Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 67-78).

During preimplantation embryonic development, imprinting genes are susceptible to methylation changes by artificial manipulation, which may lead to developmental abnormalities. In addition, environmental endocrine disruptors (EDs) in everyday household products are also found to perturb fertility development and cause epigenetic aberrations. While embryo supply is scarce and conventional epigenetic studies require embryos in vast amount, an assay was developed in this study to examine the methylation statuses of imprinting genes using DNA from single mouse blastocysts cultured in-vitro or exposed to EDs. Promoter CpG methylation patterns of three imprinting genes, small nuclear ribonucleoprotein polypeptide N (SNRPN), paternally expressed 3 (Peg3), and potassium voltage-gated channel 1 overlapping transcript 1 (Kcnq1ot1), were examined from genomic DNA of a single mouse blastocyst. The genomic DNA was isolated and treated with bisulfite modification to preserve the methylation statuses. Afterwards, the DNA was subjected to whole genome amplification (WGA). Methylation-specific polymerase chain reaction (methyl-PCR) was performed with allele-specific primers; the amplicons were cloned and sequenced. CpG methylations in SNRPN, Peg3 and Kcnq1ot1 showed no statistical significant difference (P>0.05; Mann Whitney U test) in both parental alleles between a single genomic-amplified blastocyst and 20 non-amplified blastocysts, indicating no artifact was being introduced during the WGA procedure. Using the assay, it was revealed that blastocysts cultured in-vitro expressed slight but nonsignificant deviation in methylation rates to both parental alleles of SNRPN and Kcnq1ot1 except in single blastocysts, which displayed significant loss in maternal methylation on SNRPN upon culturing. On the other hand, paternal methylation profile of Peg3 appeared unaffected, suggesting resistance to methylation perturbations induced by in-vitro culturing. Despite that there was no significant difference in overall methylation rates between in-vivo or in-vitro developed blastocysts, certain CpG residues appeared to displayed significant loss of methylation (LOM) or gain of methylation (GOM) induced by in-vitro culture in all three genes being studied. Furthermore, using the developed, assay the epigenetic effects of three endocrine disruptors, simazine, propiconazole, and cadmium chloride (CdCl2) on in-vitro cultured single blastocysts were revealed. When compared to blastocysts cultured with KSOM+AA medium as controls, CdCl2-treated blastocysts displayed the most methylation aberrations in both alleles and within particular CpG residues, possibly due to its dual effect in both hypermethylation and hypomethylation across the methylome. Both simazine- and propiconazole -treated blastocysts displayed overall methylation significant defects were observed within particular CpG residues. Overall, the assay used in this study allowed the comprehensive investigation of methylome from the DNA extracted from a single blastocyst.defects resembled to those blastocysts cultured with KSOM+AA medium alone but / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

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Thesis (M.S.)--University of Wisconsin--Madison, 1982. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 73-77).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1980. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.Med.Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 67-78). Also available in print.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1954. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 161-164).

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly those that are more stable, or, indeed, differences in expression between parental alleles within the same cell. This conundrum lead to the emergence of the field of epigenetics - the study of heritable changes in gene expression independent of DNA sequence. Such changes are dependent on “epigenetic modifications”, of which DNA methylation is one of the best characterised, and is associated with gene silencing. The establishment of correct DNA methylation patterns is particularly important during early development, leading to cell type specific and parental allele specific gene regulation. Besides DNA methyltransferases, various other proteins have recently been implicated in DNA methylation. The absence of these proteins leads to defects in DNA methylation and development that can be even more severe than those in DNA methyltransferase knockouts themselves. In this study I focus on three such accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes protein). To compare DNA methylation between WT cells and cells knocked out for each of these proteins, I used whole genome methylated DNA affinity purification and subsequent hybridization to promoter microarrays. This enabled me to compare the requirement for each protein in DNA methylation at specific genomic regions. The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the loss of DNA methylation at 20% of usually methylated promoters, and the misregulation of associated protein coding genes. This revealed a requirement for LSH in the establishment of DNA methylation at promoters normally methylated during pre-implantation as well as post-implantation development. Secondly, I identified hypomethylation at 26% of normally methylated promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is required for maintenance of DNA methylation at maternal as well as paternal imprinting control regions (ICRs). This is accompanied by expression defects of imprinted genes regulated by these ICRs. Finally, in collaboration with the Brockdorff lab at the University of Oxford I identified a role for SmcHD1 in establishing DNA methylation at promoters on the X chromosome normally methylated slowly during X chromosome inactivation. Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene promoters, contrary to previous reports that it is mainly involved in X chromosome methylation. I conclude that different accessory proteins are required to facilitate correct DNA methylation and gene repression at distinct regions of the genome, as well as at different times during development. This function of accessory proteins may be in part dependent on the prior establishment of specific chromatin signatures and developmental signals, together comprising a precisely regulated system to establish and maintain appropriate DNA methylation throughout development.

Graduation date: 1992