Epigenetics and Epigenetic Inhibitors
Dynamic cytosine methylation and demethylation control gene expression
Aberrant DNA methylation plays an important role in tumorigenesis and tumor maintenance. In tumor cells, a global loss of DNA methylation is observed together with DNA hypermethylation at specific sites such as tumor suppressor gene promoters. This may result in activation of oncogenes and silencing of tumor suppressor genes. Our laboratory is interested in further understanding the external factors that affect epigenetic regulation and the initiation of cancer.
DNA methylation and demethylation control gene expression
5-Methylcytosine (mC) is a stable epigenetic modification of DNA that is introduced enzymatically by DNA methyltransferases, playing a key role in controlling gene expression. Human tumors are characterized by large alterations in cytosine methylation patterns, leading to unregulated expression of transforming oncogenes and silencing of tumor suppressor genes. A family of ten-eleven translocation (TET) proteins (TET1-3) induce oxidation of 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Because fC and caC are excised by thymine DNA glycosylase (TDG) and are replaced with C via the base excision repair mechanism, this results in active demethylation and gene re-activation
Design and synthesis of Epigenetic Inhibitors
By oxidizing mC and removing the methylation marks, TET enzymes play a key role in a number of biological processes and have been implicated in many diseases. However, no TET-specific inhibitors have been developed thus far. N-oxalylglycine (NOG) is a α-ketoglutarate analogue that inhibits all three TET enzymes, however it also inhibits all other Fe(II)/α-ketoglutarate dependent enzymes (over 40 in humans). This lack of selectivity does not allow NOG to serve as a useful chemical tool. Our lab is working to identify direct and selective inhibitors of TET to elucidate their roles in human disease and to develop epigenetic modulators as new therapeutic agents.
Our strategy for inhibiting TET revolves around rational design based on the binding pocket of the TET catalytic domain. We are developing a series of inhibitors that would effectively combine a 5-methylcytosine and alpha-KG mimic via a synthetic linker. The idea here is to gain both potency and selectivity by using both the substrate and the cofactor as guided warheads.
Multi-omics Studies to Characterize Epigenetic Changes Leading to Cancer
We are currently working on elucidating the epigenetic changes that occur during the transition from chronic inflammation to oncogenesis. In one study, laboratory mice are subjected to long-term exposure to inflammatory agents such as lipopolysaccharide (LPS), nicotine-derived nitrosamine ketone (NNK), and tobacco smoke. Tissues from the animals are then harvested and subjected to several analyses to characterize the breadth of epigenetic, expression, and phenotypic changes that occur in response to these inflammatory stimuli. Some of the experiments performed include quantitation of global levels of 5-methylcytosine and its oxidized variants as well as analyses of differential changes in DNA methylation, gene expression, RNA transcrip levels, and protein abundance. In another study, similar multi-omics investigations are conducted in a mouse model of inflammatory bowel disease.
Characterization of Protein Readers of Epigenetic Marks in DNA
While many of the roles of 5-methylcytosine (5mC) in epigenetic signaling have been elucidated via the identification of effector proteins recognizing this DNA modification, the epigenetic functions of the oxidized methyl species- 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC)- remain poorly understood. The Tretyakova lab is actively involved in the discovery of novel epigenetic readers of these markers through affinity purification of these proteins using engineered DNA oligomers containing oxidized 5mC species paired with quantitative proteomics.
Deciphering the role of N6-methyl-deoxyadenosine (N6medA) in Aging and Alzheimer’s Disease
N6-methyl-deoxyadenosine (N6medA) is a novel DNA epigenetic mark recently discovered in mammalian genomes. Primarily found in bacteria and phage viruses, trace levels of this modification in mammalian genomes is very low, with a range from 0.6 to 7 per million modifications per dA. In humans, this modification is found to be introduced by N6AMT1 and can be erased by ALKBH1. Complimentary methylase Mettl4 and demethylase ALKBH4 have also been recently implicated in the maintenance of N6medA in mice.
The biological function of this novel epigenetic mark is largely unexplored. However, mounting evidence has demonstrated its involvement in the regulation of neurological activities. Our lab is interested in deciphering its role in aging and Alzheimer’s Disease by quantifying the global changes of N6medA via LCMS, mapping its deposition along the genome via meDIP-seq, and characterizing its protein readers through affinity proteomics.
Representative publications
Christopher L. Seiler, Jung Min Song, Delshanee Kotandeniya, Jianji Chen, Thomas J. Y. Kono, Qiyuan Han, Mathia Colwell, Benjamin Auch, Aaron L. Sarver, Pramod Upadhyaya, Yanan Ren, Christopher Faulk, Silvio De Flora, Sebastiano La Maestra, Yue Chen, Fekadu Kassie & Natalia Y. Tretyakova. (2020) Inhalation exposure to cigarette smoke and inflammatory agents induces epigenetic changes in the lung. Nature Scientific Reports. 2020 10(1), 11290. doi: 10.1038/s41598-020-67502-8. DOI: 10.1038/s41598-020-67502-8
Seiler, CL, Fernandez, J., Koerperich, Z., Andersen, MP., Kotandeniya, D., Nguyen, ME., Sham, YY., and Tretyakova, NY. “Maintenance DNA Methyltransferase Activity in the Presence of Oxidized Forms of 5-Methylcytosine: Structural Basis for TET-Mediated DNA Demethylation.” Biochemistry, 2018, pp. Biochemistry, 19 September 2018. Article.
D. Kotandeniya, C. L. Seiler, J. Fernandez, S. S. Pujari, L. Curwick, K. Murphy, S. Wickramaratne, S. Yan, D. Murphy, Yuk Y. Sham and N. Y. Tretyakova. Can 5-methylcytosine analogues with extended alkyl side chains guide DNA methylation? Chem. Commun., 2018, Advance Article. Article. DOI: 10.1039/C7CC06867K
Seiler, C.L., Song, J.M., Fernandez, J., Abrahante, J.E., Chen, Y., Ren, Y., Kassie, F., Tretyakova, N.Y. Epigenetic Effects of Inflammation in Type II Alveolar Cells of A/J Mice Treated with Lipopolysaccharide. Chem. Res. Toxicol. 2019, 32, 5, 831-839. Article.