Doctor of Philosophy, The Ohio State University, 2008, Chemistry

In the post-genome era, the regulation of gene transcription is the central topic in biomedical research. In recent years, it is found that posttranslational modifications of histones play a pivotal role in the regulation of gene expression and signal transduction. As a prominent modification linked to diverse signaling pathways and diseases, histone methylation was believed to be stable and irreversible. On the contrary, I hypothesized that the reverse demethylation reaction could occur. Using a candidate approach, I studied all five budding yeast JmjC domain containing proteins and one human JmjC protein RBP2. We used proteomic mass spectrometry techniques to study the in vivo effects of individual JmjC gene deletion and overexpression strains in yeast. We discovered four histone demethylastes out of the five JmjC proteins. Deletion and overexpression of the candidates affect histone modification patterns. Tandem MS identified the exact modification sites of the JmjC genes. In vitro enzyme assays verified Rph1, Gis1, and Jhd2 have site-specific demetylation activity. We found that the H3K36me3 demethylase activity of Rph1 is responsible for its DNA damage phenotypes. Furthermore, we identified Jhd1 and Rph1, two H3-K36 demethylases, are involved in transcription elongation. In a further effort to study transcription regulation by human histone H3K4 demethylase RBP2, a candidate trithorax group protein, and identify human trithorax response elements (TREs), I studied regulation of RBP2 by its DNA binding. PCR-assisted DNA binding selection revealed that RBP2 DNA binding domain ARID selectively binds to GC rich DNA. Subsequent electrophoretic mobility-shift assays verified it binds to BRD2 promoter via a consensus motif (CCGCCC). Solution NMR structure of RBP2 ARID showed it had 6 helices and 2 loops. Further NMR titration on ARID-DNA complex identified that the binding interface is consisted of loop 1, helix 4, loop 2, and helix 5. Importantly, we found loop 2 ma (open full item for complete abstract)

Committee: Ming-Daw Tsai (Advisor); Dehua Pei (Committee Chair); Michael Chan (Committee Member); Ross Dalbey (Committee Member); Thomas Magliery (Committee Member) Subjects: Biochemistry