No description available.

Cutaneous malignant melanoma is the deadliest form of skin cancer with a rising incidence rate. Epidemiological studies show exposure to ultraviolet radiation (UVR) cause 80% of melanomas. However, the underlying molecular mechanisms by which UVR promotes melanomagenesis are unclear. The mutagenic properties of UVR are incontrovertible; however, well-studied driver mutations of melanomagenesis (BRAF V600E and NRAS Q61L/R) do not bear UVR signature mutations and so the role UVR mutations play in the early initiation of melanoma remains controversial. This highlights the gap in knowledge of the initial critical molecular mechanisms of UVR-induced melanomas and warrant investigating non-mutational mechanisms as causal factors of UVR-induced melanomagenesis. Aberrant DNA methylation is a signature of melanoma and regulates expression of important tumor suppressors. While epigenetic dysregulation is an important aspect of melanoma etiology, it has never been investigated in the context of UVR. We hypothesize that these initial UVR-induced DNA methylation changes may sensitize a field of melanocytes to acquiring subsequent complementary spontaneous and/or UVR-induced genetic mutations and render them susceptible to melanomagenesis. My preliminary data demonstrate that UVR can modulate DNA methylation in melanocytes and suggests a pigment dependent mechanism. UVR-induced DNA methylation changes in highly pigmented melanocytes primarily in intergenic regions as areas of active transcription were protected from 5’mC changes. Additionally, UVR induced long-term transcriptional changes in both dark and light pigmented melanocytes suggesting multiple epigenetic mechanisms being altered. Evaluation of the protein regulation of the enzymes involved in writing or erasing 5’mC point towards a dysregulation in TET2. Further work is needed to determine if changes in TET2 could contribute to the observed methylation changes. To determine if these methylation changes had any significance to melanoma development, they were compared to the skin cutaneous melanoma cohort in the TCGA database which found a modest correlation in UVR-induced methylation changes and those found in melanoma patients. Interestingly, 5’mC at UVR-sensitive sites was prognostic of patient survival. A highly pigmented human melanoma cell line was UV-irradiated to see if DNA methylation can also be affected in transformed cells; however, no changes were observed. This suggests UV-induced methylation contributes to early changes in melanoma development and/or other relevant physiological changes within the melanocytes. Altogether, these data identify a novel non-mutation mechanism by which UVR may contribute of melanomagenesis. / Cancer Biology & Genetics

Tardive dyskinesia (TD) is a serious side effect of certain antipsychotic medications that are used to treat schizophrenia (SCZ) and other mental illnesses. The methylation status of the insulin receptor substrate 1 (IRS1) gene is reportedly associated with SCZ; however, no study, to the best of the authors' knowledge, has focused on the quantitative DNA methylation levels of the IRS1 gene using pyrosequencing in SCZ with or without TD. The present study aimed to quantify DNA methylation levels of 4 CpG sites in the IRS1 gene using a Chinese sample including SCZ patients with TD and without TD (NTD) and healthy controls (HCs). The general linear model (GLM) was used to detect DNA methylation levels among the 3 proposed groups (TD vs. NTD vs. HC). Mean DNA methylation levels of 4 CpG sites demonstrated normal distribution. Pearson's correlation analysis did not reveal any significant correlations between the DNA methylation levels of the 4 CpG sites and the severity of SCZ. GLM revealed significant differences between the 3 groups for CpG site 1 and the average of the 4 CpG sites (P=0.0001 and P=0.0126, respectively). Furthermore, the TD, NTD and TD + NTD groups demonstrated lower methylation levels in CpG site 1 (P=0.0003, P<0.0001 and P<0.0001, respectively) and the average of 4 CpG sites (P=0.0176, P=0.0063 and P=0.003, respectively) compared with the HC group. The results revealed that both NTD and TD patients had significantly decreased DNA methylation levels compared with healthy controls, which indicated a significant association between the DNA methylation levels of the IRS1 gene with SCZ and TD.

No description available.

No description available.

