Epigenomic landscape of 5-hydroxymethylcytosine and implications in cancer
Abstract
The addition of a methyl group (-CH3) at the C-5 position of the pyrimidine ring of cytosine nucleotide in DNA serves as an epigenetic signaling tool employed by cells to "turn off" gene expression. This epigenetic modification also known as 5-methylcytosine, 5mC, plays a critical role in numerous cellular processes including genomic imprinting, cellular differentiation and preservation of genomic stability. Aberrant 5mC patterns like hypermethylation of tumor suppressor genes and hypomethylation of oncogenes are traditional hallmarks of cancer and a testament to the role of epigenetics in oncogenesis. Recently, three other epigenetic derivatives of 5mC (enzymatically mediated by Ten-eleven-translocation dioxygenase; TET) namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) have been discovered. The biological significance of these novel DNA modifications is still unclear but it has been hypothesized that these novel derivatives serve as intermediates in the DNA demethylation pathway and confer unique transcriptional potential to genes. Understanding how these novel epigenetic marks are interpreted at discrete loci in cancers has been the goal of my doctoral study and can be broadly classified into the following domains. (i) Mapping the Epigenetic Landscape (5mC and 5hmC) of myeloid differentiation in human leukemia cells using Next Generation Sequencing (NGS) followed by exhaustive bioinformatics analysis. For the very first time, we have defined the differentiation dependent changes in the methylated and hydroxymethylated regions across the genome in the context of (a) promoters, (b) gene bodies and (c) intergenic regions. Additionally, we have correlated the changes of the methylome with the changing transcriptional profile upon HL-60 differentiation to comprehensively characterize the epigenetic landscape during myeloid differentiation. (ii) Development of a sensitive technique for the quantification of these rare epigenetic marks that constitute less than 0.05% of the genome. We have developed an enhanced Immunoassay based platform to precisely quantitate the global levels of 5mC, 5hmC, 5fC and 5caC and have evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer. We have reported a decrease in the mean level of 5hmC by 56.37% (p-value < 0.05) in blood of malignant lung cancer patients in comparison to levels in healthy controls and the clinical utility of this observation is being currently explored (iii) Elucidating a novel insight into the therapeutic activity of Decitabine (DAC), recently FDA-approved "epigenetic drug" in myeloid malignancies. Though the hypomethylating effect of DAC is attributed to the depletion of DNA-methyltransferase-1 (DNMT1), the nature of the genomic loci susceptible to DAC-induced demethylation is poorly understood. We show that DAC induces demethylation in HL60 with a paradoxical increase in 5hmC, 5fC and 5caC. Using a multi-compartmental model of DNA methylation, we propose that the observed increase in 5hmC following DAC treatment is caused by the partial selectivity of the TET dioxygenase enzymes for hemi-methylated CpG dinucleotides, whose abundance is increased during drug induced inhibition of the DNMT1-mediated maintenance methylation.
Degree
Ph.D.
Advisors
Suter, Purdue University.
Subject Area
Cellular biology
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