Hormone-dependent cancers affect millions of people worldwide and are a great burden to society, both financially and in terms of healthcare resources. The steroid hormone progesterone plays critical roles in a variety of physiological functions as well as in the carcinogenesis of hormone-dependent cancers including breast and endometrial cancers. Progesterone regulates gene expression through histone modifications and chromatin remodeling. However, the role played by DNA methylation in this context has not been determined. DNA methylation (5-methylcytosine) generally represses gene transcription via methyl-binding proteins. Changes in DNA methylation levels are observed in carcinogenesis as well as in the dynamic regulation of genes which rapidly respond to stimuli. Ten-eleven translocation (TET) proteins hydroxylate the 5-methylcytosine during the demethylation process synthesizing 5-hydroxymethylcytosine (5hmC). 5hmC inhibits the interactions of methyl-binding proteins and can influence both long- and short-term regulation of gene transcription. The goal of this proposal is to identify DNA methylation and hydroxymethylation changes induced by progesterone in breast cancer cells. We will use hMeDIP-sequencing and combined glycosylation restriction analysis to measure the differential levels of 5-methylcytosine and 5-hydroxymethylcytosine marks. Furthermore, we propose to characterize the role of TET proteins in progesterone-mediated signaling. To this end, we plan to analyze gene expression and DNA methylation/hydroxymethylation changes in hormone-treated cells in the presence and absence of TET proteins. In addition, we will investigate TET binding in untreated and hormone-treated cells by ChIP assay. The outcome of this project will clarify the biological functions of 5-hydroxymethylcytosine in hormonal-mediated gene regulation and could lead to the identification of new targets for the diagnosis and treatment of hormone-dependent cancers.