Wellcome Trust Center for Stem Cell Research

Cambridge, United Kingdom

Wellcome Trust Center for Stem Cell Research

Cambridge, United Kingdom
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Fernandez I.F.,University of Salamanca | Blanco S.,University of Salamanca | Blanco S.,Wellcome Trust Center for Stem Cell Research | Lozano J.,University of Malaga | And 2 more authors.
Molecular and Cellular Biology | Year: 2010

The epidermal growth factor (EGF)-ErbB-mitogen-activated protein kinase (MAPK) transcription signaling pathway is altered in many types of carcinomas, and this pathway can be regulated by new protein-protein interactions. Vaccinia-related kinase (VRK) proteins are Ser-Thr kinases that regulate several signal transduction pathways. In this work, we study the effect of VRK2 on MAPK signaling using breast cancer as a model. High levels of VRK2 inhibit EGF and ErbB2 activation of transcription by the serum response element (SRE). This effect is also detected in response to H-Ras(G12V) or B-Raf(V600E) oncogenes and is accompanied by a reduction in phosphorylated extracellular signal-regulated kinase (ERK) levels, p90RSK levels, and SRE-dependent transcription. Furthermore, VRK2 knockdown has the opposite effect, increasing the transcriptional response to stimulation with EGF and leading to increased levels of ERK phosphorylation. The molecular mechanism lies between MAPK/ERK kinase (MEK) and ERK, since MEK remains phosphorylated while ERK phosphorylation is blocked by VRK2A. This inhibition of the ERK signaling pathway is a consequence of a direct protein-protein interaction between VRK2A, MEK, and kinase suppressor of Ras 1 (KSR1). Identification of new correlations in human cancer can lead to a better understanding of the biology of individual tumors. ErbB2 and VRK2 protein levels were inversely correlated in 136 cases of human breast carcinoma. In ErbB2+ tumors, there is a significant reduction in the VRK2 level, suggesting a role for VRK2A in ErbB2-MAPK signaling. Thus, VRK2 downregulation in carcinomas permits signal transmission through the MEK-ERK pathway without affecting AKT signaling, causing a signal imbalance among pathways that contributes to the phenotype of breast cancer. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Tavares L.,University of Oxford | Dimitrova E.,Babraham Institute | Oxley D.,Babraham Institute | Webster J.,Babraham Institute | And 10 more authors.
Cell | Year: 2012

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors. © 2012 Elsevier Inc.


Oeztuerk-Winder F.,Wellcome Trust Center for Stem Cell Research | Ventura J.-J.,Wellcome Trust Center for Stem Cell Research
Biochemical Journal | Year: 2012

Regulation of stem cells is essential for development and adult tissue homoeostasis. The proper control of stem cell self-renewal and differentiation maintains organ physiology, and disruption of such a balance results in disease. There are many mechanisms that have been established as stem cell regulators, such as Wnt or Notch signals. However, the intracellular mechanisms that mediate and integrate these signals are not well understood. A new intracellular pathway that has been reported to be involved in the regulation of many stem cell types is that of p38 MAPK (mitogen-activated protein kinase). In particular, p38α is essential for the proper differentiation of many haematopoietic, mesenchymal and epithelial stem/progenitor cells. Many reports have shown that disruption of this kinase pathway has pathological consequences in many organs. Understanding the extracellular cues and downstream targets of p38α in stem cell regulation may help to tackle some of the pathologies associated with improper differentiation and regulation of stem cell function. In the present reviewwe present a vision of the current knowledge on the roles of the p38α signal as a regulator of stem/progenitor cells in different tissues in physiology and disease. © The Authors Journal compilation © 2012 Biochemical Society.


Leeb M.,Research Institute of Molecular Pathology | Pasini D.,Copenhagen University | Novatchkova M.,Research Institute of Molecular Pathology | Jaritz M.,Research Institute of Molecular Pathology | And 3 more authors.
Genes and Development | Year: 2010

Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, we find that genomic repeats are Polycomb targets and show that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation. © 2010 by Cold Spring Harbor Laboratory Press.


