- 1 - - 2 - Abstract

Human contain large arrays of α-satellite DNA that are thought to provide 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 -specific α-satellite probes for each of the , 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 (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 including CENtromere -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 segments. They are characteristically devoid of highly repetitive sequences. CENP-A is a 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 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 but not , providing in vivo evidence for the existence of specialised 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 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 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 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 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 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 ...... 102 Bibliography ...... 110

- 10 - Table of Contents

Abstract...... 1 Declaration...... 5 Preface...... 6 Acknowledgments...... 8 Brief Table of Contents...... 10 Table of Contents ...... 11 List of Tables and Figures ...... 14 Abbreviations...... 16 Publications arising from this study...... 21 Preamble ...... 22 CHAPTER 1 INTRODUCTION ...... 23 1.2. Human Centromere DNA...... 27 1.2.1. Alpha-satellites are variable in sequences and in array sizes ...... 27 1.2.2. Significance of α-satellite in centromere functions...... 29 1.3. Human Neocentromeres ...... 30 1.4. Human Neocentromere DNA ...... 33 1.5. Centromere chromatin ...... 34 1.5.1. CENP-A and other histone H3 homologues...... 34 1.5.2. Centromere-related DNA binding proteins ...... 39 1.5.3. Organisation of chromatin in the centromere...... 43 1.6. Aims of this study...... 48 CHAPTER 2 MATERIALS AND METHODS...... 49 2.1. Materials and Chemicals ...... 50 2.2. The minimal functional α-satellite array...... 50 2.2.1. Cell lines...... 50 2.2.2. Interphase FISH screening ...... 51 2.2.3. Metaphase FISH ...... 56 2.2.4. Pulsed-Field Gel Electrophoresis (PFGE)...... 57 2.2.5. Quantitative FISH (Q-FISH) ...... 59

- 11 - 2.2.6. Immunocytochemical analysis ...... 62 2.3. Neocentromere chromatin...... 63 2.3.1. Cell lines...... 63 2.3.2. Oligonucleosome preparation...... 64 2.3.3. Immunoprecipitation ...... 65 2.3.4. 10q25.2 BAC array...... 66 2.3.5. Data Analysis...... 68 CHAPTER 3 MINIMAL FUNCTIONAL ALPHA-SATELLITE DNA...... 69 3.1. Introduction ...... 70 3.2. Phase I: interphase FISH profiles for different α-satellite probes ...... 70 3.3. Phase II: Population screening for low-alphoid or analphoid centromeres . 73 3.4. No analphoid chromosomes were detected ...... 74 3.5. Quantitation of the low-αRI arrays...... 74 3.6. Low-alphoid centromeres bind key centromere-specific antigens ...... 77 3.7. Prevalence of low-alphoid chromosome 21 in Down syndrome (DS)...... 78 3.8. Discussion...... 79 3.8.1. No analphoid centromere was identified in ~17,000 chromosomes ...... 79 3.8.2. Low alphoid array measured by a novel Q-FISH strategy...... 81 3.8.3. Low-alphoid centromeres are functional...... 84 3.8.4. High prevalence of low-alphoid centromere in Down syndrome...... 87 CHAPTER 4 NEOCENTROMERE CHROMATIN...... 89 4.1. Introduction ...... 90 4.2. CREST#6-binding domain on mardel(10) ...... 90 4.3. CENP-A-binding along the 10q25.2 neocentromere contig ...... 91 4.4. Depletion of histone H3 but not H4 within the CENP-A-binding domain.... 92 4.5. CENP-A-binding domain can be shifted by histone deacetylase inhibitor... 93 4.6. Discussion...... 94 4.6.1. FASTSACS ...... 94 4.6.2. CENP-A binds to a ~ 350-kb domain on a neocentromere ...... 95 4.6.3. A neocentromere contains unique nucleosomes that lack histone H3 ...... 97 4.6.4. A first example of inducible centromerisation ...... 99

- 12 - CHAPTER 5 OVERVIEW AND FUTURE DIRECTIONS ...... 102 5.1. Low-alphoid centromere ...... 103 5.2. Size of a neocentromere ...... 104 5.3. Nucleosomal organisation of neocentromere...... 106 5.4. Inducible centromerisation ...... 106 5.5. Implications in biomedical research...... 107 Bibliography ...... 110

