Epigenetic Regulation of Cytosine Methylation, Hydroxymethylation, Formylation and Carboxylation in a Mouse Model of Smoking-Induced Lung Cancer Christopher L. Seiler,¥,‡ Jungmin Song,¥,† Arunkumar Anandharaj,¥,† Devon Hunter-Schlichting,¥,₤ Heather Nelson, ¥,₤ Fekadu Kassie,¥,† and Natalia Tretyakova¥,‡ Masonic Cancer Center,¥ College of Veterinary Medicine,† School of Public Health,₤ and Department of Medicinal Chemistry‡, University of Minnesota Epigenetic changes are critically important in the development of cancer. Tumors are characterized by profound changes in DNA methylation patterns, resulting in silencing of tumor suppressor genes, activation of oncogenes, and decreased chromosomal stability. The presence of 5- methyl-2′-deoxycytidine (meC) within gene promoter regions typically down-regulates gene expression, while Tet protein-catalyzed oxidation of meC to 5-hydroxymethyl-2′-deoxycytidine (hmC), 5-formyl-2′- deoxycytidine (fC), and 5-carboxyl-2′-deoxycytidine (caC) is thought to lead to gene reactivation. Recent studies have revealed that the levels of hmC are significantly reduced in all tumors. However, it is not known whether these epigenetic changes are the cause or the result of malignant transformation and whether fC and caC follow a similar trend. Smoking and inflammation are well known risk factors for lung cancer development. In the present work, we investigated the early epigenetic effects of tobacco carcinogen 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) and inflammation agent lipopolysaccharide (LPS) in a mouse model of lung cancer (A/J mouse). NNK and LPS were given intraperitoneal and intranasal, respectively, to six week old mice. An HPLC-ESI-MS/MS based method was developed to quantify global meC, hmC, fC, and caC in lung and nasal mucosa tissues. Furthermore, we employed Tab- Seq and pyrosequencing to analyze loci-specific cytosine methylation and hydroxymethylation and qRT-PCR to measure the levels of gene expression in treated and control mice. We observed significant changes in gene expression of Tet proteins after treatment with NNK and LPS tissue and tumors. Our results reveal early epigenetic changes in lung and nasal tissues of mice exposed to tobacco carcinogens and inflammatory agents. .