UBC Theses and Dissertations
Generation and characterization of a lysine (K)-specific methyltransferase 2D knockout human cell line Huff, Ryan Donald
Lysine (K)-specific methyltransferase 2D (KMT2D) encodes a histone-lysine N-methyltransferase that catalyzes the methylation of histone 3 lysine 4 (H3K4me), which is an epigenetic modification involved in transcriptional regulation. KMT2D is a recurrent target of somatic mutation in at least 27 types of cancer, with the majority of KMT2D mutations (54%) predicted to result in the loss of protein function. In particular, KMT2D is mutated in ~85% of patients with follicular lymphoma, with ~50% of cases harboring multiple mutations in KMT2D. Disruption of KMT2D function has been linked to a rare pediatric disorder named Kabuki syndrome where ~75% of patients harbour heterozygous loss of function (LOF) mutations. To investigate the impact of LOF KMT2D mutations on H3K4 methylation and transcription I inactivated KMT2D using zinc finger nuclease (ZFN) technology in the human cell line HEK293A. Consistent with previous studies, HEK293 KMT2D LOF cell lines demonstrated loss of KMT2D was sufficient to reduce bulk mono- and dimethylation of H3K4 in the cell. Previous studies have demonstrated that KMT2D’s epigenetic function is involved in nuclear hormone transactivation, and that disruption of nuclear hormone signaling via the retinoic acid receptor (RAR) leads to lymphomagenesis in mouse models. To study the link between RAR signaling and KMT2D, I investigated RAR signaling in HEK293 KMT2D LOF cell lines. I observed KMT2D was necessary for robust induction of RAR response genes RARA2, RARB2, and RARG in the presence of 9-cis-retinoic acid. These results are compatible with the notion that LOF KMT2D mutations may aid cancer cells in escaping RA induced differentiation by impairing RA dependent transcription of differentiation promoting genes.
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