The phenomenon of cell type-specific DNA methylation has received much attention in recent years and a number of DNA methylation differences have been described between cells of the immune system. Of particular interest when studying DNA methylation are CpG islands (CGIs) which are distinct from the rest of the genome due to their elevated CpG content, generally unmethylated state and promoter association. In the instances when they become methylated this is associated with gene repression although it is unclear the extent to which differential methylation corresponds to differential gene expression. I have used an immune system model to assess the role of CGI methylation and the role of the methylation reader MBD2 in regulation of gene expression. A relatively small number of DNA methylation differences were seen between immune cell types with the most developmentally related cells showing the fewest methylation differences. Interestingly, the vast majority of CGI-associated cellspecific methylation occurred at intragenic CGIs located, not at transcription start sites, but in the gene body. Increased intragenic CGI methylation tended to associate with gene repression, although the precise reason for this remains unclear. Most differentially methylated CGIs were depleted for the active chromatin mark H3K4me3 regardless of their methylation state but some of these were associated with the silencing mark H3K27me3 when unmethylated. These findings suggest that intragenic CGIs are a distinct class of genomic element particularly susceptible to cell type-specific methylation. I also looked at the effect of removing the methyl- CpG binding domain protein MBD2 from immune system cells. Immune cells from Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as a number of differences in gene expression compared to wild-type animals. Most of these genes increased their expression in the absence of MBD2 consistent with MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases in histone H3 acetylation compared to wild-type Th1 cells. This work provides an insight into the role played by cell-specific CGI methylation and MBD2 in regulating gene expression.

Zhong, Yangliu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 50-57). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Abstract (English Version) --- p.iv / Abstract (Chinese Version) --- p.V / Acknowledgement --- p.vi / Table of Contents --- p.viii / List of Tables --- p.X / List of Figures --- p.xii / List of Abbreviations and Symbols --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- DNA methylation --- p.1 / Chapter 1.2 --- Repair of m3T --- p.2 / Chapter 1.3 --- Objectives of this work --- p.3 / Chapter 1.4 --- DNA structure --- p.3 / Chapter 1.4.1 --- Nomenclature scheme for DNA --- p.3 / Chapter 1.4.2 --- Base pair scheme --- p.4 / Chapter 1.4.3 --- Sugar conformation --- p.5 / Chapter 1.4.4 --- Backbone conformation --- p.7 / Chapter 2 --- Materials and Methods --- p.9 / Chapter 2.1 --- Sample design --- p.9 / Chapter 2.2 --- Sample preparation --- p.10 / Chapter 2.3 --- NMR analysis --- p.10 / Chapter 2.3.1 --- Resonance assignment --- p.12 / Chapter 2.3.2 --- Determination of sugar conformation --- p.13 / Chapter 2.3.3 --- Determination of backbone conformation --- p.14 / Chapter 2.4 --- UV melting study --- p.15 / Chapter 3 --- Effect of m3T on Double-Helical Structures and Stabilities --- p.17 / Chapter 3.1 --- Resonance assignments --- p.17 / Chapter 3.2 --- Effect of m3T on double-helical DNA structures --- p.19 / Chapter 3.2.1 --- Base pairing mode --- p.19 / Chapter 3.2.2 --- Sugar conformation --- p.21 / Chapter 3.2.3 --- Backbone conformation --- p.22 / Chapter 3.3 --- Effect of m3T on double-helical DNA stabilities --- p.25 / Chapter 3.4 --- Discussion --- p.26 / Chapter 3.4.1 --- Single-strand requirement in FTO repair --- p.26 / Chapter 3.4.2 --- Relationship between m3T pairing structure and stability --- p.27 / Chapter 4 --- Effect of m3T Mispair on Double-Helical DNA Structures and Stabilities --- p.28 / Chapter 4.1 --- Resonance assignments --- p.28 / Chapter 4.2 --- Effect of m3T mispair on double-helical DNA structures --- p.32 / Chapter 4.2.1 --- Pairing mode of T m3T --- p.34 / Chapter 4.2.2 --- Pairing mode of G m3T --- p.35 / Chapter 4.2.3 --- Pairing mode of C.m3T --- p.35 / Chapter 4.3 --- Effect of m3T mispair on double-helical DNA stabilities --- p.36 / Chapter 4.4 --- Discussion --- p.36 / Chapter 4.4.1 --- Predominant mutation --- p.37 / Chapter 4.4.2 --- Relationship between m3T pairing structure and stabilities --- p.37 / Chapter 5 --- Conclusion and Future Work --- p.39 / Chapter Appendix I --- Proton chemical shift values (ppm) of AmT --- p.40 / Chapter Appendix II --- Proton chemical shift values (ppm) of RefAT --- p.41 / Chapter Appendix III --- Proton chemical shift values of NmT samples --- p.42 / Chapter Appendix IV --- "Σ1' and %S of TmT, GmT and CmT" --- p.45 / Chapter Appendix V --- "1H-31P HSQC spectra of (a) TmT, (b) GmT and (c) CmT" --- p.46 / Chapter Appendix VI --- "1H-31P COSY spectra of (a) TmT, (b) GmT and (c) CmT" --- p.47 / Chapter Appendix VII --- "31P chemical shifts, 3JH3'P and %Bi of TmT, GmT and CmT" --- p.48 / Chapter Appendix VIII --- "UV melting curves of RefAT, AmT, TmT, GmT and CmT" --- p.49 / References --- p.50

