DOCSLIB.ORG

Determinants of PAX3 Behavior: a Molecular and Cellular Analysis of the PAX3 Transcription Factor and Disease-Associated Mutants

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Determinants of PAX3 Behavior: a Molecular and Cellular Analysis of the PAX3 Transcription Factor and Disease-Associated Mutants University of Alberta Determinants of PAX3 behavior: A molecular and cellular analysis of the PAX3 transcription factor and disease-associated mutants by Gareth Neill Corry A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences - Medical Genetics Edmonton, Alberta Spring 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-45411-4 Our file Notre reference ISBN: 978-0-494-45411-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. •*• Canada ABSTRACT The PAX3 transcription factor is a key regulator of developmentally important processes in metazoan organisms and also carries out distinct postnatal functions. PAX3 is characterized by two DNA-binding modules, the paired domain (PD) and homeodomain (HD), which functionally interact to control target gene expression. Mutations in PAX3 cause Waardenburg syndrome in humans and the mouse Splotch phenotype, both of which are characterized by defects in neural crest-derived and myogenic cell types. Through molecular and cellular analyses, we have investigated the determinants that underlie PAX3 behavior and have shed light on how disease- causing mutations affect these determinants. We show that PAX3 uses different arrangements of its DNA-binding domains to achieve distinct modes of target sequence recognition. Notably, disease mutations exert varying effects on PAX3 target affinity, specificity, and functional cooperativity between the PD and HD. Additionally, we find that these mutations exert variable effects, including repression and enhanced expression, on the activation of PAX3-responsive reporter genes. In vivo studies show that PAX3 localizes to the interchromatin space and displays limited co-localization with sites of transcriptional activity. Importantly, each PAX3 mutant examined in live cells displayed differences in intranuclear mobility compared to the wild type protein and, based on their intranuclear compartmentalization and dynamics, we were able to separate our cohort of mutations into two distinct classes. Significantly, this classification method helps clarify the determinants that regulate PAX3 behavior in vivo and has enabled us to propose a model in which PAX3 activity is regulated by a two-step process involving compartmentalization to a particular nuclear domain, followed by identification and interaction with chromatin- embedded regulatory target sequences. Our results also suggest that conformational rearrangements during each step play a role in optimizing PAX3 performance. Together, our results provide important information regarding the determinants that regulate PAX3 behavior and the associated effects of disease mutations, and establish a foundation for integrating molecular and cellular analyses of transcription factor function. ACKNOWLEDGEMENTS Immediate thanks go to Dr. Alan Underhill for taking a chance way back in the summer of '99 and letting me do some part-time work in the lab, then keeping me on as a project student, research assistant, and finally, a graduate student. I think everything I know about scientific research is due in large part to Alan's guidance, advice, and instruction and I credit my research accomplishments to his supervision and assistance. I wish him every success for the future. Thanks to Dr. Michael Walter and Dr. Mark Glover for contributing their time to be part of my graduate committee and helping to direct my research; their feedback and suggestions over the years have been much appreciated. I also thank Dr. Jim Davie, Dr. Roseline Godbout, and Dr. Stacey Bleoo for being part of my examining committee, and Dr. Rachel Wevrick, Dr. Michael Schultz, and Dr. Moira Glerum for participating in my PhD candidacy exam. My research would not have progressed without the help of other students, postdoctoral fellows, technicians, and professors. In particular, I express thanks to Ning Hu for her technical assistance in the lab and for taking care of our tissue culture. I also appreciate the support and helpfulness of current and former members of the Underhill lab during my studies. Additional thanks go to the other Medical Genetics labs, as well as the labs of Drs. Michael Schultz, Michael Hendzel, John Greer, and Shairaz Baksh for sharing reagents, advice, and expertise. Special thanks to Jason Bush for providing primary cell cultures, Robin Clugston for providing rat livers, and Kristal Missiaen for setting aside her own work to do our FRAP experiments for us. Thanks also to Dr. Xuejun Sun and Gerry Barron at the Cross Cancer Institute microscope facility for their assistance and patience, and Dr. Colin Goding, Dr. Frederic Barr, and Dr. Peter Adams for providing