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Cancer-recurrent missense mutations in SET domain alter KMT2A methyltransferase activity

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Cancer-recurrent missense mutations in SET domain alter KMT2A methyltransferase activity

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Cancer-recurrent missense mutations in SET domain alter KMT2A
methyltransferase activity

By
Tianyang Bai

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)

August 2022

Copyright 2022 Tianyang Bai
ii

Acknowledgement
Firstly, I want to express my gratitude to my parents: Thank you, Mom and Dad. The pandemic has
separated us for two years, during which your support and words brought me relief, and now I am proud
to say that I managed to get through it. Then I want to thank my girlfriend for the help in our life. So let
us move forward to our bright future!
I have received numerous assistance and guidance throughout my master’s study and writing up this
thesis. First, I want to thank my supervisor and mentor, Professor Yali Dou, for your professional
guidance and help throughout my master’s research. Your knowledge broadens my horizon and expands
my perspective on academic life. The one and a half years in Dou lab would remain in my memory. And I
want to express my special thanks to my committee member, Dr. Oliver Bell and Dr. Judd Rice, for their
advice and suggestions on my thesis. Finally, I want to express my sincere thanks to Professor Yifan Liu
and lab members Liang Sha, Guobing Li, Hongwei Lei, David Wang, Jingqi Yu, Zi Yang, Alex Ayoub,
and Robert Zepeda. Without your help and instruction, I could never learn so many valuable techniques
and methods as a chemist undergrad who barely knows anything in biochemistry.
Then I want to thank the BMM program for the chance, and Prof. Pragna Patel, Prof. Judd Rice, Prof.
Baruch Frankel, Prof. Sita Reddy for organizing the program and the courses, and all the professors who
lectured me.
In addition, I would like to thank my BMM peers for our great memory. Special thanks to Huikang Ye
and Jiaxuan Bian, we had lunches every noon, and I will never forget the laughter we had and how you
guys helped me when I was depressed.
Per aspera ad astra.

iii

Table of Contents

Acknowledgement ........................................................................................................................................ ii
List of Tables ................................................................................................................................................ v
List of Figures .............................................................................................................................................. vi
Abstract ....................................................................................................................................................... vii
Chapter 1: Introduction ................................................................................................................................. 1
Histone Post-Translational Modification .................................................................................................. 1
Histone Methylation and Methyltransferases ........................................................................................... 1
SET Domain.............................................................................................................................................. 4
KMT2A and Cancer .................................................................................................................................. 6
Chapter 2: Methods and Material.................................................................................................................. 7
Data Analysis ............................................................................................................................................ 7
Plasmid and Vectors ................................................................................................................................. 8
Site Directed Mutagenesis ........................................................................................................................ 8
Materials ................................................................................................................................................... 9
Protein Expression and Purification ........................................................................................................ 10
In vitro HMT assay ................................................................................................................................. 11
Enzyme Assembly .............................................................................................................................. 11
In vitro HMT assay and western blot detection .................................................................................. 11
In vitro radioactive HMT assay .......................................................................................................... 12
iv

Chapter 3: Results ....................................................................................................................................... 13
Recurrent missense mutations in cancer ................................................................................................. 13
Preliminary Results ................................................................................................................................. 16
Mutations in the conserved substrate-interacting region ........................................................................ 17
KMT2A-SET mutations drastically reduced the methyltransferase activity .......................................... 20
KMT2A-SET mutants have differential effects on enzyme processivity ............................................... 20
Chapter 4: Discussion ................................................................................................................................. 24
Mutation residue analysis ....................................................................................................................... 25
Summary ................................................................................................................................................. 26
References ................................................................................................................................................... 28

v

List of Tables
Table 1 Examples of common PTM enzymes .............................................................................................. 2
Table 2 Primers for PCR-based mutagenesis ............................................................................................... 9
Table 3 Distribution of somatic mutations on KMT2A and its domains ................................................... 13
Table 4 Mutations on the conserved residues among the KMT2 methyltransferases ................................ 15
Table 5 Protein alignment of the KMT2 SET domain. .............................................................................. 19

vi

List of Figures
Figure 1 Representation of the human post-translational modifications in histone tails (Ramazi, et al.) .... 1
Figure 2 Scheme of levels of H3K4 methylation and demethylation .......................................................... 3
Figure 3 Domain architecture of the KMT2 family in human ..................................................................... 4
Figure 4 Alignment of the KMT2A counterparts among species ................................................................ 5
Figure 5 Cryo-EM structure of the SET complex on the nucleosome core particles ................................... 6
Figure 6 Number of reoccurrence of mutations on KMT2A SET domain ................................................ 14
Figure 7 Mutagenesis Results .................................................................................................................... 16
Figure 8 Coomassie staining of the purified proteins ................................................................................ 17
Figure 9 Crystal Structure Overview ......................................................................................................... 18
Figure 10 Most mutations on KMT2A SET domain has changed the methyltransferase activity. ............ 20
Figure 11 HMT result of the wild type (MLL3813IL-SET) and 10 mutations. ......................................... 21
Figure 12 H3K4 Processivity graph of R3789H, R3789C and R3968L .................................................... 22
Figure 13 H3K4 Processivity graph of R3889Q, V3865I, A3964T, R3968W and Position S3786C ........ 22
Figure 14 H3K4 Processivity graph of R3906H ........................................................................................ 23
Figure 15 H3K4 Processivity graph of R3844Q ........................................................................................ 23

vii

Abstract
KMT2A (lysine methyltransferase 2A, also known as MLL1, ALL1) tri-methylates histone 3 at lysine 4
(H3K4) to regulate gene expression. KMT2A locates on chromosome 11q23, and 10% of human
leukemia patients were reported with translocation of the gene. According to the COSMIC database,
KMT2A is also one of the most mutated genes in various cancers. KMT2A mutations include nonsense
mutations, missense mutations, frameshift mutations, and so on. Among them, missense mutations are
widely distributed throughout the gene, including the C-terminus catalytic SET domain. Somatic
mutations in KMT2A-SET domain are mainly found in small intestinal cancer, large intestinal cancer,
endometrial cancer. Interestingly, 75 percent of the SET domain mutations are missense mutations,
raising the question of whether these discrete mutations impact KMT2 methyltransferase activity.
In this study, we systematically investigate 10 missense mutations in the KMT2A SET domain that are
recurrent in different human cancers. Here we show that the R3844Q mutant retains the same enzymatic
activity on histone methylation, while the activity of R3789H, R3789C, R3889Q, R3968W, V3865I,
A1964T, S3786C, R3906H, and R398L drops drastically. We conduct enzymatic activity analyses on
KMT2A-SET mutants and hopefully could contribute to the investigation of the role that KMT2A and its
mutations play in cancer.
1