Change of DNA methylation is a hallmark of cancer. It is frequently associated with cancer progression. Testicular germ cell tumor (TGCT) is the most common malignant tumor in young males. Currently, only a limited number of genes are known to be epigenetically changed in TGCT. Genome-wide analysis of differential methylation in a previously established testicular cell line is documented here. A total of 35,208 differentially methylated regions (DMR) were identified. However, only a small number of DMRs mapped to gene promoters. Genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. Several candidate genes ( APOLD1, PCDH10 and RGAG1) were found to be dysregulated in TGCT patients. Surprisingly, APOLD1 was mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. / The majority of DMRs are located in introns or intergenic regions, but their functions are not well understood. Some of these DMRs were found to regulate non-coding RNAs (ncRNAs). In this study, differential methylation of 3 small nucleolar RNAs (snoRNA) and 3 microRNAs (miRNA) were identified. One of the miRNAs, miR-199a, is embedded in a conserved region in intron-14 of dynamin 3 at 1q24.3. Hypermethylation of miR-199a correlated with testicular cancer progression, and silencing of miR-199a. Re-expression of miR-199a in testicular cancer cells suppressed cell growth, cancer migration, invasion, and metastasis. miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with cancer progression. An embryonal carcinoma antigen, podocalyxin-like protein 1 (PODXL), was identified to be a target of miR-199a-sp. PODXL is an anti-adhesive protein overexpressed in aggressive testicular cancer. Knockdown of PODXL suppressed cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests that PODXL is one of the downstream effectors mediating cancer invasion and metastasis. This study links DNA methylation, miR-199a dysregulation, and PODXL expression as a mechanism to explain testicular cancer progression. / Cheung, Hoi Hung. / Adviser: Woi-Yee Chan. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 165-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Zhan, Yingqian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 49-54). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Abstract (English version) --- p.iv / Abstract (Chinese version) --- p.v / Acknowledgment --- p.vi / Table of Contents --- p.vii / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations and Symbols --- p.xiii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- DNA Methylation --- p.1 / Chapter 1.2 --- DNA Structure --- p.2 / Chapter 1.2.1 --- Nomenclature Scheme for DNA --- p.2 / Chapter 1.2.2 --- Conformations of DNA --- p.5 / Chapter 1.2.3 --- Base Pair Scheme --- p.5 / Chapter 1.2.4 --- Sugar Conformation --- p.7 / Chapter 1.2.5 --- Backbone Conformation --- p.7 / Chapter 1.3 --- DNA Melting --- p.7 / Chapter 1.4 --- Objective of this Work --- p.8 / Chapter 2. --- Materials and Methodology --- p.9 / Chapter 2.1 --- Sequence Design --- p.9 / Chapter 2.2 --- Sample Preparation --- p.10 / Chapter 2.3 --- Resonance Assignment --- p.10 / Chapter 2.3.1 --- Proton Resonance Assignment --- p.11 / Chapter 2.3.2 --- Phosphorous Resonance Assignment --- p.14 / Chapter 2.4 --- Determination of Sugar Conformation --- p.15 / Chapter 2.5 --- Determination of Backbone Conformation --- p.16 / Chapter 2.6 --- Thermodynamic study on DNA --- p.18 / Chapter 3. --- Effects of N1-methylated Adenine on DNA Structure and Stability --- p.21 / Chapter 3.1 --- Overview --- p.21 / Chapter 3.2 --- Resonance Assignment Results --- p.21 / Chapter 3.2.1 --- 1H Resonance Assignments --- p.21 / Chapter 3.2.2 --- 31P Resonance Assignments --- p.23 / Chapter 3.3 --- Base Pair Structures --- p.24 / Chapter 3.4 --- Sugar Conformation --- p.26 / Chapter 3.5 --- Backbone Conformation --- p.27 / Chapter 3.6 --- Thermodynamic Stability Study --- p.29 / Chapter 4. --- Effects of N1-methylated Guanine on DNA Structure and Stability --- p.31 / Chapter 4.1 --- Overview --- p.31 / Chapter 4.2 --- Resonance Assignment Results --- p.31 / Chapter 4.2.1 --- 1H Resonance Assignments --- p.31 / Chapter 4.2.2 --- 31P Resonance Assignments --- p.33 / Chapter 4.3 --- Base Pair Structures --- p.34 / Chapter 4.4 --- Sugar Conformation --- p.36 / Chapter 4.5 --- Backbone Conformation --- p.39 / Chapter 4.6 --- Thermodynamic Stability Study --- p.41 / Chapter 5. --- Conclusion and Future work --- p.43 / Appendix I H1'-H6/H8 region of NOESY spectra of TmeA at 25 °C with different mixing times --- p.44 / "Appendix II 3JH1'h2"", 3JH1'h2"", and %S of TmeA and refTA" --- p.45 / "Appendix III 3JH1' h2'，3JH1'-H2"", and %S of CmeG and refCG" --- p.46 / "Appendix IV 31P chemical shifts, 3JH3'p and %B1 of TmeA, and refTA" --- p.47 / "Appendix V 31P chemical shifts, 3JH3'P and %B1of CmeG, and refCG" --- p.48 / References --- p.49