reagents. I acknowledge the various agencies and institutions that have provided funding during my studies, including the University of Alberta, the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, and Alberta Learning. Finally, and simply, thank you to my parents and family for their continual support, advice, understanding, and love - past, present, and future. TABLE OF CONTENTS CHAPTER 1. INTRODUCTION 1 1.1 PAX3: structure, function, and disease 2 1.1.1 PAX proteins 2 1.1.2 Pax3/PAX3 gene structure and regulatory elements 3 1.1.3 PAX3 protein structure 7 1.1.4 Pax3 expression during development 9 1.1.5 PAX3-related diseases 12 1.1.6 Role of PAX3 in developmental pathways 16 1.1.7 PAX3 target genes 19 1.1.8 PAX3 protein-protein interactions 25 1.1.9 Functional interactions involving the paired domain and homeodomain 27 1.2 PAX3: from the test tube to the nucleus 29 1.3 Intranuclear organization of transcription 30 1.3.1 The eukaryotic nucleus 30 1.3.2 Subnuclear localization of transcription factors 35 1.3.3 Intrinsic determinants of transcription factor subnuclear localization 39 1.3.4 Regulation of transcription factor subnuclear localization through co-factor interactions 42 1.3.5 Intranuclear dynamics 44 1.3.6 Functional organization of eukaryotic transcription 48 1.4 Objective 51 CHAPTER 2. METHODS AND MATERIALS 53 2.1 Plasmid construction 54 2.1.1 Bacterial expression constructs 54 2.1.2 Mammalian expression constructs 61 2.1.3 Luciferase reporter constructs 62 2.2 Mutant expression constructs 64 2.3 Hexahistidine-tagged protein expression and purification 64 2.4 Electrophoretic mobility shift assays 67 2.5 Bulk chromatin isolation 70 2.6 Affinity chromatography pull-down assays 70 2.7 Cell lines and culture 72 2.8 Dual luciferase assays 72 2.9 Antibodies 73 2.10 Whole cell extracts 73 2.11 Western transfer 74 2.12 Immunocytochemistry 74 2.13 Fluorescence recovery after photobleaching 75 2.14 Bioinformatics 76 CHAPTER 3. PAX3-TARGET INTERACTION 77 3.1 Background 78 3.2 Analysis of PAX3 recognition sequences 79 3.3 Evolutionary conservation of selected PAX3 target sequences 83 3.4 DNA-binding characteristics of PAX3 86 3.5 The PAX3 homeodomain increases binding affinity for composite sequences 89 3.6 Analysis of PAX3 interaction with the Trp-1 promoter sequence 91 3.7 Sequence determinants for MTF promoter binding by PAX3 93 3.8 Sequence determinants within the MITF hd3 motif 95 3.9 Structural requirements for FAX3-MITF interaction 98 3.10 Participation of paired domain and homeodomain homologs in cooperative binding 101 3.11 Effects of disease mutations on PAX3 target recognition 105 3.12 Effects of PAX3 disease mutations on paired domain arid homeodomain cooperativity 109 3.13 Functional analysis of the MITF and Trp-1 PAX3 recognition elements Ill 3.14 Discussion 113 CHAPTER 4. CHARACTERIZATION OF PAX3 IN THE NUCLEUS 118 4.1 Background 119 4.2 Subnuclear localization of PAX3 121 4.3 Localization of PAX3 with respect to post-translational
Load more

Recommended publications
  • The Master Neural Transcription Factor BRN2 Is an Androgen Receptor–Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer
    Published OnlineFirst October 26, 2016; DOI: 10.1158/2159-8290.CD-15-1263 RESEARCH ARTICLE The Master Neural Transcription Factor BRN2 Is an Androgen Receptor–Suppressed Driver of Neuroendocrine Differentiation in Prostate Cancer Jennifer L. Bishop1, Daksh Thaper1,2, Sepideh Vahid1,2, Alastair Davies1, Kirsi Ketola1, Hidetoshi Kuruma1, Randy Jama1, Ka Mun Nip1,2, Arkhjamil Angeles1, Fraser Johnson1, Alexander W. Wyatt1,2, Ladan Fazli1,2, Martin E. Gleave1,2, Dong Lin1, Mark A. Rubin3, Colin C. Collins1,2, Yuzhuo Wang1,2, Himisha Beltran3, and Amina Zoubeidi1,2 ABSTRACT Mechanisms controlling the emergence of lethal neuroendocrine prostate cancer (NEPC), especially those that are consequences of treatment-induced suppression of the androgen receptor (AR), remain elusive. Using a unique model of AR pathway inhibitor–resistant prostate cancer, we identified AR-dependent control of the neural transcription factor BRN2 (encoded by POU3F2) as a major driver of NEPC and aggressive tumor growth, both in vitro and in vivo. Mecha- nistic studies showed that AR directly suppresses BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2. Underscoring its inverse correlation with clas- sic AR activity in clinical samples, BRN2 expression was highest in NEPC tumors and was significantly increased in castration-resistant prostate cancer compared with adenocarcinoma, especially in patients with low serum PSA. These data reveal a novel mechanism of AR-dependent control of NEPC and suggest that targeting BRN2 is a strategy to treat or prevent neuroendocrine differentiation in prostate tumors. SIGNIFICANCE: Understanding the contribution of the AR to the emergence of highly lethal, drug- resistant NEPC is critical for better implementation of current standard-of-care therapies and novel drug design.