Chapter 1: Introduction
Histone Post-Translational Modification
The basic building block of chromatin is nucleosome, which is comprised of an octamer of core histones
(H2A, H2B, H3, and H4) wrapped by a DNA fragment of about 147 bp.
[1]
Chromatin regulates DNA-
based processes like transcription, replication, and DNA repair in eukaryotes. Histone modifications
contribute significantly to chromatin regulation. Therefore, histone post-translational modification (PTM),
including phosphorylation, acetylation, ubiquitination, methylation, etc., established an epigenetic code
that regulates not only transcription, but also many other DNA-based processes, including chromosome
packaging and DNA damage/repair.
[2]
The schematic representation of common PTMs on histone tails is
shown in Figure 1.
[3]

Common histone post-translational modifications include phosphorylation, acetylation, and methylation
and play significant role in DNA damage repair, gene expression, and compacting chromatin.
[3] [4] [5]
Other modifications such as ubiquitination, ADP ribosylation, and proline isomerization also play
important roles in chromatin regulation.
[2]
Histone Methylation and Methyltransferases
Lysine methylation, especially methylation of histone H3 (K4, K9, K27, and K79) and histone H4 (K20),
are widespread.
[6]
Specifically, H3K4 methylation is associated with active transcription and open
Figure 1 Representation of the human post-translational modifications in histone tails
(Ramazi, et al.)
2

chromatin structures. H3K27, H3K9, and H4K20 methylation are repressive histone marks and are
associated with gene silencing.
[7]
Compared to enzymes conferring other modifications, the lysine
methyltransferases have higher substrate specificity, as shown in Table 1.

Table 1 Examples of common PTM enzymes
Histone PTM enzymes
Acetylation
[8]
Lysine Methylation
[9]
Arginine Methylation
[9]
Enzyme Residue(s) Enzyme Residue(s) Enzyme Residue(s)
HAT1 H4 (K5, K12) KMT2A-D H3K4 CARM1 H3 (R2, R17, R26)
CBP/P300 H3 (K14, K18)
H4 (K5, K8)
H2A (K5)
H2B (K12, K15)
SUV39H1-2 H3K9 PRMT5 H3R8, H4R3
EZH2 H3K27
SUV420H1-2 H4K20
DOT1 H3K79

Members of the histone–lysine N-methyltransferase 2 family (KMT2), also known as MLL (mixed-
lineage leukemia), or ALL (acute lymphoblastic leukemia), specifically methylates histone 3 at lysine 4
(H3K4), regulating gene expression during early development and hematopoiesis.
[10]
The catalytic SET
domain catalyzes the lysine (H3K4) methylation by transferring the methyl group from S-adenosyl-L-
methionine to the side chain lysine, as shown in Figure 2.
3

There are six enzymes in the mammalian KMT2 family. The family is made up of three pairs of
structurally similar proteins, with each pair related to a single fruit fly protein. KMT2A (Human
chromosome 11q23) and KMT2B (Human chromosome 19q13) are related with trx and mainly
trimethylated H3K4 at specific sites.
[11]
KMT2F and KMT2G can facilitate global H3K4 methylation.
[12]
KMT2 family plays an important role in various physiological paths. As described, KMT2 family are
abundant at the gene promoter and enhancer region to regulate transcription.

KMT2A can also regulates
the cell cycle by influencing expression of the homeobox-containing (Hox) gene.
[13]
The H3K4me3 level
of the relative oncogene could also serve as an indicator of gene silencing or activation in cancer cell
stemness research.
[14]
The domain architectures of the KMT2 family in humans are shown in Figure 3. Members of the KMT2
family contain different domains, some of which are highly conserved within the family. Bromodomains
are widely found in the chromatin modulating proteins, including histone acetyltransferases and
methyltransferases like KMT2A and KMT2B.
[15]
The FY-rich-C-terminal region (FYRC) and FY-rich-
N-terminal region (FYRN) are a pair of phenylalanine/tyrosine-rich regions that are presented in several
chromatin-related proteins.
[16]
Interestingly, FYRC and FYRN regions in KMT2A/B, though separated
by hundreds of amino acids, could play a role in the proteolytic process together.
[17]
They can also
Figure 2 Scheme of levels of H3K4 methylation and demethylation

4

regulate protein sub-localization.
[17]
Several conserved plant homeodomain (PHD) fingers can interfere
with the homodimerization of MLL by interacting with the nuclear cyclophilin, Cyp 33.
[19]
CXXC zinc
finger domain can bind unmethylated DNA
[20]
. Binding of the CXXC domain to unmethylated DNA is
essential for MLL-translocation-induced leukemia.
[20]
The methyltransferase activity of the KMT2 family
is carried out by the C-terminal SET domain.
[21]

SET Domain
SET domain is named after the three proteins in Drosophila melanogaster, the suppressor of variegation
3-9, Su(var)3-9, the polycomb-group chromatin regulator enhancer of zeste, E(z), and the trithorax-group
chromatin regulator Trithorax (Trx).
[22][23][24]
Majority of histone methyltransferases contain the SET
domain.
[25]
The substrate-interacting part of the SET domain is evolutionarily conserved, especially in
vertebrates. The SET domain can be traced back to ancient unicellular species like ancient yeast and
Capsaspora owczarzaki as shown in Figure 4.