DNA methylation regulates gene expression through a complex network of protein/protein and protein/DNA interactions in chromatin. The maintenance methylase, DNA methyltransferase 1 (DNMT1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications in somatic cells. The mechanistic details that explain how DNMT1 catalytic action is directed in a chromatin setting are not well understood. We hypothesize that post translational modifications and a variety of protein-protein interactions processes are key regulatory elements that set the methylation of CpG elements essential for normal growth behavior in somatic cells. These fundamental processes can be disrupted by DNA damage leading to inappropriate gene silencing and loss of growth control in somatic cells. First, we show that DNMT1 is post-translationally modified by sumoylation and we have mapped these sumoylation sites by defined mutations. Sumoylated DNMT1 is catalytically active on genomic DNA in vivo and substantially increases the enzymatic activity of DNMT1 both in vitro and in chromatin. These data establish that sumoylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in cells. Second, we investigated novel mechanisms whereby somatic cells can erase then reset DNA methylation events in somatic cells. In this study, the relationship between DNA damage and gene silencing was explored. To this end, we generated a HeLa cell line containing a specialized GFP reporter cassette (DRGFP) containing two mutated GFP genes and a unique ISceI restriction endonuclease site. These cells do not express GFP. A unique double strand break is then delivered by transfecting in the gene for I-SceI. About 4% of the cells produced a functional GFP by gene conversion and homologous recombination (HR); however roughly half iv of the GFP recombinants expressed the gene poorly and this was attributed to gene silencing. Silencing of the GFP expressing cell clones was due to DNA methylation and could be reversed using a drug that inhibits global methylation (5-aza-2'-deoxycytidine). Approximately half of the repaired genes were heavily methylated, and half were hypomethylated. That is, a key intermediate methylation state after HR repair is hemimethylated DNA, defined as methylation limited to one strand. Evidence is given that DNMT1 is acting as a de novo methylase at the HR repair patches in cells. Moreover, the DNA damage inducible protein, GADD45, interacts specifically with the catalytic domain of DNMT1 and GADD45 binds with extremely high affinity to hemimethylated DNA sites. Thus, GADD45 is a key regulatory element in silencing of HR repaired DNA segments and appears to inhibit the activity of DNMT1. Consistent with these results, we found that GADD45 increased the expression of recombinant GFP following HR repair, further suggesting its role in orchestrating strand specific DNA methylation by DNMT1. Since these experiments were performed in live cells, there is strong physiological relevance. We propose that DS DNA damage and the resulting HR process involves precise, strand selected DNA methylation mediated by the prominent methylase enzyme, DNMT1. Moreover, DS DNA break repair through HR and gene conversion, may potentially erase and reset DNA methylation patterns and therefore alter the expression of repaired genes. The overall process is tightly regulated by the DNA damage inducible protein GADD45, which may coordinate strand specific methylation by recruiting DNMT1 to HR repair templates. The ability of GADD45 to modulate DNMT1 catalytic activity may explain its role as a passive mediator of demethylation that has been reported by other groups. The overall process of silencing post DNA repair is a strong evolutionary force that may predispose cells to malignant transformation

Sex differences are widespread in both mouse and human liver, and are associated with sex differences in drug metabolism and liver pathophysiology. The secretory patterns of growth hormone (GH) is one of the major drivers of liver sex specificity, where intermittent and continuous secretion in male and female respectively lead to sex bias in the expression of more than 1000 genes in mouse liver, via a complex interplay of GH-responsive transcription factors and epigenetic mechanisms. This thesis explores three themes of molecular control in the regulation of liver sex differences: microRNAs, DNA methylation, and translational control. Studies herein identified two microRNAs, miR-1948-5p and miR-802-5p, whose expression is sex biased and regulated by GH and the transcription factor STAT5b. Small RNA sequencing confirmed the sex specificity of these two microRNAs and identified an additional 18 sex-biased microRNAs. Computational and experimental characterization of miR-1948-5p and miR-802-5p confirmed their authenticity. In vivo inhibition of these microRNAs by locked nucleic acids indicated that miR-1948-5p and miR-802-5p played a functional role in repressing female-biased genes and male-biased genes, respectively. This thesis also investigated the impact of GH and STAT5b on liver DNA methylation profiles. Reduced representation bisulfite sequencing was performed on liver tissues from four mouse models that perturbed the GH and STAT5b axis. In the wildtype liver, sex biased demethylation was positively associated with sex biased chromatin opening and gene expression. Global hypermethylation was observed in livers of mice with lit/lit mutation resulting in GH deficiency or with hepatocyte-specific deletion of the STAT5ab locus. Strikingly, these hypermethylated loci were enriched for enhancer elements and STAT5b binding sites found in wild-type mouse liver. Hypophysectomy followed by GH replacement mouse models identified differentially methylated regions that were sex-biased and rapidly methylated and demethylated in response to GH stimulation. Finally, we used ribosome profiling to validate sex-biased protein translation and identify mechanisms of translational control. In sum, this body of work provides novel insights and broadens our understanding of the diverse molecular mechanisms underlying sexual dimorphism in the liver. / 2020-10-08T00:00:00Z

