Transcript Inhibition of Lysine Methyltransferases by Crosstalk

Chatting Methyl Marks: Inhibition of Lysine Methyltransferases by Crosstalk
between K-to-M Mutations on H3 Histone
Manlu Liu1, Stefan Lundgren2,3, Siddhant Jain2,3, and Dr. Peter W. Lewis2,3
1West High School – Madison, WI
2University of Wisconsin-Madison Department of Biomolecular Chemistry, Epigenetics
3Wisconsin Institute for Discovery
Abstract
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.
• 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
Methods
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.
Crosstalk Overview
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 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.
K9 and K56 Interaction
Figure 4: Immunoblots of whole-cell extract
from lentivirus-transduced 10T1/2 cells.
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
Quad VS Individual Mutations
Purpose
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
C
C. Quad M causes a lesser
decrease in K27me3
compared to K27M
B
D
D. Similar levels of decrease
in K36me3 were observed
with K36M and Quad M.
Figure 4: Immunoblots
of whole-cell extract
from lentivirustransduced 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.
Discussion
• 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
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
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
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.