Figure 3 Domain architecture of the KMT2 family in human (Rao, Dou, 2015)
5

The KMT2 family enzymes reside in the stable core complexes for maximum enzymatic activity.
Namely, WDR5 (WD repeat protein 5), ASH2L (absent, small or homeotic 2-like histone lysine
methyltransferase complex subunit), RbBP5 (retinoblastoma binding protein 5), DPY30 (Dumpy-30) are
needed to compose the catalytic complex, as shown in Figure 5.
[26][27]
We have solved the cryo-EM
structure and revealed how KMT2A activity is regulated by the core complex on the nucleosome core
particles.
[28]
We found RbBP5 binds the NCP through both DNA and histone H4 tail and the activity of
the MLL1 core complex was dramatically reduced when the RbBP5-NCP association was disrupted. We
also found WDR5-SET-ASH2L subcomplex did not make direct contacts with nucleosome DNA.
Figure 4 Alignment of the KMT2A counterparts among species (a.a. 3884 ~ 3938) in human
KMT2A)
6

KMT2A and Cancer
KMT2A or MLL, which stands for mixed-lineage leukemia, is the founding member of the KMT2A
family. As the name suggests, MLL (on 11q23) was first found translocated in mixed-lineage leukemia
patients.
[29]
Since the initial discovery, over 80 fusion partners have been identified in patients.
[30], [31]
Some partner such as AF4, AF10 can involve in regulation of transcriptional elongation and recruitment
of the H3K79 histone methyltransferase DOT1L.
[32]
Gene expression of the KMT2A-rearranged leukemia
is different from the wild-type leukemia. The most frequently overexpressed genes in KMT2A-
rearranged leukemias are the downstream HOX genes (for instance HOXA9) and the cofactor MEIS1.
[33]
Except for leukemia, KMT2A has also been studied in other cancers, including solid tumors. Growing
numbers of KMT2A mutations have been identified in the COSMIC (Catalogue of Somatic Mutations in
Cancer) database.
[34]
There are a substantial number of mutations found for KMT2A in various cancer
types, including lung cancer, colon cancer, and stomach cancer.

Among over 2800 reported mutations on
Figure 5 Cryo-EM structure of the SET-WDR5-RbBP5-ASH2L-DPY30 on the nucleosome core
particles (Park, et al., 2019)
7

KMT2A, more than 50% are missense mutations, 121 of which are in the SET domain. In addition, there
are a total of 361 nonsense mutations, deletion, and frameshift mutations (23.95% of the total mutations)
that also lead to loss of function for the SET domain.
According to cancer genome sequencing, chromatin regulators are among the most mutated genes, and
KMT2A is no exception.
[35]
Although the majority of the mutations in other methyltransferases are loss-
of-function mutations, a portion of them can be identified as gain-of-function mutations, according to
previous research. For example, gain-of-function mutations on multiple sites in the SET domain of EZH2
were observed, and greater global H3K27me levels in the EZH2 mutant cells corresponded the results.
[36],
[37]
Such cancer-inducing gain-of-function mutations could be a viable therapeutic target. As for KMT2A,
despite the wide-spread missense mutations on the SET domain, few studies have been focused on their
functional influence. Therefore, we are interested in studying the activity of recurrent missense mutations
in the KMT2A SET domain. However, although the results of our unbiased research on the most recurrent
mutations on the KMT2A-SET domain did not reveal any gain-of-function mutations, the results will help
upcoming researchers to understand the function of KMT2 in tumorigenesis better.
Chapter 2: Methods and Material
Data Analysis
Mutational landscape and details were obtained from the public database COSMIC (Catalogue of Somatic
Mutations in Cancer, https://cancer.sanger.ac.uk/cosmic). The searching term was KMT2A, and the SET
domain range was 3771-3973, PHD domain range was 1420-1640, the bromodomain range was 1695-
1750, the FYRN domain range was 2010-2080, and the FRYC domain range was 3660-3765. KMT2B-
SET 2616-2716, KMT2C-SET 4812-4912, KMT2D-SET 5438-5538, KMT2F-SET 1608-1708, KMT2G-
SET 1867-1967.
8

Plasmid and Vectors
MLL1-SET
3813IL
was lab stock plasmid where modified SET domain was cloned into pET28a His 6-small
ubiquitin-tagged at XhoI and BamHI sites. The SET
3813
construct does not require WDR5 for in vitro
histone methylation as previously used by our lab.
[38]
SET
3771-3973
was cloned into the pET28a His 6-small
ubiquitin-tagged vector at XhoI and BamHI sites.
Site Directed Mutagenesis
SET domain with desired point mutations were synthesized through PCR based mutagenesis. Primers are
designed via QuikChange® Primer Design Program
(https://www.agilent.com/store/primerDesignProgram.jsp) and purchased from Integrated DNA
Technology™. Primers were shown below in Table 2.
S3786C, R3789H and R3789C were mutated on SET
3771
, while the rest of the mutants were mutated on
SET
3813IL
. Our protein amino acid sequence was pulled from the histone-lysine N-methyltransferase 2A
isoform 1 [homo sapiens] (NCBI index: NP_001184033.1) while the .pdb files from
https://www.rcsb.org/ and previous papers used histone-lysine N-methyltransferase 2A isoform 2
precursor 1 [homo sapiens] (NCBI index: NP_005924.2). There were 3 amino acid difference from the N-
terminus (amino acid number) for these two isoforms. The SET domains of these two isoforms were
identical.

9

Table 2 Primers for PCR-based mutagenesis
Mutation Vector Primer Sequence
S3786C SET
3771

