DOCSLIB.ORG

(Nurd) Complex

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(Nurd) Complex Understanding the Architecture, Assembly, and Interactome of the Nucleosome Remodelling and Deacetylase (NuRD) Complex Mehdi Sharifi Tabar A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy School of Life and Environmental Sciences Faculty of Science University of Sydney Sydney, Australia June 2019 i DECLARATION The work described in this Thesis was performed between August 2015 and December 2018 in the School of Life and Environmental Sciences at the University of Sydney. The experiments were carried out by the author, unless stated otherwise. This work has not been submitted, in part or in full, for a higher degree at any other institution. Mehdi Sharifi Tabar June 2019 ii ABSTRACT This thesis presents data to enhance our understanding of the composition, assembly, and structure of the nucleosome remodeling and deacetylase (NuRD) complex, a transcriptional coregulator that is strongly linked to cancer. Understanding the structure and function of the gene regulating complexes will lead to a better understanding of mechanisms of gene regulation. This should ultimately both allow insight into disease processes that originate from gene regulatory network malfunction and might enable reprogramming of the gene expression for application in health, agriculture and industry. The Thesis is broken into four sections: the first Chapter is a general introduction to gene regulation, the second chapter is materials and methods. Chapter 3 is a manuscript that is written about the stoichiometry and composition of the complex in cancer cells. Chapters 4 and 5 are results chapters investigate the structure of the subcomplexes. Chapter 6 discuss the finding of this thesis. Chapter 1 represents a general introduction to gene regulation and the NuRD complex. The NuRD complex is a 1mDa multisubunit transcriptional coregulator protein complex, which is widely expressed and harbours both chromatin remodelling and histone deacetylase activities. Abnormalities in a number of its components are associated with various types of cancers and neurodegenerative diseases. Although the core components of the complex have been well established, the structural basis of the complex has poorly been studied. Chapter 2 describes the details of the materials and methods used in this research. Chapter 3 investigates NuRD complex stoichiometry and interactome using a label-free quantitative proteomics method and provides biochemical evidence for two new direct interactors of the complex. Chapter 4 and 5 investigate the recombinantly produced subcomplexes using biochemical and a mass spectrometry-based hybrid method for structural modelling of the NuRD complex. Chapter 6 discuss the findings and outlines the future directions. iii ACKNOWLEDGEMENT Three and a half years after starting on my PhD adventure I have finally reached the end. This journey would not have been possible without the support of my supervisor Joel Mackay and lab mates. First of all, I would like to sincerely thank Joel Mackay whose guidance has been invaluable in all aspects of the thesis production. His generosity with both time and knowledge is deeply appreciated. I am highly grateful for his patience and encouragement in the face of the many bewilderment of research. It was a real privilege and an honour for not only to undertake a PhD under his supervision but also of his extraordinary human qualities. Enormous thanks are owed to Dr. Jason Low for teaching and demonstrating me mass spectrometry based proteomics and also being an inexhaustible source of useful discussions. I have no doubt that without Joel and Jason I would not be able to smoothly undertake my PhD. Special thanks also goes to Dr. Mario Torrado Delray, for his abundant assistance with protein-protein interaction and pulldown experiments, and Dr. Ana Silva for providing advice and help at various stages of my PhD and in various forms. I also would like to thank the entire Mackay & Matthews lab members and everybody in Structural Biology group and also the Mass Spectrometry core facility at CPC. The lab environment has been a fantastic place to work, with everyone always willing to help each other out. Last, but definitely not least, thank you very much to my lovely and kind wife, Diba, for her tremendous help and for always putting a smile on my face and making this journey stress free. I also extend my gratitude to my wonderful family for their unconditional support throughout my PhD, and always. iv CONTENTS Chapter1. Introduction .................................................................................... 1 1.1 Gene regulation ............................................................................................. 1 1.2 Chromatin ...................................................................................................... 2 1.3 The histone code hypothesis and chromatin remodelling .................................. 2 1.4 Histone acetylation and deacetylation .............................................................. 3 1.5 Chromatin remodelling protein complexes ........................................................ 5 1.6 Our structural understanding of chromatin remodellers ..................................... 5 1.7 The NuRD complex ......................................................................................... 8 1.8 Recruitment of the NuRD to target sites .......................................................... 9 1.9 Molecular function of the NuRD ..................................................................... 10 1.10 NuRD in pluripotency .................................................................................. 11 1.11 NuRD in disease ......................................................................................... 12 1.12 NuRD subunits ........................................................................................... 14 1.12.1 MTA1-3 ................................................................................................... 