existing hair follicles entered anagen, but no ectopic follicles formed. δK5δNβ-cateninER expressing hair follicles also entered anagen on 4OHT treatment. In addition, a subpopulation of cells at the base of the sebaceous gland readily formed ectopic follicles, resulting in complete and reversible conversion of sebaceous glands into hair follicles. Combined activation of β-catenin and the vitamin D receptor enhanced differentiation of sebaceous gland-derived hair follicles and stimulated ectopic follicle formation in the hair follicle bulb, but not in the bulge. Our results suggest that the bulge and sebaceous gland are, respectively, non-permissive and permissive niches for Wnt induced hair follicle differentiation. © 2010 Elsevier Inc.


Goldie S.J.,CRUK Cambridge Research Institute | Mulder K.W.,CRUK Cambridge Research Institute | Tan D.W.-M.,Wellcome Trust Center for Stem Cell Research | Lyons S.K.,CRUK Cambridge Research Institute | And 3 more authors.
Cancer Research | Year: 2012

New therapeutic strategies are needed to improve treatment of head and neck squamous cell carcinoma (HNSCC), an aggressive tumor with poor survival rates. FRMD4A is a human epidermal stem cell marker implicated previously in epithelial polarity that is upregulated in SCC cells. Here, we report that FRMD4A upregulation occurs in primary human HNSCCs where high expression levels correlate with increased risks of relapse. FRMD4A silencing decreased growth and metastasis of human SCC xenografts in skin and tongue, reduced SCC proliferation and intercellular adhesion, and stimulated caspase-3 activity and expression of terminal differentiation markers. Notably, FRMD4A attenuation caused nuclear accumulation of YAP, suggesting a potential role for FRMD4A in Hippo signaling. Treatment with the HSP90 inhibitor 17-DMAG or ligation of CD44 with hyaluronan caused nuclear depletion of FRMD4A, nuclear accumulation of YAP and reduced SCC growth and metastasis. Together, our findings suggest FRMD4A as a novel candidate therapeutic target in HNSCC based on the key role in metastatic growth we have identified. ©2012 AACR.


Trappmann B.,University of Cambridge | Gautrot J.E.,University of Cambridge | Connelly J.T.,University of Cambridge | Connelly J.T.,Queen Mary, University of London | And 14 more authors.
Nature Materials | Year: 2012

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal- related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions. © 2012 Macmillan Publishers Limited. All rights reserved.


Gallagher F.A.,University of Cambridge | Kettunen M.I.,University of Cambridge | Day S.E.,University of Cambridge | Hu D.-E.,University of Cambridge | And 5 more authors.
Magnetic Resonance in Medicine | Year: 2011

Dynamic nuclear polarization can be used to increase the sensitivity of solution state 13C magnetic resonance spectroscopy by four orders of magnitude. We show here that [1- 13C]glutamate can be polarized to 28%, representing a 35,000-fold increase in its sensitivity to detection at 9.4 T and 37°C. The metabolism of hyperpolarized glutamate to α-ketoglutarate, catalyzed by the enzyme alanine transaminase, was detected in vitro in human hepatoma cells (HepG2). Incubation of the cells with sodium pyruvate increased the level of the hyperpolarized label in the α-ketoglutarate pool, with an associated increase in the apparent rate constant describing flux of hyperpolarized 13C label between glutamate and α-ketoglutarate. The metabolism of hyperpolarized glutamate was observed in vivo following coadministration of pyruvate in a murine lymphoma model. This represents a new method to probe glutamate metabolism and citric acid cycle activity in vivo; as glutamate is an endogenous molecule, it has the potential to be used in the clinic. Magn Reson Med, 2011. © 2011 Wiley-Liss, Inc. Copyright © 2011 Wiley-Liss, Inc.


Gossage L.,CRUK Cambridge Research Institute | Gossage L.,Center for Protein Engineering | Eisen T.,CRUK Cambridge Research Institute | Eisen T.,Addenbrookes Hospital
Clinical Cancer Research | Year: 2010

Anticancer drugs that target protein kinases include small molecule inhibitors and monoclonal antibodies. Feedback loops and cross talk between signaling pathways impact significantly on the efficacy of cancer therapeutics, and resistance to targeted agents is a major barrier to effective treatments. Increasingly, therapies are being designed to target multiple kinase pathways. This can be achieved using a single agent that inhibits multiple signaling pathways or a combination of highly selective agents. In this review we discuss the principles of specifically targeting multiple kinase pathways with particular reference to angiogenic signaling pathways. ©2010 AACR.