GAGGCTGACGATGTTTACAAGCCAGGAAGTTAAAC
GTTTAACTTCCTGGCTTGTAAACATCGTCAGCCTC
R3789C SET
3771

CAGGAGGCTGACAATGTTTAGAAGCCAGGAAGTTA
TAACTTCCTGGCTTCTAAACATTGTCAGCCTCCTG
R3789H SET
3771

TATTCAGGAGGCTGATGATGTTTAGAAGCCAGGAAGT
ACTTCCTGGCTTCTAAACATCATCAGCCTCCTGAATA
V3865I SET
3813

GGATGGAGCGGATGATGTTGCCGGCATACTC
GAGTATGCCGGCAACATCATCCGCTCCATCC
A3964T SET
3813

CCGGCATTTCTTGGTGCCACAGTTGCAGG
CCTGCAACTGTGGCACCAAGAAATGCCGG
R3844Q SET
3813

CTTACAGAAAAGACCCTGGCCATGGATGGGAGA
TCTCCCATCCATGGCCAGGGTCTTTTCTGTAAG
D3879N SET
3813

AATGCCCTTGCTGTTGTAATACTTTTCCCGCTTGTC
GACAAGCGGGAAAAGTATTACAACAGCAAGGGCATT
R3889Q SET
3813

CTACCTCTGAGTCATCAATTTGGAACATATAGCAACCAATG
CATTGGTTGCTATATGTTCCAAATTGATGACTCAGAGGTAG
R3906H SET
3813

ACGAGTGATTGATGAAGTGTGCAGCATTTCCATGC
GCATGGAAATGCTGCACACTTCATCAATCACTCGT
R3968L SET
3813

GTTAGTTTAGGAACTTCAGGCATTTCTTGGCGCCA
TGGCGCCAAGAAATGCCTGAAGTTCCTAAACTAAC
R3968W SET
3813

TTAGTTTAGGAACTTCCAGCATTTCTTGGCGCCAC
GTGGCGCCAAGAAATGCTGGAAGTTCCTAAACTAA

Materials
Tris-HCl (ThemoFisher, lot# 206246), NaCl (ThermoFisher, lot# 200665), DTT (ThermoFisher, lot#
BP172-5), MgCl 2 (Sigma, lot# M8266), glycerol (Sigma, lot# G9012), imidazole (Sigma, lot#56748), β-
mercaptoethanol (Sigma, lot#444203), SDS (sodium dodecyl sulfate, ThermoFisher, lot# 192127),
3
H-
SAM ((S-adenosyl-L-[methyl-
3
H]-methionine, 1 mCi/mL in HCl, PerkinElmer, lot# 2774422),
bromophenol blue (Sigma, lot# GE17-1329-01), LB powder (ThermoFisher, lot#BP147), kanamycin
(ThermoFisher, lot#BP906-5), IPTG (Isopropyl β-d-l-thiogalactopyranoside, Sigma, lot#I6758).
10

Nickel NTA Agarose Beads (GoldBio, lot#H-350).
Anti-H3K4me1 antibody (Abcam, lot#ab8895), Anti-H3K4me2 antibody (Abcam, lot#ab7766), Anti-
H3K4me3 antibody (Abcam, lot#ab8580), Rabbit anti-mouse IgG H&L (HRP) antibody (Abcam,
lot#ab6728).
Protein Expression and Purification
ASH2L (1–534), RbBP5 (1–538), WDR5 (23–334), and DPY30 (1–99), and substrates NCP [(nuclear
core proteins, H3, H4, H2A, H2B)] were expressed and purified as previously described and were
obtained from lab stock from lab members Zi, Dr. Liang, and Dr. Ayoub.
[28]
All LB culture and LB agar plates described below contained 5μg/mL kanamycin as antibiotic selection.
E. coli strain BL21(DE3) transformed by expression plasmids was cultured in 5 mL LB pre-culture after
picking up from LB agar plate. The pre-culture was inoculated into the main culture (400 mL for each
mutant, 2000 mL for 3813IL), grown to 0.4-0.5 OD 600, and cooled to 20℃ for roughly 1 hour. After the
main culture was cooled down, protein expression was induced by adding 0.4mM IPTG and shaking
overnight at 100 rpm and 20℃.
The column for affinity chromatography purification was prepared by adding optimal volume of Ni-NTA
beads into a column of desired size and washed by water of 10 beads volume twice and by lysis buffer of
10 beads volume three times.
The induced culture was centrifuged at 4000 rpm, 4℃ for 15 min. Pellet was resuspended in lysis buffer
(BC500, 20mM Tris, 500mM NaCl, 5mM imidazole, 10% v/v glycerol, 2 mM β-mercaptoethanol, pH 7.4
at room temperature) in the ratio of 15mL per 400 mL culture and subjected to sonication (30s on, 30s
off, power 35%, total 20 min) until the solution became semi-transparent. The lysate was ultracentrifuged
at 22000 ×g for 20 min at 4℃, and the supernatant was run through a pre-casted Ni-NTA column twice.
The column was washed by wash buffer (BC500, 20mM Tris, 500mM NaCl, 20mM imidazole, 10% v/v
glycerol, 2 mM β-mercaptoethanol, pH 7.4 at room temperature) of 10 beads volume for three times and
11

eluted by 5 fractions of elution buffer (BC500, 20mM Tris, 500mM NaCl, 250 mM imidazole, 10% v/v
glycerol, 2 mM β-mercaptoethanol, pH 7.4 at room temperature) of 1 colume volume. The eluted protein
was dialyzed and digested by ULP1 simultaneously at 4°C overnight to remove imidazole and the SUMO
tag. The cleaved protein was further purified by collecting flowthrough from the Ni-NTA column again.
The beads could be washed by wash buffer to elute the desired purified proteins still bound to the beads if
more proteins were needed.
In vitro HMT assay
Enzyme Assembly
S3786C, R3789C, and R3789H enzyme complexes were assembled by combing core components to a
final concentration of 3 μM ASH2L, 3 μM WDR5, 3 μM RbBP5, 6 μM DPY30, and 3μM of respective
SET mutant domains in HMT buffer. R3844Q, V3865I, R3889Q, R3906H, R3968W, and R3968L
enzyme complexes were assembled by combing the core complexes to a final concentration of 3 μM
ASH2L, 3 μM RbBP5, 6 μM DPY30, and 3μM of respective SET mutant domains in HMT buffer. The
HMT buffer was 20 mM Tris-HCl , 50 mM NaCl, 1 mM DTT, 5 mM MgCl 2, and 10% v/v glycerol, pH
8.0.
In vitro HMT assay and western blot detection
Assembled recombinant 0.3 μM enzymes were treated with 0.6 μM NCP (Nuclear Core Particles) and
100 μM SAM (S-adenosyl-L-methionine) in a total volume of 20 μL HMT buffer for 1h at room
temperature. Reactions were quenched by adding 20 μL 2×SDS loading buffer (4% m/v SDS, 20% v/v
glycerol, 0.004% m/v bromophenol blue, 0.125M Tris-HCl, 10% DTT) and heating at 95℃ for 5 min.
Quenched mixtures were run on 12% SDS page gels and transferred to PVDF membranes (Bio-Rad,
cat#1620177). Western blot was performed using antibodies for H3K4me3 (1:10000 in 5% milk in
TBST), antibodies for H3K4me2 (1:10000 in 5% milk in TBST), antibodies for H3K4me1 (1:10000 in
5% milk in TBST), and antibodies for H4 (1:20000 in 5% milk in TBST) to incubate overnight at 4℃
12

