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Download, Print and Save Electronic Copies of Whole Works for Their Own Personal Non-Commercial Use - 1 - - 2 - Abstract Human centromeres contain large arrays of α-satellite DNA that are thought to provide centromere function. These arrays show size and sequence variations. However, the lower limit of the sizes of these DNA arrays in normal centromeres is unknown. Using a set of chromosome-specific α-satellite probes for each of the human chromosomes, interphase Fluorescence In Situ Hybridisation (FISH) was performed in a population screening study. This study demonstrated that extreme reduction of chromosome-specific α-satellite is unusually common in chromosome 21 (screened with the αRI probe), with a prevalence of 3.70%, compared to ≤0.12 % for each of chromosomes 13 and 17, and 0 % for the other chromosomes. No analphoid centromere was identified in over 17,000 morphologically normal chromosomes studied. All the low- alphoid centromeres are fully functional as indicated by their mitotic stability and binding to centromere proteins including CENtromere Protein-A (CENP-A), CENtromere Protein-B (CENP-B), CENtromere Protein-C (CENP-C), and CENtromere Protein-E (CENP-E). Sensitive metaphase FISH analysis of the low-alphoid chromosome 21 centromeres established the presence of residual αRI as well as other non-αRI α-satellite DNA suggesting that centromere function may be provided by (i) the residual αRI DNA, (ii) other non-αRI α-satellite sequences, (iii) a combination of i and ii, or (iv) an activated neocentromere DNA. These low-alphoid centromeres contained 51-184 kb (mean = 78 kb) of α-satellite, determined using a novel Quantitative-FISH (Q-FISH) methodology. Further delineation of the boundaries of CENP-A binding domain and the small α- satellite array, however, has been hindered by the low resolution offered by fluorescence microscopy and the lack of genomic markers. Neocentromeres belong to a different class of centromeres formed at interstitial genome segments. They are characteristically devoid of highly repetitive sequences. CENP-A is a histone H3 homologue thought to be essential for proper centromere formation. CENP-A binds to the centromere DNA and is proposed to organise DNA into specialised nucleosomal structure. This centromere-specific chromatin is essential for the - 3 - nucleation and the functioning of a centromere. Using the 10q25.2 neocentromere on the marker chromosome mardel(10) as a model system, a combined chromatin- immunoprecipitation and Bacterial Artificial Chromosome (BAC) genomic array- screening procedure, called Functional And Structural Topography Scanning Along ChromosomeS (FASTSACS), was developed to study the centromere chromatin. A region of ~350-kb CENP-A-binding domain was defined. This domain shows a depletion of normal histone H3 but not histone H4, providing in vivo evidence for the existence of specialised nucleosomes at the neocentromere. Changing acetylation status using the histone deacetylase inhibitor Trichostatin A (TSA) results in a unidirectional shift of the CENP-A domain to an adjacent position 300-400 kb away, with no significant alteration in the size of the domain or overt effect on neocentromere activity. These data suggest an optimal size requirement for the CENP-A-binding domain and provide the first example of in vivo inducible centromerisation of a previously non-centromeric DNA. - 4 - Declaration This is to certify that, (i) the thesis comprises only my original work, except where indicated in the preface and acknowlegements, (ii) due acknowledgment has been made in the text to all other materials used, (iii) the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Wing Ip Anthony Lo September, 2000 - 5 - Preface I would like to acknowledge the following people that have significantly contribute to this work. Alpha-satellite clones (pZ5.1, pX2, pAE0.68, p4n1/4, pEDZ6, pZ7.5, pZ8.4, pMR9A, pZ10-2.3, pB12, pZ16A. pZ20, pLAX and pLAY5.5) were obtained from Dr. M. Rocchi. The probe αRI, αXT (Jorgensen et al. 1987) and L1.84 (Devilee et al. 1986) were gifts from the respective authors. Clones pTRA-20, TR-17 and pTRA-7, were generated by Dr. K.H.A. Choo. BAC clone E8 was identified by Dr. M.R. Cancilla with Ms K. Tainton. Dr. R. Saffery and Dr. K.H.A. Choo, with the technical assistance of Mr. A.K. Aung, Ms. B. Griffiths, Ms. D.V. Irvine and Ms. A. Stafford, produced the 10q25.2 contig map and provided the glycerol stocks of the selected BAC clones. Anti-mouse CENPA, anti-human CENP-A and anti-CENPC antibodies were produced by Dr. P. Kalitsis. Anti-mouse CENPB antibody was a gift from Dr. D. Hudson. Anti-human CENP-E antibody was a gift from Dr. T.J. Yen. The somatic cell hybrid, WAVR-4d-F9-4a, was a gift from Dr. R.H. Riddle. The cell lines, BE2C1-18-1f and BE2C1-18-5f, were produced by Dr. D. du Sart. The combined effort of Dr. K.H.A. Choo, Ms. M.E. Earle and Mr. G.C.