DNA Methylation in Histone H3.3 Lysine to Methionine Mutants Ellie Degen with Stefan Lundgren, Siddhant Jain and Dr. Peter W. Lewis UW Department of Biomolecular Chemistry IntroductionMethodsDiscussion Results References Bender, S. et al. Cancer Cell doi: /j.ccr (31 October 2013). Das, Partha M., and Rakesh Singal. "DNA methylation and cancer." Journal of Clinical Oncology (2004): Gonzalo, Susana. "Epigenetic alterations in aging." Journal of Applied Physiology (2010): Tatton-Brown, Katrina, et al. "Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability." Nature genetics (2014). Xu, Yan‐Ming, Ji‐Ying Du, and Andy TY Lau. "Post‐translational modifications of human histone H3: An update." PROTEOMICS (2014). K4me3 K9me3 K27me3 K36me3 HA H3 Figure 4. Samples of 10T1/2 cells containing lysine to arginine mutations in the H3.3 histone were used as controls and compared to lysine to methionine mutants at the same location, for evidence has shown Arginine has no effect on methylation of the histone. HA and H3 antibodies indicated the the presence of the transgene and the histone H3.3. Focus on determining how K4M, K9M, K27M, K36M, K56M, and K64M mutations affect global DNA methylation and hydroxymethylation. Preparation of H3.3 Mutants PCR was used to both insert the H3.3 histone gene into a plasmid containing antibiotic resistance and later mutate the gene. E. Coli were transformed and incubated to multiply the DNA. 293T cells were transfected and 10T1/2 cells transduced and selected with puromycin. Western Blots As a control, we verified the function of various lysine mutations on histone methylation. Samples were run on 15% SDS-PAGE gels and transferred onto a nitrocellulose membrane. A ponceau stain was used to check for even loading of samples. After application of primary and secondary antibodies, the blots were developed with chemiluminescence to display methylation levels. Figure 3. Binding of primary and secondary antibodies enable Me3 detection by chemiluminescence Dot Blots Bound 10T1/2 DNA to a membrane, applied primary and secondary antibodies, and developed with chemiluminescence. Epigenetics and Histones Histones organize genomic DNA to form nucleosomes and are subject to post-translational modifications (PTMs). Epigenetics, the study of heritable changes in gene expression without changes to the DNA sequence, centers around this chromatin modifying process. Figure 1. Histones wrap DNA into a nucleosome DNA Methylation DNA methylation at a specific genetic locus regulates transcription of the gene by physically blocking RNA polymerase or by attaching to methyl-binding proteins (MBDs), which recruit further gene activity altering enzymes. Figure 2. A methyl group added to the 5 carbon of cytosine Histone and DNA Modification The de novo methyltransferase DNMT3A methylates DNA based on the methylation/demethylation of specific sites on the H3.3 histone. Goal To pinpoint the role of H3.3 lysine methylation in gene repression and activation through monitoring DNA methylation. By determining the effect of lysine to methionine mutations in the H3.3 histone on global and site-specific DNA methylation, we will better understand the function of lysine methylation at different points on the histone. Predictions → global decrease in DNA methylation as the result of K9M and K36M mutant transgenes because DNMT3A interacts with H3K36me3 to methylate DNA at a specific location → no effect on DNA methylation caused by K4M mutant transgenes because K4M mutants do not affect K4me3 → global decrease in DNA methylation as the result of K27M mutants for prior evidence has shown K27me3 and DNA methylation patterns overlap. Conclusion Are results were largely inconclusive due to difficulties with the dot blot protocol. It is possible our DNA was not binding to the membrane, or that the antibodies were not effective indicators of DNA methylation/hydroxymethylation. Future Directions Aberrant methylation patterns have been linked to genomic instability and seen as hallmarks to disease. DNA methylation prevents differentiation through alteration of gene expression in dividing cells and thus plays a crucial role in the development of cancer. Additionally, hypermethylation of tumor suppressor genes and/or hypomethylation of oncogenes are associated with tumorigenesis. Understanding the genomic effects of histone modifications will hopefully provide paths for treatment. Wild Type K4M K4R K9M K9R K27M K27R K36M K36R