15 1.12.2 HDAC1/2 ................................................................................................. 16 1.12.3 CHD3-5 ................................................................................................... 17 1.12.4 GATAD2A/B ............................................................................................. 19 1.12.5 MBD2/3 .................................................................................................. 20 1.12.6 RBBP4/7 ................................................................................................. 20 1.12.7 CDK2AP1/DOC-1 ...................................................................................... 22 1.13 Aims of this thesis ...................................................................................... 22 Chapter 2: Materials and Methods ................................................................ 24 2.1 Materials ...................................................................................................... 24 2.2 Methods ...................................................................................................... 26 v 2.2.1 Cloning ..................................................................................................... 26 2.2.1.1 Primer design ......................................................................................... 26 2.2.1.2 RNA isolation & cDNA synthesis ............................................................... 26 2.2.1.3 Polymerase chain reaction (PCR) ............................................................. 26 2.2.1.4 Gibson assembly ..................................................................................... 27 2.2.1.5 Colony PCR ............................................................................................ 27 2.2.2 Cell culture ................................................................................................ 28 2.2.2.1 Expi293 cells .......................................................................................... 28 2.2.2.2 NTERA2, PC3 and MCF7 cell culture ......................................................... 28 2.2.3 siRNA knock down experiment ................................................................... 28 2.2.4 Quantitative real-time PCR (qRT-PCR, qPCR) ............................................... 29 2.2.5 Protein production and FLAG-affinity purification ......................................... 29 2.2.5.1 Protein production in mammalian EXPI293 cells ........................................ 30 2.2.5.2 Protein production and purification in cell free system (Rabbit Reticulocyte Lysate) .............................................................................................................. 30 2.2.5.3 Preparing sucrose gradients (tilting method) ............................................ 31 2.2.5.4 Protein concentration and buffer exchange .............................................. 31 2.2.6 Native NuRD complex pull down ................................................................. 31 2.2.6.1 Preparation of bead-bound-GST-FOG ....................................................... 31 2.2.6.2 NuRD extraction from nucleus ................................................................. 32 2.2.6.3 NuRD pulldown experiment ..................................................................... 32 2.2.7 Protein analysis ......................................................................................... 32 2.2.7.1 SDS-PAGE .............................................................................................
Load more

Recommended publications
  • DYRK1A Protein Kinase Promotes Quiescence and Senescence Through DREAM Complex Assembly
    Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly Larisa Litovchick,1,2 Laurence A. Florens,3 Selene K. Swanson,3 Michael P. Washburn,3,4 and James A. DeCaprio1,2,5,6 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA; 2Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA; 3Stowers Institute for Biomedical Research, Kansas City, Missouri 64110, USA; 4Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas City, Kansas 66160, USA; 5Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma] , E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly.
    [Show full text]
  • Genetic Variants in A
    Neurobiology of Aging 35 (2014) 2881.e7e2881.e10 Contents lists available at ScienceDirect Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging Brief communication Genetic variants in a ‘cAMP element binding protein’ (CREB)-dependent histone acetylation pathway influence memory performance in cognitively healthy elderly individuals Sandra Barral a,b, Christiane Reitz a,b, Scott A. Small a,b, Richard Mayeux a,b,* a Department of Neurology, Columbia University Medical Center, New York, NY, USA b Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, New York, NY, USA article info abstract Article history: The molecular pathways underlying age-related memory changes remain unclear. There is a substantial Received 5 March 2014 genetic contribution to memory performance through life span. A recent study has implicated RbAp48, Received in revised form 17 June 2014 which mediates its effect on age-related memory decline by interacting with cyclic adenosine mono- Accepted 24 June 2014 phosphate responsive element binding protein (CREB)1 binding protein and influencing this histone Available online 28 June 2014 acetylation pathway. To validate these findings, we tested whether genetic variants in RbAp48, CREB1, and CREBBP are associated with memory performance in 3 independent data sets consisting of 2674 Keywords: cognitively healthy elderly individuals. Genetic variant rs2526690 in the CREBBP gene was significantly Histone metabolism ¼ Â À4 Meta-analysis associated with episodic memory performance (pmeta 3.7 10 ) in a multivariate model adjusted for Episodic memory performance age, sex, and apolipoprotein E status. Identifying genetic variants that modulate mechanisms of cognitive aging will allow identifying valid targets for therapeutic intervention.
