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1.2.3 the Role O F Hox Genes in Regulating Haematopoiesis ------32 2809585471 REFERENCE ONLY UNIVERSITY OF LONDON THESIS Year ^ Name of Author £ H ,0 V n ^ P H O R COPYRIGHT \ cnAA*2_v v\C \ This is a thesis accepted for a Higher Degree of the University of London. It is an unpublished typescript and the copyright is held by the author. All persons consulting this thesis must read and abide by the Copyright Declaration below. COPYRIGHT DECLARATION I recognise that the copyright of the above-described thesis rests with the author and that no quotation from it or information derived from it may be published without the prior written consent of the author. LOANS Theses may not be lent to individuals, but the Senate House Library may lend a copy to approved libraries within the United Kingdom, for consultation solely on the premises of those libraries. Application should be made to: Inter-Library Loans, Senate House Library, Senate House, Malet Street, London WC1E 7HU. REPRODUCTION University of London theses may not be reproduced without explicit written permission from the Senate House Library. Enquiries should be addressed to the Theses Section of the Library. Regulations concerning reproduction vary according to the date of acceptance of the thesis and are listed below as guidelines. A. Before 1962. Permission granted only upon the prior written consent of the author. (The Senate House Library will provide addresses where possible). B. 1962-1974. In many cases the author has agreed to permit copying upon completion of a Copyright Declaration. C. 1975-1988. Most theses may be copied upon completion of a Copyright Declaration. D. 1989 onwards. Most theses may be copied. This thesis comes within category D. This copy has been deposited in the Library of LLC.L- This copy has been deposited in the Senate House Library, Senate House, Malet Street, London WC1E 7HU. Investigation of the role of MLL-ENL in leukaemogenesis Tanzina Chowdhury Institute of Child Health University College London A thesis submitted for the Degree of Doctor of Philosophy UMI Number: U591882 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591882 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declaration I, Tanzina Chowdhury, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Acute leukaemia is the most common form of childhood cancer and is the primary cause of cancer-related mortality in children. The major initiating event in infant leukaemia is rearrangement of the Mil gene at 1 lq23, and such leukaemias have a poor survival rate. Chromosomal translocations occur commonly in leukaemia and an in-frame fusion gene is created with altered properties to the original genes involved. Translocations of chromosome 1 lq23 result in the fusion of Mil to a variety of partners genes. Mil-Aft results from t(9;l 1), Mll-A/4 from t(4; 11) and Mll-Enl from t( 11; 19) and are thought to cause aberrant transcriptional regulation. To identify candidate target genes of t( 11; 19), MLL-ENL has been over­ expressed in 32D cells, an immortalised myeloid progenitor cell line. The actions of MLL-ENL on cell survival following growth factor withdrawal, and on G-CSF mediated differentiation have been studied. Potential transcriptional targets of MLL-AF9 and MLL-ENL have been identified using microarray technology. Ten genes were identified as possible candidate target genes of the MLL-ENL protein that might be involved in leukaemogenesis. Of particular interest were Gatal and HTm4 which have a role in haematopoiesis and cell cycle regulation. shRNA constructs were generated to knock down MLL-ENL and allow validation of the potential target genes. The mechanism of leukaemic transformation by MLL-ENL has also been investigated. In leukaemia, fusion proteins may contribute to haematological malignancy in various ways, though the main routes are thought to be either a gain-of-function or dominant negative action. In order 3 to determine the mechanism by which a MLL fusion protein immortalizes murine haematopoietic progenitor cells (HPC), a system was developed where MLL-ENL was expressed in murine HPCs in the absence of both alleles of Mil. The experiments were carried out using murine HPCs from conditional Mil knockout mice generated in our laboratory where the wild- type Mil allele is flanked by LoxP sites at exons 9 and 10, and can be inactivated following Cre-mediated recombination. The ability of cells to undergo sequential re-plating in methylcellulose was used as an indication of immortalization. These experiments now definitively show that the MLL-ENL fusion protein acts in a gain-of-function manner. 