while shaking. The blots were then incubated by rabbit IgG-HRP secondary antibodies (1:2000) for 45
min. The membranes were then developed by ECL (Bio-Rad, cat#170-5061) and visualized by Bio-Rad
ChemDoc™ XRS+ molecular imager®.
In vitro radioactive HMT assay
Assembled recombinant 0.6 μM enzymes were treated with 0.5μM NCP (Nuclear Core Particles) and 100
μM SAM in a total volume of 15 μL HMT buffer and incubated on shaker at 1000 rpm for 1h at 25℃.
Reactions were quenched by adding 15 μL 2×SDS loading buffer (4% m/v SDS, 20% v/v glycerol,
0.004% m/v bromophenol blue, 0.125M Tris-HCl, 10% DTT) and heating at 95℃ for 5 min. Quenched
mixtures were run on 12% SDS page gel. The gel was stained by Coomassie blue staining buffer (30%
v/v MeOH, 10% v/v acetic acid, 60% v/v ddH 2O, 0.04% m/v Coomassie blue) and de-stained by de-
staining buffer (30% v/v MeOH, 10% v/v acetic acid, 60% v/v ddH 2O) several times until the bands were
clear to observe. Pictures of the gel were taken, and the gel was applied to Amersham™ Amplifier (Cat#
17167543) for 1 hour followed by vacuum gel drying. The dried gel was exposed to an X-ray film in an
autoradiography cassette in -80℃ for at least 1 week. The X-ray film was developed in an X-ray
developer.

13

Chapter 3: Results
Recurrent missense mutations in cancer
Upon investigating the COSMIC Database, multiple somatic KMT2A mutations were reported in various
types of cancers, including skin cancer, lung cancer, stomach cancer, etc. Most KMT2A mutations did not
fall in any known functional domains and had relatively low frequency with unknow significance. In
contrast, mutations in the well-defined secondary structures were intriguing, because of potential
functional impacts.
Table 3 Distribution of somatic mutations on KMT2A and its domains
Number of KMT2A
missense mutations
Frequent Cancer
Tissues
Number of KMT2A-SET
domain missense
mutations
Frequent Cancer Tissues
1589 Meninges 121 Small Intestine
Skin Large Intestine
Urinary Tract Endometrium
Stomach Skin
Number of KMT2A-PHD
domain missense
mutations
Frequent Cancer
Tissues
Number of KMT2A-
Bromo domain missense
mutations
Frequent Cancer Tissues
140 Cervix 78 Skin
Urinary Endometrium
Skin Salivary Gland
Endometrium Upper Aerodigestive
Tract
Number of KMT2A-
FYRN domain missense
mutations
Frequent Cancer
Tissues
Number of KMT2A-
FYRC domain missense
mutations
Frequent Cancer Tissues
54 Skin 105 Small Intestine
Endometrium Endometrium
Stomach Skin
Urinary Tract Large Intestine

As stated in Table 3Table 3, 152 somatic mutations were reported in the SET domain of KMT2A.
Among them, 121 of which were missense mutations. Missense mutations are often highly correlated
with or even drive various cancers,

which drew our attention.
[40]
Analyzing COSMIC database helped us
14

reveal ten recurrent mutations in the KMT2A SET domain that had frequency of greater or equal to three,
namely S3786C, R3789H, R3789C, R3844Q, V3865I, R3889Q, E3906H, A3964T, R3968W, and
R3968L (Figure 6).

Among these mutations, S3786C, R3789C, and R3789H were in the N-flanking region of the SET
domain, which was essential for maintaining the domain architecture but away from the catalytic center.
[39]
R3844Q and V3865I were located at the SET-N region. R3889Q and R3906H are in the SET-I and
SET-C regions, respectively. A3964T, R3968W, and R3968L are in the post-SET region. We decided to
perform a systematic analysis on these ten recurrent SET mutations for their impact on the
methyltransferase activity.
Additionally, we conducted a thorough analysis of the selected mutations among other KMT2
methyltransferases. The results were summarized in Table 4Table 4. For R3844 (KMT2A), mutations of
R2587Q (KMT2B, once) and R5454Q (KMT2D, five times) was reported. No counterparts of V3865
(KMT2A) were reported mutated in cancer. For R3886 (KMT2A), mutations of R4828H (KMT2C, three
times), R4828C (KMT2C, 3 times), R1626Q (KMT2F, once) and R1885W (KMT2G, once) were
reported. For R3906 (KMT2A), mutations of R2649H (KMT2B, once), R4845K/T/M (KMT2C, once,
Figure 6 a) Number of reoccurrence of mutations on KMT2A SET domain. (R3789H, R3789C: 5; R3889Q,
R3988W: 4; V3865I, A3964T, S3786C, R3844Q, R3906H, R3968L: 3) b) Distribution of the mutations on the
SET domain.
15

respectively), R5741M (KMT2D, once), and R1885W (KMT2G, once) were reported. For A3964T
(KMT2A), mutations of A2707T (KMT2B, once) and A5529V (KMT2D, once) were reported. For
R3968 (KMT2A), mutations of R2712Q (KMT2B, three times), R4907W (KMT2C, twice), R4907Q
(KMT2C, three times), R5533Q (KMT2D, twice) and R1962Q (KMT2G, once) were reported.

Table 4 Mutations on the conserved residues among the KMT2 methyltransferases. (Mut. Resi.: Mutation
residue, Num.: mutation number.)

Mut. Resi. Num. Mut. Resi. Num. Mut. Resi. Num. Mut. Resi. Num.
SET_KMT2A S3786C 3 R3789H/C 5 R3844Q 3 V3865I 3
SET_KMT2B R2587Q 1
SET_KMT2C
SET_KMT2D R5454Q 5
SET_KMT2F
SET_KMT2G

Mut. Resi. Num. Mut. Resi. Num. Mut. Resi. Num Mut. Resi. Num.
SET_KMT2A R3886Q 4 R3906H 3 A3964T 4 R3968W/L 3/5
SET_KMT2B R2649H 3 A2707T 1 R2712Q 3
SET_KMT2C R4828H/C 3 R4845K/T/M 1 R4907W/Q 2/3
SET_KMT2D R5741M 2 A5529V 1 R5533Q 2
SET_KMT2F R1626Q 1
SET_KMT2G R1885W 3 R1902C 2 R1962Q 1
16

Preliminary Results
Mutations were successfully introduced into the plasmid via mutagenesis, and the blast results were
demonstrated in Figure 7. Point mutations were labelled red.

