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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
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.
K36R
K36M
K27R
K27M
K9R
Predictions
K9M
Focus on determining how K4M, K9M, K27M, K36M, K56M,
and K64M mutations affect global DNA methylation and
hydroxymethylation.
K4R
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.
Discussion
Results
K4M
Epigenetics and Histones
Methods
Wild Type
Introduction
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.
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.
→ 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.
References
Bender, S. et al. Cancer Cell doi: 10.1016/j.ccr.2013.10.006 (31 October 2013).
Das, Partha M., and Rakesh Singal. "DNA methylation and cancer." Journal of Clinical Oncology 22.22 (2004): 4632-4642.
Gonzalo, Susana. "Epigenetic alterations in aging." Journal of Applied Physiology 109.2 (2010): 586-597.
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).