Gho Wai-Man. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-123). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENT --- p.VII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / ABBREVIATIONS --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Epidemiology --- p.2 / Chapter 1.3 --- Etiological factors --- p.6 / Chapter 1.3.1 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3.1.1 --- Hepatitis B Virus (HBV) --- p.7 / Chapter 1.3.1.2 --- Hepatitis C Virus (HCV) --- p.9 / Chapter 1.3.2 --- Aflatoxin Exposure --- p.10 / Chapter 1.3.3 --- Alcohol Abuse --- p.11 / Chapter 1.3.4 --- Liver Cirrhosis --- p.12 / Chapter 1.4 --- Genetic alterations in hcc --- p.16 / Chapter 1.4.1 --- Chromosomal Gain --- p.16 / Chapter 1.4.2 --- Chromosomal Loss --- p.17 / Chapter 1.5 --- Discovery of common activating transcription factor 5 (atf5) down-regulations in hcc --- p.19 / Chapter 1.5.1 --- Chromosome 19 Aberration in HCC --- p.19 / Chapter 1.5.2 --- Discovery of High Frequency of　ATF5 Down-regulations --- p.19 / Chapter 1.5.3 --- Activating Transcription Factor Family --- p.20 / Chapter 1.6 --- Aim of thesis --- p.28 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.3 --- Cell culture --- p.31 / Chapter 2.1.4 --- Nucleic acids --- p.32 / Chapter 2.1.5 --- Enzymes --- p.32 / Chapter 2.1.6 --- Equipment --- p.32 / Chapter 2.1.7 --- Kits --- p.33 / Chapter 2.1.8 --- Software and Web Resource --- p.33 / Chapter 2.2 --- Dna extraction --- p.34 / Chapter 2.2.1 --- Cell Lines --- p.34 / Chapter 2.2.2 --- Primary HCC --- p.34 / Chapter 2.2.3 --- Lymphocytic DNA --- p.35 / Chapter 2.3 --- Rna extraction --- p.36 / Chapter 2.4 --- Dna sequencing --- p.38 / Chapter 2.4.1 --- Polymerase Chain Reaction (PCR) --- p.38 / Chapter 2.4.2 --- Cycle Sequencing --- p.39 / Chapter 2.5 --- Dual-labeled fluirescence in situ hybridization (fish) --- p.41 / Chapter 2.5.1 --- FISH Probe Preparation --- p.41 / Chapter 2.5.1.1 --- Preparation of Human Bacterial Artificial Chromosome (BAC) --- p.41 / Chapter 2.5.1.2 --- Nick Translation --- p.41 / Chapter 2.5.2 --- FISH --- p.42 / Chapter 2.6 --- 5-aza-2'-deoxycytidine & trichostatin a treatment on cell lines --- p.43 / Chapter 2.7 --- Bisulfite modificaiton of dna --- p.43 / Chapter 2.8 --- Methylation-specific pcr (msp) --- p.44 / Chapter 2.9 --- Bisulfite dna sequencing --- p.44 / Chapter 2.10 --- Quantitative reverse transcription pcr (qrt-pcr) --- p.46 / Chapter 2.11 --- In-vitro and in-vivo functinal examination --- p.49 / Chapter 2.11.1 --- ATF5 Transfection --- p.49 / Chapter 2.11.2 --- Cell Growth Assay --- p.50 / Chapter 2.11.3 --- Xenograft Development --- p.51 / Chapter 2.12 --- codelink expression microarray --- p.51 / Chapter 2.13 --- Statistical analysis --- p.53 / Chapter CHAPTER 3 --- INACTIVATION OF MECHANISMS UNDERLYING ATF5 DOWN-REGULATION --- p.54 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and methods --- p.58 / Chapter 3.2.1 --- Cell Lines --- p.58 / Chapter 3.2.2 --- Mutational Analysis --- p.58 / Chapter 3.2.3 --- Copy Number Loss --- p.59 / Chapter 3.2.4 --- Epigenetic Control --- p.59 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Sequencing Analysis of A TF5 Gene --- p.67 / Chapter 3.3.2 --- FISH Analysis of ATF5 Copy Number --- p.73 / Chapter 3.3.3 --- Epigenetic Control of A TF5 Expression --- p.73 / Chapter 3.4 --- Discussion --- p.82 / Chapter CHAPTER 4 --- FUNCTIONAL EXAMINATION AND INVESTIGATION OF DOWNSTREAM TARGETS MODULATED BY ATF5 --- p.85 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Cell Lines --- p.88 / Chapter 4.2.2 --- Plasmids and Transfection --- p.88 / Chapter 4.2.3 --- Cell Growth Assay --- p.88 / Chapter 4.2.4 --- Xenograft Development --- p.88 / Chapter 4.2.5 --- CodeLink Expression Microarray --- p.89 / Chapter 4.2.6 --- Quantitative RT-PCR --- p.90 / Chapter 4.2.7 --- Statistical analysis --- p.90 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Cell Proliferation --- p.91 / Chapter 4.3.1.1 --- In-Vitro Examination --- p.91 / Chapter 4.3.1.2 --- In-Vivo Examination --- p.91 / Chapter 4.3.2 --- Microarray A nalysis --- p.91 / Chapter 4.3.3 --- Correlation of A TF5 with Id-1 Expression --- p.103 / Chapter 4.4 --- Discussion --- p.106 / Chapter CHAPTER 5 --- PROPOSED FUTURE INVESTIGATIONS --- p.110 / Chapter 5.1 --- inactivation mechanisms of atf5 gene --- p.111 / Chapter 5.2 --- Molecular pathways modulated by atf5 --- p.112 / Chapter CHAPTER 6 --- REFERENCES --- p.114

Indiana University-Purdue University Indianapolis (IUPUI) / The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.