    [Show full text]
  • Engaging Chromatin: PRC2 Structure Meets Function
    www.nature.com/bjc REVIEW ARTICLE Engaging chromatin: PRC2 structure meets function Paul Chammas1, Ivano Mocavini1 and Luciano Di Croce1,2,3 Polycomb repressive complex 2 (PRC2) is a key epigenetic multiprotein complex involved in the regulation of gene expression in metazoans. PRC2 is formed by a tetrameric core that endows the complex with histone methyltransferase activity, allowing it to mono-, di- and tri-methylate histone H3 on lysine 27 (H3K27me1/2/3); H3K27me3 is a hallmark of facultative heterochromatin. The core complex of PRC2 is bound by several associated factors that are responsible for modulating its targeting specificity and enzymatic activity. Depletion and/or mutation of the subunits of this complex can result in severe developmental defects, or even lethality. Furthermore, mutations of these proteins in somatic cells can be drivers of tumorigenesis, by altering the transcriptional regulation of key tumour suppressors or oncogenes. In this review, we present the latest results from structural studies that have characterised PRC2 composition and function. We compare this information with data and literature for both gain-of function and loss-of-function missense mutations in cancers to provide an overview of the impact of these mutations on PRC2 activity. British Journal of Cancer (2020) 122:315–328; https://doi.org/10.1038/s41416-019-0615-2 BACKGROUND and embryonic ectoderm development (EED) (Table 1). These Transcriptional diversity is one of the hallmarks of cellular three proteins form the minimal core that confers histone identity. It is largely regulated at the level of chromatin, where methyltransferase (HMT) activity. A fourth factor, retinoblastoma- different protein complexes act as initiators, enhancers and/or binding protein (RBBP)4/7 (also known as RBAP48/46), has a repressors of transcription.
    [Show full text]
  • Retinoblastoma Binding Protein 4 Maintains Cycling Neural Stem Cells and Prevents DNA Damage and Tp53-Dependent Apoptosis in Rb1 Mutant Neural Progenitors Laura E
    Genetics, Development and Cell Biology Genetics, Development and Cell Biology Publications 9-25-2018 Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors Laura E. Schultz-Rogers Iowa State University Maira P. Almeida Iowa State University, [email protected] Wesley a. Wierson Iowa State University Marcel Kool Hopp Children’s Cancer Center at the NCT (KiTZ) MFoallourwa MthicsGr andail additional works at: https://lib.dr.iastate.edu/gdcb_las_pubs IowaP Satrate of U ntheiversitCya,nc mmcgrer Baiiol@ilogasyt aCteommon.edu s, and the Genetics and Genomics Commons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ gdcb_las_pubs/208. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Genetics, Development and Cell Biology at Iowa State University Digital Repository. It has been accepted for inclusion in Genetics, Development and Cell Biology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors Abstract Retinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells.
    [Show full text]
  • Rabbit Mab A
    Revision 1 C 0 2 - t SIN3A (D1B7) Rabbit mAb a e r o t S Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) 1 9 Web: [email protected] 6 www.cellsignal.com 7 # 3 Trask Lane Danvers Massachusetts 01923 USA For Research Use Only. Not For Use In Diagnostic Procedures. Applications: Reactivity: Sensitivity: MW (kDa): Source/Isotype: UniProt ID: Entrez-Gene Id: WB, ChIP H M R Mk Endogenous 145 Rabbit IgG Q96ST3 25942 Product Usage Information nucleosome binding of the complex. The SIN3 complex functions to repress transcription, in part, by deacetylating histones at target gene promoters (3,4). In addition, recent For optimal ChIP results, use 5 μl of antibody and 10 μg of chromatin (approximately 4 x studies have shown that SIN3 is recruited to the coding regions of repressed and active 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic genes, where it deacetylates histones and suppresses spurious transcription by RNA Chromatin IP Kits. polymerase II (3,5). In addition to histone deacetylase activity, the SIN3 complex associates with histone methyltransferase (ESET), histone demethylase Application Dilution (JARID1A/RBP2), ATP-dependent chromatin remodeling (SWI/SNF), methylcytosine dioxygenase (TET1), and O-GlcNAc transferase (OGT) activities, all of which appear to Western Blotting 1:1000 contribute to the regulation of target genes (5-9). The SIN3 complex is critical for proper Chromatin IP 1:100 regulation of embryonic development, cell growth and proliferation, apoptosis, DNA replication, DNA repair, and DNA methylation (imprinting and X-chromosome inactivation) Storage (3,4).