Chromatin is decorated by a large array of biochemical modifications made to DNA and histone proteins. These modifications—and the broader organizational structure of chromatin—provide an important additional layer of information that is superimposed upon genome sequence and thus are widely referred to as the epigenome. The epigenome helps control which genes are expressed in a given context to produce the gene expression patterns that underlie the many different cellular phenotypes that arise during an organism’s development, and determine how these gene expression patterns are subsequently maintained for the life of an organism. The epigenetically heritable states are maintained and transmitted by self-propagating epigenetic mechanisms that persist in the absence of an initial stimulus. These epigenetic programs are generally thought to be controlled by core regulatory networks involving molecular writers and readers of chromatin marks. Guided by these principles, in this dissertation, we establish an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system consists of synthetic factors that can write and read m6A, and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we use our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits are able to program epigenetic functions de novo, conceptually validating “read-write” architectures. This dissertation outlines a synthetic framework for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells. / 2021-08-30T00:00:00Z

Epigenetic alterations are observed in virtually all cancer types, yet there is limited understanding of their role in tumorigenesis and evolution. The role of DNA methylation has been particularly elusive in this context. While this epigenetic mark has been extensively profiled in healthy and cancerous samples, our ability to understand its relationship with underlying biological processes is still limited. Moreover, recent advancements in the profiling of cell-free DNA in circulation have sparked renowned attention toward tissue-specific and cancer-specific DNA methylation patterns. In this thesis, I present results to improve and refine the computational characterization of DNA methylation in cancer, focusing on metastatic castration-resistant prostate cancer. The first contribution is the development and performance assessment of Rockermeth, a computational methodology to leverage large-scale DNA methylation profiling data to nominate robust differentially methylated regions (DMRs). Rocker-meth can retrieve biologically relevant DNA methylation changes, as demonstrated by extensive integrative analyses with gene expression, chromatin states, and genomic annotations. The second contribution is the generation of a map of DNA methylation changes across prostate cancer progression. The application of Rockermeth and other tailored methodologies can be used to trace the critical evolutionary steps of this disease, from the healthy tissue to the most lethal metastatic AR-independent counterpart. The main result is the evidence of the ability of DNA methylation to capture a snapshot of the active transcription factors in each state of the disease, offering orthogonal information compared to standard genomic sequencing. The third contribution is the design and development of NEMO, a tailored liquid biopsy sequencing panel approach to allow non-invasive neuroendocrine castration-resistant prostate cancer detection in patients with metastatic disease. Based on previous results and the comprehensive analysis of multiple datasets, I designed a set of informative genomic regions to estimate disease burden and evidence of neuroendocrine transdifferentiation. The actual implementation of the NEMO panel produced a scalable and cost-effective strategy, which has been extensively benchmarked using both in silico and in vitro approaches. The application of NEMO to patient-derived cfDNA samples demonstrated accurate tumor content estimation and robust detection of neuroendocrine disease, making it a promising instance of liquid biopsy for CRPC.

Prenatal exposure to mixture N1, a chemical mixture consisting of four phthalate diesters,three pesticides and Bisphenol A, has been associated with behavioural changes as well as changes in gene expression in mice. In this study it was investigated whether the changes in gene expression could be explained by changes in DNA methylation. Mixture N1 was found to significantly change DNA methylation in three different genes (Nr3c1, Nr3c2 and Crhr1) on totally eight different Cytosine Guanine dinucleotides (CpG) positions. To further investigate whether these changes could be induced already during differentiation of cells, a hippocampal cell model HT22 was exposed to mixture N1 during differentiation. In this mode, mixture N1 induced a statistically significant change in the promoter region of Nr3c1. Unfortunately, this change could however not be validated, and the experiments would need to be repeated. In conclusion, this study showed that exposure to mixture N1 can result in changes in DNA methylation.