    [Show full text]
  • Core Transcriptional Regulatory Circuitries in Cancer
    Oncogene (2020) 39:6633–6646 https://doi.org/10.1038/s41388-020-01459-w REVIEW ARTICLE Core transcriptional regulatory circuitries in cancer 1 1,2,3 1 2 1,4,5 Ye Chen ● Liang Xu ● Ruby Yu-Tong Lin ● Markus Müschen ● H. Phillip Koeffler Received: 14 June 2020 / Revised: 30 August 2020 / Accepted: 4 September 2020 / Published online: 17 September 2020 © The Author(s) 2020. This article is published with open access Abstract Transcription factors (TFs) coordinate the on-and-off states of gene expression typically in a combinatorial fashion. Studies from embryonic stem cells and other cell types have revealed that a clique of self-regulated core TFs control cell identity and cell state. These core TFs form interconnected feed-forward transcriptional loops to establish and reinforce the cell-type- specific gene-expression program; the ensemble of core TFs and their regulatory loops constitutes core transcriptional regulatory circuitry (CRC). Here, we summarize recent progress in computational reconstitution and biologic exploration of CRCs across various human malignancies, and consolidate the strategy and methodology for CRC discovery. We also discuss the genetic basis and therapeutic vulnerability of CRC, and highlight new frontiers and future efforts for the study of CRC in cancer. Knowledge of CRC in cancer is fundamental to understanding cancer-specific transcriptional addiction, and should provide important insight to both pathobiology and therapeutics. 1234567890();,: 1234567890();,: Introduction genes. Till now, one critical goal in biology remains to understand the composition and hierarchy of transcriptional Transcriptional regulation is one of the fundamental mole- regulatory network in each specified cell type/lineage.
    [Show full text]
  • PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain
    Published OnlineFirst April 26, 2017; DOI: 10.1158/2159-8290.CD-16-1297 RESEARCH ARTICLE PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability Berkley E. Gryder 1 , Marielle E. Yohe 1 , 2 , Hsien-Chao Chou 1 , Xiaohu Zhang 3 , Joana Marques 4 , Marco Wachtel4 , Beat Schaefer 4 , Nirmalya Sen 1 , Young Song 1 , Alberto Gualtieri 5 , Silvia Pomella 5 , Rossella Rota5 , Abigail Cleveland 1 , Xinyu Wen 1 , Sivasish Sindiri 1 , Jun S. Wei 1 , Frederic G. Barr 6 , Sudipto Das7 , Thorkell Andresson 7 , Rajarshi Guha 3 , Madhu Lal-Nag 3 , Marc Ferrer 3 , Jack F. Shern 1 , 2 , Keji Zhao8 , Craig J. Thomas 3 , and Javed Khan 1 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. 16-CD-16-1297_p884-899.indd 884 7/20/17 2:21 PM Published OnlineFirst April 26, 2017; DOI: 10.1158/2159-8290.CD-16-1297 ABSTRACT Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and ado- lescent young adults, driven primarily by the chimeric transcription factor PAX3–FOXO1. The mechanisms by which PAX3–FOXO1 dysregulates chromatin are unknown. We fi nd PAX3–FOXO1 repro- grams the cis -regulatory landscape by inducing de novo super enhancers. PAX3–FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3–FOXO1-occupied super enhancers.
