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Arsenic, an established carcinogen and toxicant, occurs in drinking water and food and affects millions of people worldwide. Arsenic appears to interfere with gene expression through epigenetic processes, such as DNA methylation and post-translational histone modifications. We investigated the effects of arsenic on histone residues in vivo as well as in vitro. Analysis of H3K9Ac and H3K9me3 in CD4+ and CD8+ sorted blood cells from individuals exposed to arsenic through drinking water in the Argentinean Andes showed a significant decrease in global H3K9me3 in CD4+ cells, but not CD8+ cells, with increasing arsenic exposure. In vitro studies of inorganic arsenic-treated T lymphocytes (Jurkat and CCRF-CEM, 0.1, 1, and 100 μg/L) showed arsenic-related modifications of H3K9Ac and changes in the levels of the histone deacetylating enzyme HDAC2 at very low arsenic concentrations. Further, in vitro exposure of kidney HEK293 cells to arsenic (1 and 5 μM) altered the protein levels of PCNA and DNMT1, parts of a gene expression repressor complex, as well as MAML1. MAML1 co-localized and interacted with components of this complex in HEK293 cells, and in silico studies indicated that MAML1 expression correlate with HDAC2 and DNMT1 expression in kidney cells. In conclusion, our data suggest that arsenic exposure may lead to changes in the global levels of H3K9me3 and H3K9Ac in lymphocytes. Also, we show that arsenic exposure affects the expression of PCNA and DNMT1—proteins that are part of a gene expression silencing complex.

Inorganic arsenic is a toxic metalloid that occurs naturally on the earth’s crust. Millions of people worldwide are exposed to inorganic arsenic via drinking water, and more recently also via contaminated food, in particular rice. Many studies have focused on exploring the adverse health effects of arsenic as well as its mode of action once entering the body. Still, little is known about the intracellular processes that drive arsenic toxicity. In the present thesis we investigated the effects of arsenic on transcriptional and epigenetic processes both in normal and cancer cell lines. In order to understand how exogenous compounds affect transcriptional regulators such as MAML1, which is involved in many different signaling pathways and has been related to developmental processes and human diseases (e.g. cancer), it’s important to understand which cellular processes regulate the protein levels. Consequently, we studied the transcriptional co-activator MAML1 and the ubiquitination and degradation process. We show that MAML1 protein levels are regulated via ubiquitination and that this process is enhanced by p300 and repressed by N1ICD. On top of that, we also investigated MAML1 involvement in cell proliferation and epigenetic regulation as well as arsenic effect on MAML1 expression and kidney cell proliferation. We show that MAML1 interacts with DNMT1 and PCNA, both members of the DNMT1-PCNA-HDAC2 repressive complex, which is involved in epigenetic regulation. We further report that arsenic decreases kidney cell proliferation and we suggest this occurs via MAML1 downregulation. In order to further explore the effects of arsenic on the epigenome, we studied arsenic exposure in relation to the post-translational histone modifications (PTHMs) H3K9me3 and H3K9Ac in lymphocytes isolated from exposed individuals as well as in vitro in cell culture system. We report arsenic-related changes in H3K9me3 epigenetic mark in CD4+ lymphocytes isolated from the arsenic exposed indiv

Immortalized cell lines are widely used in genetic and epigenetic studies, from exploration of basic molecular pathways to evaluation of disease-specific cellular properties. They are also used in biotechnology, e.g., in drug toxicity tests and vaccine production. Cellular and genetic uniformity is the main feature of immortalized cell lines and it has been particularly advantageous in functional genomic research, which has in recent years been expanded to include epigenetic mechanisms of gene expression regulation. Using the MS-HRM technique, we demonstrated heterogeneity in locus-specific methylation patterns in different cell cultures of four human cell lines: HEK293, HEK293T, LCL and DU145. Our results show that some human immortalized cell lines consist of cells that differ in the methylation status of specific loci, i.e., that they are epigenetically heterogeneous. We show that even two cultures of the same cell line obtained from different laboratories can differ in the methylation status of the specific loci. The results indicated that epigenetic uniformity of the cell lines cannot be assumed in experiments which utilize cell cultures and that the methylation status of the specific loci in the immortalized cell lines should be re-characterized and carefully profiled before epigenetic studies are performed.

Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1). MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

Mastermind-like 1 (MAML1) is a transcriptional coregulator of diverse/multiple activators, such as Notch, p53, myocyte enhancer factor 2C, NF-κB, beta-catenin, papillomavirus E6 proteins, early growth response 1 and runt-related transcription factor 2. Thus, MAML1 functions in various signaling pathways, most of them connected to cell proliferation, which suggests that MAML1 might play a potential role as a cell proliferation marker. In this study we show that MAML1 expression in the kidney correlates in silico with established cell proliferation markers including PCNA, CDC2 and XRCC5 (Ku80). Over-expression of MAML1 increased proliferation of human embryonic kidney (HEK) 293 cells, while MAML1 downregulation by siRNA decreased cell proliferation. Exposure of HEK293 cells to inorganic arsenic (arsenite) showed reduced levels of MAML1, in combination with a decreased proliferation rate. Our findings provide evidence that arsenic can inhibit proliferation of embryonic kidney cells, possibly through reduction of MAML1 gene expression.