    [Show full text]
  • RBBP4 Protein Full-Length Recombinant Human Protein Expressed in Sf9 Cells
    Catalog # Aliquot Size R314-30G-20 20 µg R314-30G-50 50 µg RBBP4 Protein Full-length recombinant human protein expressed in Sf9 cells Catalog # R314-30G Lot # U1595-3 Product Description Purity Full-length recombinant human RBBP4 was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. The gene accession number is BC075836. The purity of RBBP4 was determined Gene Aliases to be >90% by densitometry. Approx. MW 75 kDa. NURF55, RBAP48 Formulation Recombinant protein stored in 50mM Tris-HCl, pH 7.5, 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM DTT, 0.1mM PMSF, 25% glycerol. Storage and Stability Store product at –70oC. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. Scientific Background Histone-binding protein RBBP4 (RBBP4) was initially identified as one of the strongest interactions when a CREB-CREB binding protein (CBP) complex was used as bait to screen a mouse embryo cDNA library in yeast. May target chromatin assembly factors, chromatin remodeling factors and histone deacetylases to their RBBP4 Protein histone substrates in a manner that is regulated by Full-length recombinant human protein expressed in Sf9 cells nucleosomal DNA. Catalog # R314-30G References Lot # U1595-3 Purity >90% Concentration 0.1 µg/µl 1. Zhang Q, et al: Histone binding protein RbAp48 interacts Stability 1yr at –70oC from date of shipment with a complex of CREB binding protein and phospho- Storage & Shipping Store product at –70oC. For optimal rylated CREB.
    [Show full text]
  • PWWP2A Binds Distinct Chromatin Moieties and Interacts with an MTA1-Specific Core Nurd Complex
    ARTICLE DOI: 10.1038/s41467-018-06665-5 OPEN PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex Stephanie Link1,2, Ramona M.M. Spitzer1,2, Maryam Sana3, Mario Torrado3, Moritz C. Völker-Albert1, Eva C. Keilhauer4,9, Thomas Burgold5,10, Sebastian Pünzeler1,11, Jason K.K. Low3, Ida Lindström 3, Andrea Nist6, Catherine Regnard1, Thorsten Stiewe 6,7, Brian Hendrich5, Axel Imhof 1,8, Matthias Mann 4,8, Joel P. Mackay 3, Marek Bartkuhn2 & Sandra B. Hake 2,8 1234567890():,; Chromatin structure and function is regulated by reader proteins recognizing histone mod- ifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A. Z-containing nucleosomes and is involved in mitotic progression and cranial–facial devel- opment. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels. 1 Department of Molecular Biology, BioMedical Center (BMC), Ludwig-Maximilians-University Munich, 82152 Planegg-Martinsried, Germany.
    [Show full text]
  • Aberrations of EZH2 in Cancer
    Published OnlineFirst March 2, 2011; DOI: 10.1158/1078-0432.CCR-10-2156 Clinical Cancer Molecular Pathways Research Aberrations of EZH2 in Cancer Andrew Chase and Nicholas C.P. Cross Abstract Control of gene expression is exerted at a number of different levels, one of which is the accessibility of genes and their controlling elements to the transcriptional machinery. Accessibility is dictated broadly by the degree of chromatin compaction, which is influenced in part by polycomb group proteins. EZH2, together with SUZ12 and EED, forms the polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3). PRC2 may recruit other polycomb complexes, DNA methyltransferases, and histone deacetylases, resulting in additional transcriptional repressive marks and chromatin compaction at key developmental loci. Overexpression of EZH2 is a marker of advanced and metastatic disease in many solid tumors, including prostate and breast cancer. Mutation of EZH2 Y641 is described in lymphoma and results in enhanced activity, whereas inactivating mutations are seen in poor prognosis myeloid neoplasms. No histone demethylating agents are currently available for treatment of patients, but 3-deazaneplanocin (DZNep) reduces EZH2 levels and H3K27 trimethylation, resulting in reduced cell proliferation in breast and prostate cancer cells in vitro. Furthermore, synergistic effects are seen for combined treatment with DNA demethylating agents and histone deacetylation inhibitors, opening up the possibility of refined epigenetic treatments in the future. Clin Cancer Res; 17(9); 2613–8. Ó2011 AACR. Background trithorax homolog myeloid-lymphoid leukemia (MLL), is associated with transcriptional activation (Fig. 1A). Impor- DNA is wrapped around histone complexes termed tantly, many genes involved in development, stem cell nucleosomes, and gene accessibility is determined largely maintenance, and differentiation are targets of H3K27 by the local chromatin configuration.