    [Show full text]
  • PAX3-FOXO1A Expression in Rhabdomyosarcoma Is Driven by the Targetable Nuclear Receptor NR4A1 Alexandra Lacey1, Aline Rodrigues-Hoffman2, and Stephen Safe1
    Published OnlineFirst November 18, 2016; DOI: 10.1158/0008-5472.CAN-16-1546 Cancer Therapeutics, Targets, and Chemical Biology Research PAX3-FOXO1A Expression in Rhabdomyosarcoma Is Driven by the Targetable Nuclear Receptor NR4A1 Alexandra Lacey1, Aline Rodrigues-Hoffman2, and Stephen Safe1 Abstract Alveolar rhabdomyosarcoma (ARMS) is a devastating pediatric PAX3-FOXO1A. Mechanistic investigations revealed a requirement disease driven by expression of the oncogenic fusion gene PAX3- for the NR4A1/Sp4 complex to bind GC-rich promoter regions FOXO1A. In this study, we report overexpression of the nuclear to elevate transcription of the PAX3-FOXO1A gene. In parallel, receptor NR4A1 in rhabdomyosarcomas that is sufficient to NR4A1 also regulated expression of b1-integrin, which with PAX3- drive high expression of PAX3-FOXO1A there. RNAi-mediated FOXO1A, contributed to tumor cell migration that was blocked by silencing of NR4A1 decreased expression of PAX3-FOXO1A and C-DIM/NR4A1 antagonists. Taken together, our results provide a its downstream effector genes. Similarly, cell treatment with the preclinical rationale for the use of NR4A1 small-molecule antago- NR4A1 small-molecule antagonists 1,1-bis(3-indolyl)-1-(p- nists to treat ARMS and other rhabdomyosarcomas driven by hydroxy or p-carbomethoxyphenyl)methane (C-DIM) decreased PAX3-FOXO1A. Cancer Res; 77(3); 1–10. Ó2016 AACR. Introduction activity for NR4A1. In contrast, NR4A1 exhibits tumor promoter activity (6, 7) in solid tumors. NR4A1 is also overexpressed in Rhabdomyosarcoma is the most common soft-tissue sarco- tumors from patients with breast, lung, pancreatic, colon, and ma that is primarily observed in children and adolescents and ovarian cancer and is a negative prognostic factor for patients with accounts for 50% of all pediatric cancers and 50% of soft-tissue breast, lung, and ovarian cancer (9–15).
    [Show full text]
  • PAX3-FOXO1A Expression in Rhabdomyosarcoma Is Driven by the Targetable Nuclear Receptor NR4A1 Alexandra Lacey1, Aline Rodrigues-Hoffman2, and Stephen Safe1
    Published OnlineFirst November 18, 2016; DOI: 10.1158/0008-5472.CAN-16-1546 Cancer Therapeutics, Targets, and Chemical Biology Research PAX3-FOXO1A Expression in Rhabdomyosarcoma Is Driven by the Targetable Nuclear Receptor NR4A1 Alexandra Lacey1, Aline Rodrigues-Hoffman2, and Stephen Safe1 Abstract Alveolar rhabdomyosarcoma (ARMS) is a devastating pediatric PAX3-FOXO1A. Mechanistic investigations revealed a requirement disease driven by expression of the oncogenic fusion gene PAX3- for the NR4A1/Sp4 complex to bind GC-rich promoter regions FOXO1A. In this study, we report overexpression of the nuclear to elevate transcription of the PAX3-FOXO1A gene. In parallel, receptor NR4A1 in rhabdomyosarcomas that is sufficient to NR4A1 also regulated expression of b1-integrin, which with PAX3- drive high expression of PAX3-FOXO1A there. RNAi-mediated FOXO1A, contributed to tumor cell migration that was blocked by silencing of NR4A1 decreased expression of PAX3-FOXO1A and C-DIM/NR4A1 antagonists. Taken together, our results provide a its downstream effector genes. Similarly, cell treatment with the preclinical rationale for the use of NR4A1 small-molecule antago- NR4A1 small-molecule antagonists 1,1-bis(3-indolyl)-1-(p- nists to treat ARMS and other rhabdomyosarcomas driven by hydroxy or p-carbomethoxyphenyl)methane (C-DIM) decreased PAX3-FOXO1A. Cancer Res; 77(3); 1–10. Ó2016 AACR. Introduction activity for NR4A1. In contrast, NR4A1 exhibits tumor promoter activity (6, 7) in solid tumors. NR4A1 is also overexpressed in Rhabdomyosarcoma is the most common soft-tissue sarco- tumors from patients with breast, lung, pancreatic, colon, and ma that is primarily observed in children and adolescents and ovarian cancer and is a negative prognostic factor for patients with accounts for 50% of all pediatric cancers and 50% of soft-tissue breast, lung, and ovarian cancer (9–15).