Pullirsch D.,Research Institute of Molecular Pathology | Hartel R.,Research Institute of Molecular Pathology | Kishimoto H.,Research Institute of Molecular Pathology | Kishimoto H.,Wellcome Trust Center for Stem Cell Research | And 4 more authors.
Development | Year: 2010

Mammals compensate X chromosome gene dosage between the sexes by silencing of one of the two female X chromosomes. X inactivation is initiated in the early embryo and requires the non-coding Xist RNA, which encompasses the inactive X chromosome (Xi) and triggers its silencing. In differentiated cells, several factors including the histone variant macroH2A and the scaffold attachment factor SAF-A are recruited to the Xi and maintain its repression. Consequently, in female somatic cells the Xi remains stably silenced independently of Xist. Here, we identify the Trithorax group protein Ash2l as a novel component of the Xi. Ash2l is recruited by Xist concomitantly with Saf-A and macroH2A at the transition to Xi maintenance. Recruitment of these factors characterizes a developmental transition point for the chromatin composition of the Xi. Surprisingly, expression of a mutant Xist RNA that does not cause gene repression can trigger recruitment of Ash2l, Saf-A and macroH2A to the X chromosome, and can cause chromosome-wide histone H4 hypoacetylation. This suggests that a chromatin configuration is established on non-genic chromatin on the Xi by Xist to provide a repressive compartment that could be used for maintaining gene silencing. Gene silencing is mechanistically separable from the formation of this repressive compartment and, thus, requires additional pathways. This observation highlights a crucial role for spatial organization of chromatin changes in the maintenance of X inactivation.


Arthold S.,Wellcome Trust Center for Stem Cell Research | Kurowski A.,Wellcome Trust Center for Stem Cell Research | Wutz A.,Wellcome Trust Center for Stem Cell Research
Human Genetics | Year: 2011

In mammals, one of the two X chromosomes in female cells is transcriptionally silenced for dosage compensation between the sexes. Chromosome-wide silencing is initiated by the non-coding Xist RNA that accumulates within the inactive X chromosome territory and triggers gene repression and chromatin modifications. Epigenetic changes of the inactive X chromosome in a developmentally regulated manner result in stable gene repression in female somatic cells. X inactivation is a model for understanding the formation of facultative heterochromatin in mammalian development and represents a paradigm for RNA mediated regulation of gene expression. In this review, we summarize the present knowledge of the mechanism of chromosome-wide silencing and give an outlook on future directions. © 2011 Springer-Verlag.


Berta M.A.,CR UK Cambridge Research Institute | Baker C.M.,CR UK Cambridge Research Institute | Cottle D.L.,Wellcome Trust Center for Stem Cell Research | Watt F.M.,CR UK Cambridge Research Institute | Watt F.M.,Wellcome Trust Center for Stem Cell Research
EMBO Molecular Medicine | Year: 2010

Myc is activated in many tumours, yet, paradoxically, stimulates differentiation in mammalian epidermis. To test whether the epidermis responds differently to different levels of Myc, we treated K14MycER transgenic mice with a range of concentrations of the inducing agent, 4-hydroxy-tamoxifen (4OHT). Proliferation was stimulated at all levels of Myc activity; sebocyte differentiation was stimulated at low and intermediate levels; and interfollicular epidermal differentiation at intermediate and high levels. Mutational inactivation of the Myc p21 activated kinase 2 (PAK2) phosphorylation sites increased Myc activity and further enhanced epidermal differentiation. We conclude that Myc induced differentiation acts as a fail-safe device to prevent uncontrolled proliferation and neoplastic conversion of epidermal stem cells expressing high levels of Myc. © 2009 EMBO Molecular Medicine.


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.


Duszczyk M.M.,Helmholtz Center Munich | Duszczyk M.M.,TU Munich | Duszczyk M.M.,ETH Zurich | Wutz A.,Wellcome Trust Center for Stem Cell Research | And 3 more authors.
RNA | Year: 2011

X-chromosome inactivation (XCI) in female mammals depends on the noncoding RNA X inactivation specific transcript (Xist). The mechanism of chromosome-wide silencing by Xist is poorly understood. While it is established that the 5′ region of Xist RNA, comprising the A-repeats and holding 7.5-8.5 copies of a conserved 26-mer sequence, is essential for Xist-mediated silencing, high-resolution structural information for the A-repeats is not available. Here, we report the three-dimensional solution structure of a 14-mer hairpin in the 5′ region of a human A-repeat. This hairpin is remarkably stable and adopts a novel AUCG tetraloop fold, the integrity of which is required for silencing. We show that, contrary to previous predictions, the 3′ region of single or tandem A-repeats mediates duplex formation in vitro. Significantly, mutations in this region disrupt the interrepeat duplex formation in vitro and abrogate the silencing function of Xist A-repeats in vivo. Our data suggest that the complete A-repeat region may be stabilized by inter-repeat duplex formation and, as such, may provide a platform for multimerization and specific recognition of the AUCG tetraloops by trans-acting factors. Copyright © 2011 RNA Society.

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