Figure 7 Mutagenesis Results
17

The proteins (wild type and mutants) were expressed and purified according to the methods. The
Coomassie staining of the purified proteins were demonstrated in Figure 8Figure 8.

Mutations in the conserved substrate-interacting region
To evaluate whether ten selected mutations were pathogenetic or just passenger mutations, we first
considered the potential changes in enzymatic function. KMT2A mainly methylates Histone 3 at lysine 4
methylation.

Therefore, we hypothesized that mutations on the KMT2A-SET domain may affect histone
methylation. We first examined the crystal structure using PyMol® to better understand the three-
dimensional structure of the mutations within the SET domain and whether they are involved in the
interaction with the substrates.
[41]
By comparing the sequence alignment (Figure 4) and crystal structure
of the SET domain (Figure 9, pdb: 2w5z), we identified the conserved region in SET domain that are
Figure 8 Coomassie staining of the purified proteins
18

involved in substrate interactions.

Although there were slight differences between the structure of the SET domain interacting with histone
H3, they largely overlaid well with each other. Among the mutations examined, V3865I, R3889Q,
R3906H, R3844Q are located on the “conserved” region as shown in Figure 9a, while A3964T,
R3968W, and R3968L are located on the post-SET region surrounding the Zinc ion. S3786C, R3789H.
and R3789C were located away from the substrate binding pocket, likely not involved in direct substrate
interactions.
As for the residues among the KMT2 family, we conducted similar protein alignment analysis and the
results were summarized in Table 5. Mutations on S3786 and R3789 residues were in the N-flanking
region of KMT2A and conserved in KMT2A and KMT2B. V3865 was conserved in KMT2A and
KMT2B. A3964 was conserved in KMT2A, KMT2B, KMT2C and KMT2D. R3886, R3906 and R3968
were conserved in all KMT2 methyltransferases.
Figure 9 Crystal Structure Overview a) Crystal structure of SET domain bound to H3 and co-factor
SAM. Evolutional Conservative region in orange, other part in semi-transparent cyan. b) Crystal
structure of SET domain bound to H3 and co-factor SAM. The MLL-SET domain is shown in light-
blue, mutations in red, Histone 3 in pink, and SAM in yellow.
19

Table 5 Protein alignment of the KMT2 SET domain.

S3786 R3789

SET_KMT2A -------------NPHGSARAEVHLRKSAFDMF---NFLASK---HRQPPEY-------- 33
SET_KMT2B ---------EPPLNPHGAARAEVYLRKCTFDMF---NFLASQ---HRVLPEG-------- 37
SET_KMT2C ----------LAVNPTGCARSEPKMSAHVKRFVLRPHTLNSTS----------------- 33
SET_KMT2D YGRHPLMELPLMINPTGCARSEPKILTHYK----RPHTLNSTS----------------- 39
SET_KMT2F ---------------TGSARSEGYYPISKKEKD---KYLDVCPVSARQLE----G----V 34
SET_KMT2G ----------------------GFYTIDKKDKL---RYLNSSRASTDEPPADTQGMSIPA 35
:
------------------------------------------------------------
R3844 V3865
SET_KMT2A RFRHLKKTSKEAVGVYRSPIHGRGLFCKRNIDAGEMVIEYAGNVIRSIQTDKREKYYDSK 120
SET_KMT2B RFRHLKKTSKEAVGVYRSAIHGRGLFCKRNIDAGEMVIEYSGIVIRSVLTDKREKFYDGK 65
SET_KMT2C QYRKMKTEWKSNVYLARSRIQGLGLYAARDIEKHTMVIEYIGTIIRNEVANRKEKLYESQ 85
SET_KMT2D QYRRLRTEWKNNVYLARSRIQGLGLYAAKDLEKHTMVIEYIGTIIRNEVANRREKIYEEQ 49
SET_KMT2F KLNQL-KFRKKKLRFGRSRIHEWGLFAMEPIAADEMVIEYVGQNIRQMVADMREKRYVQE 50
SET_KMT2G KFNQL-KFRKKKLKFCKSHIHDWGLFAMEPIAADEMVIEYVGQNIRQVIADMREKRYEDE 43
: .:: . *. : . :* *: **:. . : ***** * **. :: :** * :
R3889 R3906
SET_KMT2A GIGC-YMFRIDDSEVVDATMHGNAARFINHSCEPNCYSRVINIDGQKHIVIFAMRKIYRG 179
SET_KMT2B GIGC-YMFRMDDFDVVDATMHGNAARFINHSCEPNCFSRVIHVEGQKHIVIFALRRILRG 124
SET_KMT2C NRGV-YMFRMDNDHVIDATLTGGPARYINHSCAPNCVAEVVTFERGHKIIISSSRRIQKG 144
SET_KMT2D NRGI-YMFRINNEHVIDATLTGGPARYINHSCAPNCVAEVVTFDKEDKIIIISSRRIPKG 108
SET_KMT2F GIGSSYLFRVDHDTIIDATKCGNLARFINHCCTPNCYAKVITIESQKKIVIYSKQPIGVD 110
SET_KMT2G GIGSSYMFRVDHDTIIDATKCGNFARFINHSCNPNCYAKVITVESQKKIVIYSKQHINVN 103
. * *:**::. ::*** *. **:***.* *** :.*: .: .:*:* : : * .
A3964 R3968
SET_KMT2A EELTYDYKFPIEDASNKLPCNCGAKKCRKFLN 211
SET_KMT2B EELTYDYKFPIEDASNKLPCNCGAKRCRRFLN 156
SET_KMT2C EELCYDYKFDFEDDQHKIPCHCGAVNCRKWMN 176
SET_KMT2D EELTYDYQFDFEDDQHKIPCHCGAWNCRKWMN 140
SET_KMT2F EEITYDYKFPLED--NKIPCLCGTESCRGSLN 140
SET_KMT2G EEITYDYKFPIED--VKIPCLCGSENCRGTLN 133
**: ***:* :** *:** **: ** :*

20

KMT2A-SET mutations drastically reduced the methyltransferase activity
We first examined the methyltransferase activity for KMT2A-wild type SET 3813
IL
protein using
radioactive-fluorography Histonemethyltransferase (HMT) assay. As shown in Figure 10a, the
methyltransferase activity of the R3844Q mutant remained as robust as the control while the
methyltransferase activities of mutants of R3789H, R3789C, R3889Q, R3968W, V3865I, A3964T,
S3786C, R3906H, and R3968L were completely abolished.