    [Show full text]
  • In Mediating Transcriptional Activity of Androgen Receptor Splice Variants
    ROLE OF TRANSCRIPTIONAL ACTIVATION UNIT 5 (TAU5) IN MEDIATING TRANSCRIPTIONAL ACTIVITY OF ANDROGEN RECEPTOR SPLICE VARIANTS A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY SARITA KUMARI MUTHA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE SCOTT DEHM & JIM McCARTHY DECEMBER 2012 © Sarita Kumari Mutha 2012 ACKNOWLEDGEMENTS I would like to thank the Dehm Lab: Scott Dehm, Yingming Li, Siu Chiu Chan, and Luke Brand for the support, discussions, journal clubs, and training. I would like to thank MCBD&G faculty Kathleen Conklin and Meg Titus for their support. Finally, I would also to acknowledge my committee Scott Dehm, Jim McCarthy, and Kaylee Schwertfeger for their time and support. i DEDICATION This thesis is dedicated to my mentor and advisor Scott Dehm. This work would not have been possible without his support. I am very grateful for my time under his mentorship and the opportunity to learn from him. ii ABSTRACT The standard treatment for advanced prostate cancer is chemical castration, which inhibits the activity of the androgen receptor (AR). Eventually, prostate cancer reemerges with a castration-resistant phenotype (CRPC) but still depends on AR signaling. One mechanism of AR activity in CRPC is the synthesis of AR splice variants, which lack the ligand binding domain. These splice variants function as constitutively active transcription factors that promote expression of endogenous AR target genes and support androgen independent prostate cancer cell growth. Previous work has shown transcriptional activation unit 5 (TAU5) is necessary for ligand independent activity of the full length AR in low or no androgen conditions and that this activation is mediated by the WHTLF motif.
    [Show full text]
  • Functional Impact of a Germline RET Mutation in Alveolar Rhabdomyosarcoma
    Downloaded from molecularcasestudies.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Follow-up Report for CSHL Molecular Case Studies Functional impact of a germline RET mutation in alveolar rhabdomyosarcoma Mutant RET in alveolar rhabdomyosarcoma Noah E. Berlow1*, Kenneth A. Crawford1, Carol J. Bult2, Christopher Noakes3, Ido Sloma3, Erin R. Rudzinski4, Charles Keller1* 1 Children’s Cancer Therapy Development Institute, Beaverton, OR 97005 USA 2 The Jackson Laboratory, Bar Harbor, ME 04609 USA 3 Champions Oncology, One University Plaza, Hackensack, NJ 07601 USA 4 Seattle Children's Hospital, Seattle, WA 98105 USA * Correspondence: Noah E. Berlow, Children's Cancer Therapy Development Institute, 12655 SW Beaverdam Road-West, Beaverton OR 97005 USA, Tel (806) 370-8119, Fax (270) 675-3313, email [email protected] Charles Keller, Children's Cancer Therapy Development Institute, 12655 SW Beaverdam Road-West, Beaverton OR 97005 USA, Tel (801) 232-8038, Fax (270) 675-3313, email [email protected] Keywords: RET, alveolar rhabdomyosarcoma, endotypes in rhabdomyosarcoma, genetic profiles, transcriptomic profiles, drug screening, combination therapy word count (2333 words in main text, excluding Abstract, Methods, References and figure legends) 1 Downloaded from molecularcasestudies.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract Specific mutations in the RET proto-oncogene are associated with multiple endocrine neoplasia type 2A, a hereditary syndrome characterized by tumorigenesis in multiple glandular elements. In rare instances, MEN2A- associated germline RET mutations have also occurred with non-MEN2A associated cancers. One such germline mutant RET mutation occurred concomitantly in a young adult diagnosed with alveolar rhabdomyosarcoma, a pediatric and young adult soft-tissue cancer with a generally poor prognosis.