Aldridge S.,CRUK Cambridge Research Institute | Hadfield J.,CRUK Cambridge Research Institute
Methods in Molecular Biology | Year: 2012

MicroRNA analysis has been widely adopted for basic and applied science. The tools and technologies available for quantifying and analysing miRNAs are still maturing. Here, we give an introductory overview of the main tools and the challenges in their use. We also discuss the importance of basic experimental design, sample handling and analysis methods as the impact of these can be as profound as the choice of miRNA analysis platform. Whether the reader is interested in a gene-by-gene or genome-wide approach choosing the platform to use is not trivial. Careful thought given before starting an experiment will make the execution much easier. © 2012 Springer Science+Business Media, LLC.


Schroeder M.A.,University of Oxford | Swietach P.,University of Oxford | Atherton H.J.,University of Oxford | Gallagher F.A.,University of Cambridge | And 5 more authors.
Cardiovascular Research | Year: 2010

Aims: Technological limitations have restricted in vivo assessment of intracellular pH (pHi) in the myocardium. The aim of this study was to evaluate the potential of hyperpolarized [1-13C]pyruvate, coupled with 13C magnetic resonance spectroscopy (MRS), to measure pH i in the healthy and diseased heart. Methods and results: Hyperpolarized [1-13C]pyruvate was infused into isolated rat hearts before and immediately after ischaemia, and the formation of 13CO2 and H13CO3 - was monitored using 13C MRS. The HCO3 -/CO 2 ratio was used in the Henderson-Hasselbalch equation to estimate pHi. We tested the validity of this approach by comparing 13C-based pHi measurements with 31P MRS measurements of pHi. There was good agreement between the pHi measured using 13C and 31P MRS in control hearts, being 7.12 ± 0.10 and 7.07 ± 0.02, respectively. In reperfused hearts, 13C and 31P measurements of pHi also agreed, although 13C equilibration limited observation of myocardial recovery from acidosis. In hearts pre-treated with the carbonic anhydrase (CA) inhibitor, 6-ethoxyzolamide, the 13C measurement underestimated the 31P-measured pHi by 0.80 pH units. Mathematical modelling predicted that the validity of measuring pHi from the H 13CO3 -/13CO2 ratio depended on CA activity, and may give an incorrect measure of pHi under conditions in which CA was inhibited, such as in acidosis. Hyperpolarized [1-13C]pyruvate was also infused into healthy living rats, where in vivo pHi from the H13CO3 -/ 13CO2 ratio was measured to be 7.20 ± 0.03. Conclusion: Metabolically generated 13CO2 and H 13CO3 - can be used as a marker of cardiac pHi in vivo, provided that CA activity is at normal levels. © The Author 2009.


Connelly J.T.,University of Cambridge | Gautrot J.E.,University of Cambridge | Trappmann B.,University of Cambridge | Tan D.W.-M.,University of Cambridge | And 5 more authors.
Nature Cell Biology | Year: 2010

Epidermal homeostasis depends on a balance between stem cell renewal and differentiation and is regulated by extrinsic signals from the extracellular matrix (ECM). A powerful approach to analysing the pathways involved is to engineer single-cell microenvironments in which individual variables are precisely and quantitatively controlled. Here, we employ micropatterned surfaces to identify the signalling pathways by which restricted ECM contact triggers human epidermal stem cells to initiate terminal differentiation. On small (20m diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50m diameter) islands. Differentiation did not depend on ECM composition or density. Rather, the actin cytoskeleton mediated shape-induced differentiation by regulating serum response factor (SRF) transcriptional activity. Knockdown of SRF or its co-factor MAL inhibited differentiation, whereas overexpression of MAL stimulated SRF activity and involucrin expression. SRF target genes FOS and JUNB were also required for differentiation: c-Fos mediated serum responsiveness, whereas JunB was regulated by actin and MAL. Our findings demonstrate how biophysical cues are transduced into transcriptional responses that determine epidermal cell fate. © 2010 Macmillan Publishers Limited. All rights reserved.

Loading CRUK Cambridge Research Institute collaborators
Loading CRUK Cambridge Research Institute collaborators