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

DNA methylation serves as a major epigenetic modification crucial to the normal organismal development and the onset and progression of complex diseases such as cancer. Computational predictions for DNA methylation profiling serve multiple purposes. First, accurate predictions can contribute valuable information for speeding up genome-wide DNA methylation profiling so that experimental resources can be focused on a few selected while computational procedures are applied to the bulk of the genome. Second, computational predictions can extract functional features and construct useful models of DNA methylation based on existing data, and can therefore be used as an initial step toward quantitative identification of critical factors or pathways controlling DNA methylation patterns. Third, computational prediction of DNA methylation can provide benchmark data to calibrate DNA methylation profiling equipment and to consolidate profiling results from different equipments or techniques. This thesis is written based on our study on the computational analysis of the DNA methylation patterns of the human genome. Particularly, we have established computational models (1) to predict the methylation patterns of the CpG islands in normal conditions, and (2) to detect the CpG islands that are unmethylated in normal conditions but aberrantly methylated in cancer conditions. When evaluated using the CD4 lymphocyte data of Human Epigenome Project (HEP) data set based on bisulfite sequencing, our computational models for predicting the methylation status of CpG islands in the normal conditions can achieve a high accuracy of 93-94%, specificity of 94%, and sensitivity of 92-93%. And, when evaluated using the aberrant methylation data from the MethCancerDB database for aberrantly methylated genes in cancer, our models for detecting the CpG islands that are unmethylated in normal conditions but aberrantly methylated in colon or prostate cancer can achieve an accuracy of 92-93%, specificity of 98-99%, and sensitivity of 92-93%.

Trimethylation of histone H3 lysine 9 (H3K9me3) and DNA methylation mark heterochromatin, contributing to gene silencing and normal cellular functions. My research investigated the control of H3K9me3 and DNA methylation in the filamentous fungus Neurospora crassa. The H3K9 methyltransferase complex, DCDC, consists of DIM-5, DIM-7, DIM-9, DDB1, and CUL4. Each component of DCDC is required for H3K9me3. The DIM-9/DDB1/CUL4 subunits are reminiscent of known cullin E3 ubiquitin ligases. I showed that core features of CUL4-based E3 ubiquitin ligases are not required for H3K9me3 and DNA methylation in Neurospora. H3K9me3 is bound by heterochromatin protein 1 (HP1) to recruit the DIM-2 DNA methyltransferase and the HCHC histone deacetylase complex. HCHC consists of HP1, CDP-2, HDA-1, and CHAP. Both HP1 and CDP-2 harbor conserved chromodomains that bind H3K9me3, and CHAP contains two putative AT-hook domains that bind A:T-rich DNA. To test the contributions of these domains to HCHC function, I deleted the chromodomains of HP1 and CDP-2. Deletion of the HP1 chromodomain resulted in a reduction of DNA methylation, which was not exacerbated by deletion of the CDP-2 chromodomain. A strain with deletions of chap and the HP1 chromodomain showed a DNA methylation phenotype comparable to the loss of the HDA-1 catalytic subunit. These findings support a model in which recognition of H3K9me3 and A:T-rich DNA by HP1 and CHAP, respectively, are required for proper HCHC function. To examine the relationships between H3K9me3, DNA methylation, and histone acetylation, I utilized in vivo protein tethering of core heterochromatin components. The requirement of DIM-7 for native heterochromatin, previously implicated in localizing the H3K9 methyltransferase DIM-5, was not bypassed by DIM-5 tethering, indicating that DIM-7 has additional roles within the DCDC. Artificial localization of the HCHC histone deacetylase, by tethering HP1 or HDA-1, resulted in induction of H3K9me3, DNA methylation, and gene silencing, but silencing did not require H3K9me3 or DNA methylation. HCHC-mediated establishment of H3K9me3 was not required for de novo heterochromatin formation at native heterochromatic loci suggesting a role in heterochromatin spreading. Together, this work implicates HDA-1 activity as a key driver of heterochromatin spreading and silencing. This dissertation includes previously published co-authored material.

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