    [Show full text]
  • Balance Between Retroviral Latency and Transcription: Based on HIV Model
    pathogens Review Balance between Retroviral Latency and Transcription: Based on HIV Model Aneta Pluta 1,* , Juan P. Jaworski 2 and César N. Cortés-Rubio 3 1 Department of Biochemistry, National Veterinary Research Institute, 24-100 Puławy, Poland 2 Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de Virología, Nicolás Repetto y De los Reseros (s/n), CP1686 Hurlingham, Buenos Aires, Argentina; [email protected] 3 Centre for Research in Infectious Diseases, National Institute of Respiratory Diseases, Tlalpan 4502, 14080 Mexico City, Mexico; [email protected] * Correspondence: [email protected] Abstract: The representative of the Lentivirus genus is the human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). To date, there is no cure for AIDS because of the existence of the HIV-1 reservoir. HIV-1 infection can persist for decades despite effective antiretroviral therapy (ART), due to the persistence of infectious latent viruses in long-lived resting memory CD4+ T cells, macrophages, monocytes, microglial cells, and other cell types. However, the biology of HIV-1 latency remains incompletely understood. Retroviral long terminal repeat region (LTR) plays an indispensable role in controlling viral gene expression. Regulation of the transcription initiation plays a crucial role in establishing and maintaining a retrovirus latency. Whether and how retroviruses establish latency and reactivate remains unclear. In this article, we describe what is known about the regulation of LTR-driven transcription in HIV-1, that is, the cis-elements present in the LTR, the role of LTR transcription factor binding sites in LTR-driven transcription, the role of HIV-1-encoded transactivator protein, hormonal effects on virus transcription, impact of LTR variability on transcription, and epigenetic control of retrovirus LTR.
    [Show full text]
  • The Kinesin Spindle Protein Inhibitor Filanesib Enhances the Activity of Pomalidomide and Dexamethasone in Multiple Myeloma
    Plasma Cell Disorders SUPPLEMENTARY APPENDIX The kinesin spindle protein inhibitor filanesib enhances the activity of pomalidomide and dexamethasone in multiple myeloma Susana Hernández-García, 1 Laura San-Segundo, 1 Lorena González-Méndez, 1 Luis A. Corchete, 1 Irena Misiewicz- Krzeminska, 1,2 Montserrat Martín-Sánchez, 1 Ana-Alicia López-Iglesias, 1 Esperanza Macarena Algarín, 1 Pedro Mogollón, 1 Andrea Díaz-Tejedor, 1 Teresa Paíno, 1 Brian Tunquist, 3 María-Victoria Mateos, 1 Norma C Gutiérrez, 1 Elena Díaz- Rodriguez, 1 Mercedes Garayoa 1* and Enrique M Ocio 1* 1Centro Investigación del Cáncer-IBMCC (CSIC-USAL) and Hospital Universitario-IBSAL, Salamanca, Spain; 2National Medicines Insti - tute, Warsaw, Poland and 3Array BioPharma, Boulder, Colorado, USA *MG and EMO contributed equally to this work ©2017 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2017.168666 Received: March 13, 2017. Accepted: August 29, 2017. Pre-published: August 31, 2017. Correspondence: [email protected] MATERIAL AND METHODS Reagents and drugs. Filanesib (F) was provided by Array BioPharma Inc. (Boulder, CO, USA). Thalidomide (T), lenalidomide (L) and pomalidomide (P) were purchased from Selleckchem (Houston, TX, USA), dexamethasone (D) from Sigma-Aldrich (St Louis, MO, USA) and bortezomib from LC Laboratories (Woburn, MA, USA). Generic chemicals were acquired from Sigma Chemical Co., Roche Biochemicals (Mannheim, Germany), Merck & Co., Inc. (Darmstadt, Germany). MM cell lines, patient samples and cultures. Origin, authentication and in vitro growth conditions of human MM cell lines have already been characterized (17, 18). The study of drug activity in the presence of IL-6, IGF-1 or in co-culture with primary bone marrow mesenchymal stromal cells (BMSCs) or the human mesenchymal stromal cell line (hMSC–TERT) was performed as described previously (19, 20).