4 Contents INVESTIGATION OF THE ROLE OF MLL-ENL IN LEUKAEMOGENESIS---------------- 1 DECLARATION------------------------------------------------------------------------------------------------------2 ABSTRACT------------------------------------------------------------------------------------------------------------- 3 CONTENTS------------------------------------------------------------------------------------------------------------ 5 LIST OF FIGURES-------------------------------------------------------------------------------------------------- 9 LIST OF TABLES--------------------------------------------------------------------------------------------------- 11 ACKNOWLEDGEMENTS--------------------------------------------------------------------------------------- 12 ABBREVIATIONS-------------------------------------------------------------------------------------------------- 13 1 INTRODUCTION--------------------------------------------------------------------------------------------17 1.1 Haematopoiesis ----------------------------------------------------------------------------------------------------------17 1.2 T ranscriptional r e g u l a t io n o f haematopoietic development -------------------------- 22 1.2.1 Transcription factors involved in myeloid and lymphoid lineage determination - 23 1.2.2 The structure andfunction o f the MLL gene in normal haematopoiesis ------------ 26 1.2.3 The role o f Hox genes in regulating haematopoiesis -----------------------------------32 1.3 C h r o m o s o m a l translocations in p a e d ia t r ic a c u t e l e u k a e m ia -------------------------- 37 1.4 N e o p l a s t ic transformation a n d leukaemogenesis ---------------------------------------------- 39 1.4.1 Evolution o f the malignant clone --------------------------------------------------------- 39 1.4.2 The cell of origin o f the malignant clone ------------------------------------------------ 42 1.5 M L L -translocations in a c u t e l e u k a e m ia -------------------------------------------------------------- 46 1.5.1 Structure o f MLL fusion genes------------------------------------------------------------ 46 1.5.2 Structure andfunction of MLL partner proteins ---------------------------------------- 4 7 1.5.3 Mechanisms of transcriptional deregulation by MLL-fusion genes ----------------- 50 1.6 P o t e n t ia l t a r g e t s o f M L L -f u s io n g e n e s in a c u t e l e u k a e m ia ------------------------------- 56 1.6.1 The role o f Hox genes in MLL-fusion leukaemia --------------------------------------- 56 1.6.2 The role of other candidate targets o f MLL-fusion proteins --------------------------60 1.7 DOWN-REGULATION OF GENE EXPRESSION BY SH RN A ---------------------------------------------------- 61 1.8 A im s ------------------------------------------------------------------------------------------------------------------------------- 65 2 MATERIALS AND METHODS--------------------------------------------------------------------------66 2.1 Culture o f 32Dcl3 cell lines ------------------------------------------------------------------------------------68 2 .2 P l a s m id c o n s t r u c t s ----------------------------------------------------------------------------------------------------69 2.3 E lectroporation o f c e l l s w it h p l a s m id D N A ----------------------------------------------------------71 2 .4 I s o l a t io n o f s in g l e c e l l c l o n e s -------------------------------------------------------------------------------- 71 2.5 L u c if e r a s e r e p o r t e r c o n s t r u c t s a n d a s s a y s ----------------------------------------------------------72 2.6 Preparation of cellular extracts for SDS-PAGE------------------------------------------------ 72 2.7 P r o t e i n t r a n s f e r t o PVDF----------------------------------------------------------------------72 2.8 W e s t e r n a n a l y s is f o r p r o t e in e x p r e s s io n --------------------------------------------------------------- 73 2.9 Isolation of genomic DNA f r o m c e l l s ----------------------------------------------------------------------75 2 .10 S o u t h e r n b l o t t in g a n a l y s is -------------------------------------------------------------------------------- 75 2.11 IL-3 WITHDRAWAL EXPERIMENTS-------------------------------------------------------------
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