- 13 - List of Tables and Figures

Table 1.1 Chromosomal rearrangement and mechanism of formation of neocentromeres Table 2.1 Chromosome-specific α-satellite probes used in this study Table 3.1 Interphase FISH screening of low-alphoid cell lines Table 4.1 Mitotic indices of 5f cell line grown in the presence or absence of 33 nM TSA Figure 1.1 Schematic diagram of human centromere Figure 1.2 Evolution and structural organisation of α-satellite DNA Figure 1.3 Formation of mardel(10) Figure 1.4 Organisation of centromere elements Figure 1.5 Histone H3 and homologues Figure 2.1 Slide templates for 20 specimens Figure 2.2 FISH of uncultured human lymphoblasts and archival cytogenetic samples Figure 2.3 Physical mapping of the 10q25.2 contig Figure 3.1 Relative frequency distribution curves of interphase FISH performed with chromosome specific α-satellite probes on cytogenetically normal subjects Figure 3.2 Relative frequency distribution curves of interphase FISH in reconstruction experiments Figure 3.3 Interphase and metaphase FISH analysis of low-alphoid centromeres Figure 3.4 Pulsed-field gel electrophoresis of low-alphoid cell lines Figure 3.5 Validation of Q-FISH analysis on chromosome 21 somatic cell hybrid Figure 3.6 Q-FISH analysis of a low-alphoid cell line Figure 3.7 Immunocytochemical-FISH studies of a low-alphoid cell line Figure 3.8 CENP-A/FISH studies of cell line with the smallest αRI array Figure 3.9 Relative frequency distribution curves of interphase FISH using the probe αRI performed on a cohort of 73 Down syndrome cell lines Figure 3.10 Organisation of α-satellite in chromosome 21 and unequal crossing over. Figure 4.1 10q25.2 BAC genomic array in the FASTSAC study

- 14 - Figure 4.2 Distribution of CREST#6-binding signals along the 10q25.2 BAC contig Figure 4.3 Distribution of CENP-A-binding signals along the 10q25.2 BAC contig Figure 4.4 Distribution of histone H3 along the 10q25.2 BAC contig Figure 4.5 Distribution of histone H4 along the 10q25.2 BAC contig Figure 4.6 Distribution of CENP-A along the 10q25.2 BAC contig with and without treatment of 33 nM Trichostatin A (TSA)

- 15 - Abbreviations 1f BE2C1-18-1f, somatic cell hybrid containing the normal human 5f BE2C1-18-5f, somatic cell hybrid containing the marker chromosome mardel(10) A Adenine Å Angstrom Abp1p Actin Binding Protein 1 Arp-1 Actin Related Protein-1 BAC Bacterial Artificial Chromosome bp BSA Bovine Serum Albumin BUB1 Budding Uninhibited by Benzimidazoles 1 BUB3 Budding Uninhibited by Benzimidazoles 3 C Cytosine C-terminus Carboxyl-terminus CBF CCAAT-Binding Proteins Cbh+ Centromere Proten B Homologue CCA Close Aposition CDEI Centromere DNA Element I CDEII Centromere DNA Element II CDEIII Centromere DNA Element III CENP Centromere Protein (different centromere proteins are named with different letters separated by a hyphen, e.g., CENP-A) CENPA Mouse Centromere Protein A* Cenpa Mouse CENP-A Gene or * CENPB Mouse * Cenpb Mouse CENP-B Gene or locus* CENPC Mouse Centromere Protein C* Cenpc Mouse CENP-C Gene or locus* CenpcA Homologue of CENP-C coded by the CenpcA gene in Zea mays

- 16 - CenpcB Homologue of CENP-C coded by the CenpcB gene in Zea mays CenpcC Homologue of CENP-C coded by the CenpcC gene in Zea mays Cenpf Mouse CENP-F Gene or locus* Cenph Mouse CENP-H Gene or locus* CHO Chinese Hamster Ovary Cid Centromere Identifier CLIP-170 Cytoplasmic Linker Protein-170 CREST Calcinosis, Raynaud’s phenomenon, oEsophagetasia, Sclerodactyly, Telangectasia CREST#6 A specific batch of serum from an anonymous patient suffering from CREST syndrome known to be reactive to CENP-A and CENP-B Cse4p Gene product of cse4-1 in Saccharomyces cerevisiae, homologue of CENP-A DAPI 4,6-diamindo-2-phenylindole DMEM Dulbecco’s Modified Eagle’s Medium DNA Deoxyribonucleic Acid DNase Deoxyribonuclease dNTPs Deoxyribonucleotide triphosphate DOP-PCR Degenerate Oligonucleotide Primed-Polymerase Chain Reaction DS Down syndrome EBV Epstein Barr Virus EDTA Sodium Ethylenediaminetetraacetic Acid ERK Extracellular Signal-Regulated Kinase EST Expressed Sequence Tag FASTSACS Functional And Structural Topography Scanning Along ChromosomeS FISH Fluorescence in situ Hybridisation FITC Fluorescein isothiocynate G Guanine GFP Green Fluorescent Protein

- 17 - HBUB1 Human Homologue of the Budding Uninhibited by Benzimidazoles 1 HBUB3 Human Homologue of the Budding Uninhibited by Benzimidazoles 3 hBUBR1 Human BUB-Related Protein Kinase 1 HCP-3 Holocentric protein-3 HP1 Heterochromatin Protein 1 HPLC High Performance Liquid Chromatography HSR Homogenous Staining Region hZW10 Human Homologue of the Drosophila –1(1)zw10 gene production INCENP Inner Centromere Protein IS Inner Symmetry KAO Kao and Michayluk Medium kb Kilobase KB- Tris-NaCl-BSA KCM Potassium Chromosome Medium kDa Kilodalton L1H Human Long Inserted Elements-1 retrotransposon LB Luria Bertani Broth LINE-1 Long Inserted Element-1 M Molar M31 Human Homologue of Heterochromatin Protein HP1 MAD1 Mitotic Arrest-Deficient 1 MAD2 Mitotic Arrest-Deficient 2 MAPK Mitogen Activated Protein Kinase Mb Megabase MCAK Mitotic Centromere-Associated Mif2 Mitotic Fidelity Gene 2 Mis6 A Gene coding a Centromere Connector Protein in yeast species Mis12 A Gene coding a Centromere Connector Protein in yeast species ml Millilitre mM Millimolar MUGS in cell with Unreplicated