    [Show full text]
  • Targeting Human Retinoblastoma Binding Protein 4 (RBBP4) and 7 (RBBP7)
    bioRxiv preprint doi: https://doi.org/10.1101/303537; this version posted April 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Targeting Human Retinoblastoma Binding Protein 4 (RBBP4) and 7 (RBBP7) Megha Abbey1, Viacheslav Trush1, Elisa Gibson1, and Masoud Vedadi1,2* 1Structural Genomics Consortium, University of Toronto, Toronto, Ontario, M5G 1L7, Canada 2Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada To whom correspondence should be addressed: Masoud Vedadi; Tel.: 416-432-1980; E-mail: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/303537; this version posted April 18, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract RBBP4 and RBBP7 (RBBP4/7) are highly homologous nuclear WD40 motif containing proteins widely implicated in various cancers and are valuable drug targets. They interact with multiple proteins within diverse complexes such as NuRD and PRC2, as well as histone H3 and H4 through two distinct binding sites. FOG-1, PHF6 and histone H3 bind to the top of the donut shape seven-bladed β-propeller fold, while SUZ12, MTA1 and histone H4 bind to a pocket on the side of the WD40 repeats. Here, we briefly review these six interactions and present binding assays optimized for medium to high throughput screening. These assays enable screening of RBBP4/7 toward the discovery of novel cancer therapeutics.
    [Show full text]
  • Global Analysis of Chromosome X Gene Expression in Primary Cultures of Normal Ovarian Surface Epithelial Cells and Epithelial Ovarian Cancer Cell Lines
    5-17 6/12/06 18:21 Page 5 INTERNATIONAL JOURNAL OF ONCOLOGY 30: 5-17, 2007 5 Global analysis of chromosome X gene expression in primary cultures of normal ovarian surface epithelial cells and epithelial ovarian cancer cell lines MARIE-HÉLÈNE BENOÎT1, THOMAS J. HUDSON1,2,3, GEORGES MAIRE4, JEREMY A. SQUIRE4,5, SUZANNA L. ARCAND6, DIANE PROVENCHER7,8, ANNE-MARIE-MES-MASSON7,9 and PATRICIA N. TONIN1,3,6 1Department of Human Genetics, McGill University, Montreal, Quebec H3A 1A1; 2McGill University and Genome Quebec Innovation Centre, Montreal, Quebec H3A 1A4; 3Department of Medicine, McGill University, Montreal, Quebec H3G 1A4; 4Applied Molecular Oncology, The Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Ontario M5G 2M9; 5Departments of Medical Biophysics and Laboratory of Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5; 6The Research Institute of McGill University Health Center, Montreal, Quebec H3G 1A4; 7Centre de Recherche du Centre Hospitalier de l'Université de Montreal (CR-CHUM)/Institut du cancer de Montréal, Montreal, Quebec H2L 4M1; 8Division de gynécologie et obstétrique, and 9Départément de médicine, Université de Montréal, Montreal, Quebec H3C 3J7, Canada Received June 2, 2006; Accepted August 3, 2006 Abstract. The interpretation of loss of heterozygosity (LOH) and TOV21G. The combined evidence is consistent with two in cancers is complicated as genes that map to LOH regions proposed mechanisms to account for absence of Xi in female may be transcriptionally active (Xa) or inactive (Xi) due to X cancers: Xi loss followed by Xa duplication (exemplified by chromosome inactivation (XCI). We have analyzed the TOV112D) and transcriptional reactivation of Xi (exemplified chromosome X transcriptome in four epithelial ovarian by TOV21G).
    [Show full text]