KMT2A-SET mutants have differential effects on enzyme processivity
According to the results from the last session, majority of the mutations in KMT2A-SET reduced the
methyltransferase activity on histone H3. We then investigated how the mutations affect KMT2A
processivity of methylation. Previous report suggested that KMT2A is a non-processive enzyme,
requiring capture and release of H3K4 for each methylation step. Therefore, it is intriguing and important
to investigate whether our selected mutations change the processivity.
[41]
Similar HMT assay based on
western blot were conducted for H3K4me3, H3K4me2, and H3K4me1.

Figure 10 Most mutations on KMT2A SET domain has changed the methyltransferase activity. a)
Recombinant KMT2A SET with core components were treated with NCP and
3
H-SAM, and analyzed by SDS-
PAGE, Coomassie Blue staining, exposed to X-ray film in -80℃ for 7 days, and developed. b) Quantification
of the fluorograph in 8a.
21

The western blot results was presented in Figure 11a, and quantified by ImageJ® as in Figure 11c, d, e.
[42]
We observed and identified some pattern of the processivity of different mutants. R3789H, R3789C,
and R3968L mutants lost more than 90% signal in tri-, di-, and mono-methylation suggesting that the
mutations affect total SET activities regardless of methylation level.

Figure 11 HMT result of the wild type (MLL3813IL-SET) and 10 mutations. a) Western blot of HMT
reaction of different SET mutations and W.T. probed to antibodies specific to H3K4me3, H3K4me2,
H3K4me1, and H4. b) Coomassie Blue Stain of assembled complex, from top to bottom: PbBP5,
ASH2L, WDR5, SET and DPY30 c, d, e) Quantification of the western blot result stated in a).

22

R3889Q, V3865I, A3964T, R3968W and S3786C mutants show robust signals in mono-methylation but
relatively low di-methylation and tri-methylation signal (Figure 11a: 5-9, summarized in Figure 12). The
results suggest these mutations decrease the di-methylating and tri-methylating activity, therefore halting
the methyltransferase activity in the first monomethylating step, affecting processivity of the enzyme.

In the contrast, R3906H showed relatively strong di-methylation signal, while low tri-methylation and
mono-methylation signal, suggesting that the mutation mainly affects di-methylation step (Figure 13).
Figure 13 a) H3K4 Processivity graph of R3889Q, V3865I, A3964T, R3968W and Position
S3786C. b) and distribution of the five mutants
Figure 12 a) H3K4 Processivity graph of R3789H, R3789C and R3968L. b) Position and
distribution of the three mutants
23

R3906H is also evolutional conserved, and it locates on an alpha helix in SET-C region. Previous results
from Weirich et al. showed the R3906H mutant's linkages with the RbBP5, ASH2L and WDR5 are
unaffected, but the author then argued that such substitution of arginine for histidine at the interface may
modify the conformation of this crucial area, resulting in activity loss.

R3844Q, which was reported in three independent studies on large intestinal cancer, is located on at the
adenosyl methionine binding pocket. Antibody signals (Figure 11a: panel 1,10) show this mutation,
however, does not affect any methylation activity, performing similar to wide type KMT2A-SET.
Figure 14 a) H3K4 Processivity graph of R3906H. b) Position and distribution of the mutant.
Figure 15 a) H3K4 Processivity graph of R3844Q. b) Position and distribution of the mutant.

24

Chapter 4: Discussion
The KMT2 family plays a role in regulating genome accessibility and transcription by facilitating overall
H3K4 methylation. The members differ in terms of genome-wide localization and specificity. KMT2A
and KMT2B bind to de novo enhancer sites and catalyze dimethylation and trimethylation of H3K4 at
specific sites, while ChIP data showed KMT2A is only responsible for around 5% of global H3K4
trimethylation.
[13], [43]
KMT2F and KMT2G, on the other hand, are the predominant H3K4
methyltransferases responsible for global H3K4me3 levels.
[44]
H3K4 monomethylation at enhancers is
mediated by KMT2C and KMT2D.
[45]
Functions of KMT2C are found to be partially redundant with that
of KMT2D.
[46]
KMT2A’s functional redundancy among the KMT2 family makes it interesting to
investigate its unique role in the H3K4 methylation.
The function of KMT2A has been extensively studied in leukemia. About 10 percent of acute leukemia
has KMT2A translocation, which is associated with extremely poor prognoses. KMT2A is also reported
in a variety of cancers beyond mixed lineage leukemia. For example, KMT2A has been shown to be
essential for pancreatic tumor growth, salivary gland tumor growth and intestinal tumorigenesis.
[47], [48],
[49]

However, whether the KMT2A-SET domain plays an obligatory role in tumorigenesis remains unclear.
Weirich et al. previously investigated four cancer-derived KMT2A-SET mutations. The authors as well
utilized the COSMIC database seeking potential candidates. However, the selection of Weirich et al. was
based on their positions relative to the substrate-binding pockets. In comparison, we have taken an
unbiased approach and analyzed all recurrent KMT2A-SET mutations as curated by COSMIC. Our
results on R3906H (which is equivalent to R3903H in isoform 2) are consistent with that reported by
Weirich et al.
[49]
We further investigate the recurrent mutations that have not been reported previously.
25