    [Show full text]
  • TBX3 Represses TBX2 Under the Control of the PRC2 Complex In
    Oh et al. Oncogenesis (2019) 8:27 https://doi.org/10.1038/s41389-019-0137-z Oncogenesis ARTICLE Open Access TBX3 represses TBX2 under the control of the PRC2 complex in skeletal muscle and rhabdomyosarcoma Teak-Jung Oh1, Abhinav Adhikari 2, Trefa Mohamad3, Aiysha Althobaiti2 and Judith Davie2 Abstract TBX2 and TBX3 function as repressors and are frequently implicated in oncogenesis. We have shown that TBX2 represses p21, p14/19, and PTEN in rhabdomyosarcoma (RMS) and skeletal muscle but the function and regulation of TBX3 were unclear. We show that TBX3 directly represses TBX2 in RMS and skeletal muscle. TBX3 overexpression impairs cell growth and migration and we show that TBX3 is directly repressed by the polycomb repressive complex 2 (PRC2), which methylates histone H3 lysine 27 (H3K27me). We found that TBX3 promotes differentiation only in the presence of early growth response factor 1 (EGR1), which is differentially expressed in RMS and is also a target of the PRC2 complex. The potent regulation axis revealed in this work provides novel insight into the effects of the PRC2 complex in normal cells and RMS and further supports the therapeutic value of targeting of PRC2 in RMS. Introduction phenotypes and the presence of myogenic markers such 4 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Rhabdomyosarcoma (RMS) is the most common soft as myogenic regulatory factors (MRFs) , yet these factors tissue pediatric sarcoma, which is thought to largely arise appear to be inactive in RMS5. from the skeletal muscle lineage1. The more common The T-box family of transcription factors are highly form of the disease is the embryonal subtype (ERMS), conserved and related throughout all metazoan lineages.
    [Show full text]
  • Mechanisms of 4-Hydroxytamoxifen- Induced Apoptosis in Rhabdomyosarcoma Cells
    MECHANISMS OF 4-HYDROXYTAMOXIFEN- INDUCED APOPTOSIS IN RHABDOMYOSARCOMA CELLS by Kevin Min Chen A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Medical Biophysics University of Toronto © Copyright by Kevin Min Chen (2011) Mechanisms of 4-hydroxytamoxifen-induced apoptosis in rhabdomyosarcoma cells Kevin Min Chen Master of Science Department of Medical Biophysics University of Toronto 2011 ABSTRACT Rhabdomyosarcoma (RMS) is a malignant soft-tissue sarcoma in children, accounting for about 40% of pediatric soft-tissue tumours. Five-year survival for metastatic RMS is only about 25%. Furthermore, there has been no significant improvement in RMS survival since 1975, pointing to a need for improved therapy. Previous work in our lab has shown that 4-hydroxytamoxifen (4OHT) leads to increased apoptosis and decreased viability in RMS cells. Expanding on this work, the current project aims to elucidate the mechanisms behind 4OHT-induced apoptosis in RMS cells, focusing on the roles of estrogen receptors (ER) and MAP kinases (MAPK). We found that: 1) 4OHT-induced apoptotic signaling was associated with increased MAPK phosphorylation, 2) Inhibition of MAPK protected cells against 4OHT, 3) Inhibition of ER also protected against 4OHT, and 4) ER inhibition blocked 4OHT- associated MAPK phosphorylation. ii ACKNOWLEDGEMENTS This work would not have been possible without the generous support and assistance received from the individuals and organizations below: Funding was provided in part by a Frederick Banting and Charles Best Canada Graduate Scholarship (CGS) - Master's Award, from the Canadian Institutes of Health Research (CIHR). Much gratitude goes to Dr. David Malkin for his guidance, and for cultivating a healthy and stimulating research environment.
