NOVEL ROLES FOR TEN-ELEVEN TRANSLOCATION 1 (TET1) 5-METHYLCYTOSINE DIOXYGENASE IN THE CELLULAR RESPONSE TO STRESS

Cells maintain exquisite control over gene expression in the struggle to preserve homeostasis. While some genes remain constitutively active to function in energy production or provide mechanical support, other batteries of genes respond only in the context of specific stresses, and must be silenced when homeostasis is achieved. Numerous transcription factors have been identified as responders to stress, whose gene targets aid in maintaining homeostasis. An additional layer of regulation over the relatively static genetic code involves chemical modifications of histones and DNA, particularly in or near gene promoters. Recent advances in the understanding of such epigenetic changes show that methylation at the 5-carbon position of cytosines neighboring guanosines (CpG) is an important player in the regulation of mammalian genes and chromatin architecture. Moreover, methyl groups can be further modified by in a reaction catalyzed by Ten-Eleven Translocation (TET) 5-methylcytosine dioxygenases, whose products were shown to be intermediates in DNA demethylation as well as stable, final products that may have functional relevance. DNA methylation has therefore become increasingly viewed as a dynamic process, yet the proteins regulating epigenetic marks and gene expression remain poorly-understood. Because changes in CpG methylation underlie numerous threats to health, including cancer and neurodegenerative diseases, these proteins are potential targets in developing biomarkers and in drug development. This dissertation explores the role of TET1 in response to cellular stress. Toward this end, an in vitro approach was taken in order to manipulate TET1 levels and measure the response to toxicant-induced stress. Two major roles for TET1 were elucidated. First, reactive oxygen species generated by exposure to the benzene metabolite hydroquinone (HQ) led to genome-wide and gene-specific CpG demethylation in a TET1-dependent manner. Moreover, cytoprotective genes induced by HQ were dependent TET1, suggesting the protein is involved in transcriptional responses to stress. Secondly, a role for TET1 in the response to DNA damage was uncovered and found to be unrelated to catalytic activity, indicating a novel, non-enzymatic role for TET1. Taken together, these data suggest that TET1 represents a heretofore unappreciated interface between the environment and the cellular response to stress. The findings presented are used to support the underlying hypothesis that TET1 is involved in mediating responses to stresses which would be harmful to the genome if left unconstrained. The idea that TET1 plays a protective role linked to disease is strengthened by reports of nearly uniformly low levels of TET proteins and their products in cancers. Highlighting the role of TET1 as a critical protector of the genome may serve as a foundation upon which a better understanding of the role of DNA methylation in disease may be built. Ultimately, unraveling the protein’s functions – both enzymatic and non-enzymatic - will lead to improved prevention and treatment strategies in diseases involving inappropriate DNA methylation