- 18 - N-terminus Amino-terminus nm Nanometre OMIM Online Mendelian Inheritance In Man OS Outer Symmetry p Short arm of a normal chromosome p’ Short arm of marker chromosome p150Glued A dynactin subunit, p55cdc p55 related to cell division cycle protein PARP Poly(ADP-Ribose) Polymerase PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PFGE Pulsed-Field Gel Electrophoresis PMSF Phenylmethylsulphonyl Fluoride PRINS Primed in situ Labelling q long arm of a normal chromosome q’ long arm of marker chromosome Q-FISH Quantitative Fluorescence In Situ Hybridisation RNA Ribonucleic Acid RNase A Ribonuclease A SDS Sodium Dodecyl Sulphate SSC Saline Sodium Citrate SpCENP-A Homologue of CENP-A in Schizosaccharomyces pombe STS Sequence Tagged Site T Thymidine TACT Telomere-Associated Chromosomal Truncation

TAFII60 TATA-binding-proteins associated factors TEEN Triethanolamine-EDTA-NaCl TNB Tris-NaCl-Blocking Agent TopoII Topoisomerase II TSA Trichostatin A Tsg24 Meiotic checkpoint regulator

- 19 - U Unit YAC Yeast Artificial Chromosome YPD Yeast extract, Peptone, Dextrose Medium µg Microgram µl Microlitre µM Micromolar

* Gene symbols for centromere proteins of mouse are slightly different to homologues of other organisms due to the adoptation of the guidelines published by the International Committee on Standardized Genetic Nomenclature for Mice in October, 2000 (http://www.informatics.jax.org/mgihome/nomen).

- 20 - Publications arising from this study

1. Lo A.W.I., Craig J.M., Saffery R., Kalitsis P., Irvine D.V., Magliano D.J., Earle E., Choo K.H.A. A 330-kb CENP-A binding domain and altered replication timing at a human neocentromere. (submitted to the European Molecular Biology Organisation Journal).

2. Lo A.W.I., Magliano D.J., Kalitsis P., Sibson M.C., Choo K.H.A. (in press). CENP- A binding domain and sequence analysis of a neocentromere. Genome Research.

3. Lo A.W.I., Liao G.C.-C., Rocchi M., Choo K.H.A. (1999). Extreme reduction of chromosome-specific α-satellite array is unusually common in human chromosome 21. Genome Research 9:895-908.

4. Lo A.W.I., Choo K.H.A. (1999). Fluorescence in situ hybridisation (FISH) screening of frozen cell lines in large numbers. BioTechniques 26:408-412.

5. Choo K.H.A., Craig J.M., Cutts S.M., Lo A.W.I. (in press). In situ hybridisation. In “Encyclopaedia of Life Sciences”. Mcmillan Publishing. London. (Embryonic version available online: www.els.net/elsonline/html/A0002646.html).

- 21 - Preamble

DNA is a long linear molecule that contains the basic blueprint and the information required for the whole life process. The total length of a DNA molecule is estimated to be meters long, yet it is packaged into the nuclei of all living cells, which have sizes of 5-10 µm in diameter. Packaging is achieved by wrapping DNA around nucleosomal particles forming the chromatin fibres and, subsequently, supercoiling these chromatin fibres into higher order structures. Throughout the cell cycle, the chromatin undergoes various stages of condensation and reorganisation. The metaphase chromosomes at the stage of mitosis can be considered as the highest order of packaging.

The centromere is a specialised structure on a chromosome that plays an essential role for the correct segregation during mitosis and . It is a distinct primary constriction on the metaphase human chromosome. Unlike satellite sites or fragile sites, the primary constriction is heavily stained with DNA-binding dye suggesting the abundance of some specialised DNA. The tightly constricted appearance of the centromere suggests a unique chromatin configuration with even higher degree of packaging than the general genome at metaphase. An understanding of the chromatin of the centromere will allow us to study the interactions of various centromere proteins in the coordination of chromosome segregation. This is essential for the understanding of the basic life process of cell division and pathological situations such as missegregation and malignant changes.

- 22 -

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Lo, Wing Ip Anthony

Title: Human centromeric and neocentromeric chromatin

Date: 2000-09

Citation: Lo, W. I. A. (2000). Human centromeric and neocentromeric chromatin. PhD thesis, Department of Paediatrics, The University of Melbourne.

Publication Status: Unpublished

Persistent Link: http://hdl.handle.net/11343/39474

File Description: Front

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