    [Show full text]
  • Role of SOX10 in the Development of Neural Crest-Derived Melanocytes and Glia
    Oncogene (2003) 22, 3024–3034 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc The importance of having your SOX on: role of SOX10 in the development of neural crest-derived melanocytes and glia Ramin Mollaaghababa1 and William J Pavan*,1 1National Human Genome Research Institute, National Institutes of Health, 49 Convent Drive, Bethesda, MD 20892-4472, USA SOX10w is a member of the high-mobility group-domain differentiate to form melanocytes of the skin, hair, and SOX family of transcription factors, which are ubiqui- inner ear while others move ventrally, either through the tously found in the animal kingdom. Disruption of neural somites or in the space between the somites and the crest development in the Dominant megacolon (Dom) neural tube, and contribute to the formation of mice is associated with a Sox10 mutation. Mutations in additional distinct lineage. These include sensory human Sox10 w gene have also been linked with the neurons and glia, neurons and glia of cranial ganglia, occurrence of neurocristopathies in the Waardenburg– cartilage and bone, connective tissue, and neuroendo- Shah syndrome type IV (WS-IV), for which the Sox10Dom crine cells (Le Douarin and Kalcheim, 1999). mice serve as a murine model. The neural crest disorders The specification of neural crest to distinct lineage in the Sox10Dom mice and WS-IV patients consist of and their proper differentiation is dependent on both hypopigmentation, cochlear neurosensory deafness, and intrinsic factors and environmental interactions (La- enteric aganglionosis. Consistent with these observations, Bonne and Bronner-Fraser, 1998). The use of mouse a critical role for SOX10 in the proper differentiation of neural crest mutants has been instrumental in the neural crest-derived melanocytes and glia has been identification and analysis of genes essential for proper demonstrated.
    [Show full text]
  • Regulation of Target Genes of PAX3−FOXO1 in Alveolar Rhabdomyosarcoma
    ANTICANCER RESEARCH 33: 2029-2036 (2013) Regulation of Target Genes of PAX3−FOXO1 in Alveolar Rhabdomyosarcoma EUN HYUN AHN1,2 1Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.; 2Department of Pathology, School of Medicine, University of Washington, Seattle, WA, U.S.A. Abstract. Background: The majority of alveolar major subtypes based on their histological appearance: rhabdomyosarcoma (ARMS) are distinguished through the embryonal rhabdomyosarcoma (ERMS) and alveolar paired box 3−forkhead box protein O1 (PAX3−FOXO1) rhabdomyosarcoma (ARMS) (1). ARMS has a higher fusion oncoprotein, being generated by a 2;13 chromosomal frequency of metastases at the initial diagnosis than ERMS, translocation. This fusion-positive ARMS is the most commonly conferring a poorer prognosis than ERMS (2, 3). clinically difficult type of rhabdomyosarcoma. The present A common characteristic of ERMS is a loss of study characterized four genes [gremlin 1 (GREM1), death- heterozygosity at 11p15, however ERMS has not been associated protein kinase-1 (DAPK1), myogenic reported to exhibit a diagnostic genetic alteration. In contrast, differentiation-1 (MYOD1), and hairy/enhancer-of-split chromosomal translocation is frequently observed for ARMS related with YRPW motif-1 (HEY1)] as targets of (4, 5). The translocation t(2;13)(q35;q14) generating the PAX3−FOXO1. Materials and Methods: The expression of paired box 3−forkhead box protein O1 (PAX3−FOXO1) gene the four genes, PAX3−FOXO1, and v-myc myelocytomatosis fusion was found to occur in 55% of ARMS cases, while the viral-related oncogene, neuroblastoma-derived (avian) translocation t(1;13)(q36;q14) generating the paired box (MYCN) was determined in various ARMS cell models and 7−forkhead box protein O1 (PAX7−FOXO1) gene fusion primary tumors.
    [Show full text]
  • Functional Study of Transcriptional Corepressor GPS2 and Tumor
    THE FUNCTIONAL STUDY OF TRANSCRIPTIONAL COREPRESSOR G- PROTEIN SUPRESSOR 2 (GPS2) AND TUMOR SUPRESSOR PROMYELOCYTIC LEUKEMIA (PML) by XIWEN CHENG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Hung-Ying Kao Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of __Xiwen Cheng__________________ candidate for the Ph.D. degree*. (signed)_______David_Samols__Ph.D._________________ (chair of the committee) _____________Edward Stavnezer__Ph.D._____________ _____________Gary Landreth___Ph.D.________________ _____________Hung-Ying Kao___Ph.D.________________ _____________Youwei Zhang____Ph.D._______________ ________________________________________________ (date) __May 26, 2010____________ *We also certify that written approval has been obtained for any proprietary material contained therein. To my parents and family Table of Contents LIST OF TABLES ................................................................................................. 7 LIST OF FIGURES ............................................................................................... 8 ACKNOWLEDGEMENTS ................................................................................... 12 LIST OF ABBREVIATIONS ................................................................................ 13 CHAPTER 1: INTRODUCTION ...................................................................... 19
    [Show full text]