-C. Liao significantly contributed to approximately 5 % of the interphase FISH screening for low- alphoid/analphoid centromeres. Cytogenetic analysis and specimen preparations were performed by the staff of the Victorian Clinical Genetics Services as part of their routine clinical laboratory services. Retrieval and re-freezing cell lines in Phase II studies were performed by the staff of Tissue Culture Facilities of the Murdoch Childrens Research Institute operated by Ms. M. Crawford. - 6 - Dr. J.M. Craig performed cell preparation and counting in the mitotic analysis of the marker chromosome mardel(10) in the study of the effect of trichostatin A to the CENP- A binding domain in neocentromere. This work has been accredited by the Royal College of Pathologists of Australasia (RCPA) and the Hong Kong College of Pathologists (HKCP) as training, equivalent for 2 years and 1 year, respectively. - 7 - Acknowledgments I would like to thank all the wonderful people in the Murdoch Childrens Research Institute for creating such a stimulating environment for quality research. I thank the support of University of Melbourne and MCRI for offering me the Melbourne International Research Scholarship, International Postgraduate Research Scholarship and an MCRI award. My special thanks also go to the director of MCRI, Prof. Bob Williamson, who has been very encouraging since I first applied for Ph.D. to MCRI in early 1996. I am very grateful to my supervisor, Dr. K.H. Andy Choo, for all his help throughout these 3.5 years, being very understanding and most importantly, directing a research team which is both friendly, resourceful and powerful. I would also like to thank members of my Ph.D. committee: Dr. P. Ioannou (chairman), Dr. S. la Fontaine (1997- 98), Dr. A. McCall (1998-99), Dr. J. Mercer (1997-98), Dr. K. Nararyanan, Dr. D. Newgreen (1999-2000) and Dr. H. Slater. Hearty thanks to all the past and present laboratory members of the Chromosome Research Group who have constituted such a great team and making me proud to be associated with. Special thanks to Dr. Suzi Cutts, Ms. Liz Earle and Dr. Lee Wong, who have been advising me on a lot of my everyday technical challenges. Besides those acknowledged in the “Preface” for contribution to the work reported in this publication, collaboration works have been set up with nearly all the members of the laboratory, from whom I have learned a lot and be most grateful, 1. Production of HAC from chromosome 21 with Dr. Richard Saffery; 2. Study of truncation product on mardel(10) with Mr. Andrew MacDonald; 3. Characterisation of telomere and α-satellite in a bottom-up E8 HAC with Dr. Michael Cancilla, Dr. Suzi Cutts, Ms. Linda Hii and Ms Kelly Tainton; 4. Study of a ring marker chromosome 1p32-p36.1 with Ms. Liz Earle; 5. Study of a chromosome 21 with diminished heterochromatin at the centromere with Ms. Anne Robertson (VCGS); - 8 - 6. Characterisation of mouse CENPA/GFP embryonal stem cells with Ms. Saara Redwood and Dr. Emily Howman; 7. Characterisation of Cenpf knock out mouse with Ms. Kerry Fowler and Dr. Richard Saffery; 8. Mapping of mouse Cenph gene with Ms. Kerry Fowler and Mr. Dave Longmuir; 9. Study of histone acetylation status of the 10q25.2 neocentromere with Dr. Alyssa Barry and Dr. Jeff Craig; 10. FASTSACS to identify the neocentromere on invdup(20p) with Dr. Dianna Magliano and Ms. Mandy Sibson; 11. Characterisation of a chromosome 18 derived marker chromosome with Dr. Jane Craig and Dr. Howard Slater (VCGS). Special thanks also to Prof. Ed Janus who acted as my clinical supervisor from 1997- 99 and offered me a post of “Honorary Registrar” in the core laboratory of Clinical Biochemistry of the Royal Children’s Hospital for continuation of my training for FRCPA and FHKCP. Thanks to Ms. Wai Kit Lam for producing the originals of the images of my wife, Dr. Wai Ming Lam. Finally, I would also like to thank Ms Sarah Chan, Dr. J. Craig and Dr. R. Saffery for critical reading of this thesis and helpful discussions. awilo 16th September, 2000 Melbourne, Australia - 9 - Brief Table of Contents Abstract........................................................................................................................1 Preamble .................................................................................................................... 22 CHAPTER 1 INTRODUCTION ........................................................................... 23 CHAPTER 2 MATERIALS AND METHODS.................................................... 49 CHAPTER 3 MINIMAL FUNCTIONAL ALPHA-SATELLITE DNA............ 69 CHAPTER 4 NEOCENTROMERE CHROMATIN........................................... 89 CHAPTER 5 OVERVIEW AND FUTURE DIRECTIONS
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