published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Mechanisms of arsenic toxicity are not yet clear. Arsenite has effects on methylation pathways, by decreasing expression of DNA methylases and depletion of S-adenosylmethionine. Histones are DNA packing proteins that regulate gene expression modulating chromatin accessibility. Methylation at Lysine 9 of Histone H3 (K9H3) is a hallmark of heterochromatin. Dimethyl K9H3 is a mark of facultative heterochromatin and trimethyl K9H3 is present on constitutive heterochromatin. HeLa cells exposed for 24 hrs to 1 uM or 5 uM Sodium Arsenite were fixed and different posttranslational modifications of histones were detected by indirect immunofluorescence. Images were analyzed to assess the change on average methylated species of K9H3 in cell nuclei. Interestingly Arsenite (1 uM and 5 uM) treated cells had a significant increase in the trimethylated and dimethylated of K9H3, evaluated throught the comparison of average nuclei brightness and pixel value analysis between treatments.

Introduction: Body Mass Index (BMI) is a relative measure of whether an individual’s weight is at a healthy level for their height. A higher BMI is associated with an increased risk of cancer and cardiovascular disease (CVD). However, the biologic mechanisms are not well understood. One proposed mechanism is through changes in global DNA methylation levels, particularly global DNA hypomethylation. Global DNA hypomethylation refers to lower levels of DNA methylation across the entire genome and hypermethylation refers to higher levels of DNA methylation across the entire genome. Changes in methylation levels can affect gene expression, genomic stability, and chromosomal structure. The methylation status of repetitive sequences in the DNA, such as LINE-1, is commonly used to represent a surrogate measure of global DNA methylation levels. Objectives: 1. Quantify and describe LINE-1 DNA methylation in leukocytes in a large sample of healthy volunteers. 2. Examine the relationship between BMI and LINE-1 DNA methylation levels. 3. Assess if sex is an effect modifier of the relationship between BMI and LINE-1 DNA methylation levels. Methods: A nested cross-sectional study was composed of 502 healthy volunteers between the ages of 20 and 50. Subjects completed a study questionnaire and provided blood samples for laboratory analyses. For each subject, DNA was isolated, underwent bisulfite conversion, and LINE-1 DNA methylation levels were measured by Polymerase Chain Reaction (PCR) High-Resolution Melting Curve analysis. For the main analysis, a multivariate linear regression model was used to examine the relationship between BMI and LINE-1 DNA methylation levels, while controlling for confounders. Results: LINE-1 DNA methylation was normally distributed with a mean of 84.52% and a standard deviation of 3.19%. BMI (normal, overweight, and obese categories) was not significantly associated with LINE-1 DNA methylation levels in the adjusted linear regression model (p=0.41) and the interaction term between BMI and sex was not significant (p=0.50). Conclusions: LINE-1 DNA methylation was measured with a high degree of reliability in a sample of healthy volunteers. This research provided a description of LINE-1 DNA methylation levels in a large healthy population and showed that BMI was not associated with global DNA methylation. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2012-04-03 17:23:59.843

Haematopoiesis requires strict regulation in order to maintain a balanced production of the various blood cell components. Escape from this regulation contributes to the development of cancers such as leukemia. SOCS2 is a member of the Suppressor of cytokine signalling (SOCS) family, and normally functions as a negative regulator of the JAK/STAT pathway. I examined gene expression and promoter methylation in acute myeloid leukemia (AML) cell lines and patient samples. SOCS2 expression was quite variable in AML patients, and very low in acute promyelocytic leukemia (APL) patients. Promoter hyper-methylation was found in these patients, particularly those with high white blood cell count and a FLT3-ITD. I speculate that SOCS2 interacts with an aspect of the signalling complex to inhibit cell growth in these patients, and silencing SOCS2 is necessary for leukemia progression. Treating these patients with a de-methylating agent, such as decitabine, may show promise in the clinic.

Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Microbiology and Immunology. Title from resource description page. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 297-327).

In eukaryotes, the SET1 family of methyltransferases carry out the methylation of Lysine 4 on Histone H3. Alone, these enzymes exhibit low enzymatic activity and require the presence of additional regulatory proteins, which include RbBP5, Ash2L, WDR5 and DPY-30, to stimulate their catalytic activity. While previous structural studies established the structural basis underlying the interaction between RbBP5, Ash2L and WDR5, the formation of the Ash2L/DPY-30 complex remains elusive. Here we report the crystal structure of the Ash2L/DPY-30 complex solved at 2.2Å. Our results show that a Cterminal amphipathic α-helix on Ash2L makes several hydrophobic interactions with the DPY-30 homodimer. Moreover, the structure reveals that a tryptophan residue on Ash2L, which directly precedes its C-terminal amphipathic α-helix, makes key interactions with one of DPY-30 α-helix. Finally, biochemical studies of Ash2L revealed a hitherto unknown ability of this protein to bind anionic lipids.

No description available.

Breast cancer is the most commonly diagnosed cancer amongst Canadian women. Though numerous treatments are available, in many instances tumours become refractory or recur. Therefore, understanding the biological events that lead to the progression and therapeutic resistance of breast cancer is essential for the development of novel treatment options for this disease. Numerous members of the protein arginine methyltransferase (PRMT) family, which are the enzymes responsible for catalyzing methylation on arginine residues are aberrantly regulated in breast cancer. Hence, understanding the precise contribution of PRMTs to the development and progression of breast cancer is important. This Thesis will present my findings on the alternatively spliced PRMT1 isoform, PRMT1v2, previously identified to be overexpressed in breast cancer cell lines and here shown to promote breast cancer cell survival and invasion. Second, a novel role is ascribed to PRMT6, another PRMT aberrantly expressed in breast cancer. PRMT6 promotes chemoresistance to the drug bortezomib by mediating stress granule formation through down-regulation of eIF4E. Increased stress granule formation in bortezomib-resistant cancer cells promotes cell survival. Third, DDX3, a prototypical PRMT substrate which is overexpressed in breast cancer cell lines and stimulates transformation of mammary epithelial cells is a novel substrate of PRMT1, CARM1, and PRMT6. Lastly, TDRD3, a reader/effector of arginine methylation also overexpressed in breast tumours regulates breast cancer cell proliferation, anchorage-independent growth and cell motility and invasion.

Lysine methylation modulates diverse biological processes and is catalyzed by SET domain methyltransferases such as the SMYDs (SMYD1-5), which possess a SET domain split by a MYND motif. Through association with NCoR, the H4 Lys20 methyltransferase activity of SMYD5 represses inflammation by restricting TLR-4 mediated expression in macrophages, yet biochemical and structural features required for SMYD5 methylation activity remain elusive. To determine how SMYD5 catalyses methylation, crystallization screens were conducted with SMYD5 in complex with the co-factor AdoMet and histone H4. Screens yielded lead conditions but no crystals. To determine the motif recognized by SMYD5 and decipher its methylome, peptide arrays were conducted to produce a methylation motif used to identify putative substrates. Surprisingly, arrays revealed that substitution of Lys16, not Lys20, is detrimental to SMYD5 activity. Further enzymatic assays are required to determine if SMYD5 methylates residues other than Lys20 on the H4 tail, or if structural determinants or interacting partners restrict methylation of target lysines.