1. Chatting Methyl Marks: Inhibition of Lysine Methyltransferases by Crosstalk between K-to-M Mutations on H3 Histone Manlu Liu 1, Stefan Lundgren 2,3, Siddhant Jain 2,3, and Dr. Peter W. Lewis 2,3 1 West High School – Madison, WI 2 University of Wisconsin-Madison Department of Biomolecular Chemistry, Epigenetics 3 Wisconsin Institute for Discovery Abstract Purpose Discussion Lysine to methionine (K-to-M) substitutions in histone H3 proteins have been shown to inhibit methyltransferase activity, leading to global decreases in trimethylation at specific sites along the N-terminus of histone H3. As a loss of methylation at various lysine residues correlates with tumorigenesis, experiments were performed to better understand the interactions between the sensory mechanisms of histone methyltransferases (HMTs) and the methionine mutants. Immunoblotting using whole-cell extracts from HEK293-T and C3H-10T1/2 cell lines was implemented to explore the connection between K-to-M modification crosstalk and HMT G9a, SUV39h, and PRC2 behaviors. Experiments compared effects of concurrent K-to-M mutations at the K4, K9, K27, and K36 loci on the respective lysine trimethylation levels to the changes in the trimethylation levels caused by individual K-to-M mutations at each loci. Additionally, tests were performed to examine the relationships between K9M and K56me3, K9M and K56me1, K56M and K9me3, as well as K56M and K9me1. Results show that the four concurrent K-to-M mutations have significant effects on the HTMs for K4 and K27 but almost no effect on K9me3 and K36me3. No crosstalk effect was observed between the K9 and K56 variations. Further experimentation involving specific combinations of K-to-M mutations could help pinpoint their discrete inhibitory effects on HTMs which may eventually lead to more targeted and effective treatment for specific cancers. Introduction Part 1 – Epigenetic Modifications Post-translational modifications to chromatin structure include the addition of methyl groups by methyltransferases to the amino-acid chains of histones. Figure 1: Epigenetic modifications that can occur on the H3 Histone Research has shown that mutations within the “histone code” could affect enzymatic activity. Certain lysine to methionine (K-to-M) mutations on the H3 histone, in particular, inhibits the active sites of methyltranferases, leading a loss of methylation. As methylation on these sites of N-terminal tails of H3.3 functions to silence genes, the loss of methyl groups opens up genes for transcription, resulting in an aberrant epigenetic landscape. Part 2 – Effects of K-to-M on Methyltransferases Figure 2: Inhibition of SET domain of PRC2 methyltransferase by methionine at position 27 (instead of lysine) K4M: has methyltransferase with atypical SET domain; previous research shows K4M decreases K9me3 only slightly K9M: an effect similar to that of K27M occurs with SUV39h enzymes; the SET domain is significantly inhibited by the methionine mutant K36M: a similar effect occurs; also has been shown to increase K27me3 levels K56M: published papers suggest that K9 and K56 are methylated by the same enzymes K64M: published papers suggest a correlation between K9 and 64 trimethylation Selected References Jack, Antonia PM, et al. "H3K56me3 is a novel, conserved heterochromatic mark that largely but not completely overlaps with H3K9me3 in both regulation and localization." PloS one 8.2 (2013): e51765. Lewis, Peter W., et al. "Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma." Science 340.6134 (2013): 857-861. Tropberger, Philipp, and Robert Schneider. "Going global." Epigenetics 5.2 (2010): 112-117. Yuan, Wen, et al. "H3K36 methylation antagonizes PRC2-mediated H3K27 methylation." Journal of Biological Chemistry 286.10 (2011): 7983-7989. Acknowledgements Ellie Degen for all the help, fun, and “Tea @3”s Rachel Egan, Lisa Wachtel, Carmen Lombard, and the Madison Metropolitan School District for providing this extraordinary opportunity Conclusions K4 methyltransferase is inhibited by K9M, K27M, and/or K36M mutations K9 and K56 may not be trimethylated by the same enzymes; there could be a lack of association between K9 and K64 methyltransferases Methods To discover the effects of combined K4M, K9M, K27M, and K36M (Quad M) mutations on K4me3, K9me3, K27me3, and K36me3, and how such effects differ if the mutations were present separately (i.e. one sample would only have the K4M mutation, a second sample would only have the K9M mutation, etc) To verify the relationships between K9, K56, and K64 methyltranferases Crosstalk Overview A series of Western Blots using modification-specific antisera were performed on whole-cell extracts from HEK293-T and C3H-10T1/2 cell lines with different mutations, including the H3.3 gene as a control. R mutations were used as controls for specific sites. Preparation of Samples: Using primers containing specific mutations, PCR Mutagenesis was implemented on plasmids containing the H3.3 gene. Bacteria were transformed with the mutated gene and then incubated to multiply. After extraction of the bacterial plasmid, transfection and transduction were performed on 293T and 10T1/2 cells. Mutated cells were selected with puromycin and subsequently prepared for Western Blotting. Western Blots: Proteins were run on 15% SDS-PAGE gels and transferred onto nitrocellulose membranes for probing. Blots were developed using chemiluminescence. Figure 3: Preliminary immunoblots of whole-cell extract from lentivirus-transduced 293T cells expressing indicated H3.3 transgenes. The “Quad” mutants refers to simultaneous mutations at the K4, K9, K27, and K36 sites. The ponceau stain serves as a control for even loading and shows the presence of the H3 histone. No effects were observed with K56M/K56me3, Quad M/K56me3, K56M/K9me3, and K64M/K9me3 combinations. Quad M exhibits a significant decrease in K4me3. K27me3 was only slightly decreased in Quad M, but severe decreases in K9me3 and K36me3 were found. Quad VS Individual Mutations K9 and K56 Interaction Figure 4: Immunoblots of whole-cell extract from lentivirus-transduced 10T1/2 cells. Inconclusive lack of effects was found with K9M/K9me1, K56M/K9me1, and K64M/K9me1. Similarly, unverified evidence show no change in K56me1 in samples with K9M, K56M, or K64M. Confirmed results show no decrease in K56me3 from K56M mutations; K9me3 from K56M mutations; K9me3 from K64M mutations; K56me3 from K9M mutations. A depletion in K9me3 is observed in K9M mutant cells. A B C D Figure 4: Immunoblots of whole-cell extract from lentivirus- transduced 10T1/2 cells. A. The Quad M exhibits a significant decrease in K4me3 as opposed to the slight/no decrease with K4M B. Similar levels of decrease in K9me3 were observed with K9M and Quad M. C. Quad M causes a lesser decrease in K27me3 compared to K27M D. Similar levels of decrease in K36me3 were observed with K36M and Quad M. A confirmation of unchanged trimethylation levels in different K9, 56, 64 combinations implies no association between K9, K56, and K64 methyltransferases, contrary to hypothesis based on previous published research An unexpected decrease in K4me3 with Quad M suggests that the K9M, K27M, and/or K36M mutations could inhibit the SET domain of the K4 methyltranferase Supporting previous research, the slight decrease in K27me3 caused by Quad M indicates that the presence of K4M, K9M, and/or K36M mutations reduces the inhibitory effects of K27M on PRC2 Similar levels of decrease observed with K36me3/K36M/Quad M and K9me3/K9M/Quad M suggest that the other respective mutations in the Quad have no effect on the interaction between methionine and methyltransferases at these sites