Mutation residue analysis
R3789 and S3786 locate in the N-flanking region of the SET domain, which is away from the catalytic
center. Both S3786C and R3789C introduce the cysteine with a sulfhydryl group, which may change the
protein folding and thus interfere with protein-protein interactions.
[50]
R3789H substitute an arginine to a
histidine, which may result in the total rise of pH and can implement cancer cell behavior.
[51]
R3789 and
S3786 are all evolutionally conserved residues. However, they are only conserved in KMT2A and
KMT2B. There are no mutations reported on their counterparts in the family. The uniqueness makes it
more intriguing to study their role in tumorigenesis further. While it remains to be investigated, we
hypothesize that these two mutations may change SET domain interaction with other core components.
Experiments such as GST pull-down assay can be designed to investigate whether the mutants change the
interaction.
V3865, R3886 and R3906 are only conserved in vertebrates. Despite separation in the linear sequence, all
reside around the catalytic center in the three-dimension structure. Their positions suggest that their loss-
of-function may directly result from interference with catalysis. R3889 and R3906 are conserved among
the KMT2 family, and there are plenty of mutations reported on their counterparts in the family (Table
4). R3889Q mutation change the positive charged arginine to non-charged glutamine, so we can
hypothesize that loss of negative charge could contribute to the loss of methyltransferase activity.
R3906H changed the arginine to a histidine which contains an imidazole group. Therefore, we may
hypothesize the mutation can bring conformational change that leads to the loss of activity. Future
researchers can introduce the mutations in Table 4 to the respective residues and conduct experiments to
investigate the impact of those mutations and the role of those residues. In contrast, V3865 is only
conserved in KMT2A and KMT2B; and there are no mutations reported in its counterpart in KMT2B.
Additionally, valine to isoleucine is a trivial mutation compared to other mutations we chose. Therefore,
the V3865I is interesting for future investigation.
26

A3964 and R3968 are evolutionarily conserved. They are in the post-SET region which regulate a zinc
atom.
[41]
A3964 is conserved in KMT2A, B, C and D, and R3968 is conserved in all KMT2
methyltransferases. There are mutations on their counterparts among the family reported. R3968W and
R3968L also result in the change in the charge of the side chain, we may also suggest the change in
charge leads to loss of activity. Additionally, the interaction with the zinc ion might also be altered and
can lead the loss of activity.
Summary
KMT2A is frequently reported in various cancers, and it is functionally redundant in global H3K4
trimethylation. Therefore, it is interesting to investigate the potentially unique role that KMT2A plays in
tumorigenesis. Our project conducts an unbiased analysis of the cancer-recurrent mutations on KMT2A to
investigate the impact of the cancer-recurrent mutation and in search of potential therapeutic targets.
Among the residues of the mutations we select, S3786, R3789, R3844, and V3865 are conserved in
KMT2A and KMT2B. A3964 is conserved in KMT2A, KMT2B, KMT2C, and KMT2D. R3889, R3906,
and R3968 are conserved in all KMT2 methyltransferases. (Table 5) There are no mutations reported on
the counterparts of the S3786, R3789, and A3964, while mutations on counterparts of other residues have
been widely reported (Table 4). Future investigations can focus on unique mutations such as S3786C,
R3789H, R3789C and A3964T. With the update of the database, researchers can also focus on the non-
conserved residue (among the KMT2 family) and mutations with fewer counterparts in other KMT2
members.
The main method we utilize is in vitro histone methyltransferase assay. It is a well-established method for
qualification or quantification of the methyltransferase activity based on the detection method. Tritium
can provide us with good quantification of the methyltransferase activity. In contrast, antibody-based
western blot provides less quantified results, yet specific antibodies can monitor three-step methylation,
which reflects the processivity of the KMT2A enzyme.
27

Our data points out that most of the chosen mutants experienced a loss of methyltransferase activity. They
also show distinct effects on the processivity of H3K4 methylation. Though no gain-of-function
mutations are discovered, our results show the overall decrease in methyltransferase activity of the most
recurrent missense cancer mutations on the KMT2A-SET domain. Taken together with frameshift and
nonsense mutations in KMT2A, a significant subset of KMT2A mutations lose its methyltransferase
activity. Taken together the functional redundancy of KMT2A in global H3K4 trimethylation, our results
strongly argue that KMT2A methyltransferase activity plays an important role in tumorigenesis. Future
studies are needed to inspect the potential role of the mutations and their relevance in cancer using
cellular and in vivo models.

28

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Abstract (if available)

Abstract KMT2A (lysine methyltransferase 2A, also known as MLL1, ALL1) tri-methylates histone 3 at lysine 4 (H3K4) to regulate gene expression. KMT2A locates on chromosome 11q23, and 10% of human leukemia patients were reported with translocation of the gene. According to the COSMIC database, KMT2A is also one of the most mutated genes in various cancers. KMT2A mutations include nonsense mutations, missense mutations, frameshift mutations, and so on. Among them, missense mutations are widely distributed throughout the gene, including the C-terminus catalytic SET domain. Somatic mutations in KMT2A-SET domain are mainly found in small intestinal cancer, large intestinal cancer, endometrial cancer. Interestingly, 75 percent of the SET domain mutations are missense mutations, raising the question of whether these discrete mutations impact KMT2 methyltransferase activity.
In this study, we systematically investigate 10 missense mutations in the KMT2A SET domain that are recurrent in different human cancers. Here we show that the R3844Q mutant retains the same enzymatic activity on histone methylation, while the activity of R3789H, R3789C, R3889Q, R3968W, V3865I, A1964T, S3786C, R3906H, and R398L drops drastically. We conduct enzymatic activity analyses on KMT2A-SET mutants and hopefully could contribute to the investigation of the role that KMT2A and its mutations play in cancer.

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Asset Metadata

Creator Bai, Tianyang (author)

Core Title Cancer-recurrent missense mutations in SET domain alter KMT2A methyltransferase activity

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2022-08

Publication Date 07/06/2022

Defense Date 04/25/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag H3K4,H3K4me1.,H3K4me2,H3K4me3,HMT assay,KMT2A,lysine methyltransferase,OAI-PMH Harvest,SET domain

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Dou, Yali (committee chair), Bell, Oliver (committee member), Rice, Judd (committee member)

Creator Email bait@usc.edu,baitianyang327@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111369228

Unique identifier UC111369228

Legacy Identifier etd-BaiTianyan-10810

Document Type Thesis

Format application/pdf (imt)

Rights Bai, Tianyang

Type texts

Source 20220708-usctheses-batch-951 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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