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Structural and biochemical studies of a MEF2 cancer mutant

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Structural and biochemical studies of a MEF2 cancer mutant

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Content STRUCTURAL AND BIOCHEMICAL STUDIES OF A MEF2 CANCER
MUTANT

by

Xiao Lei

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY

(Genetics, Molecular and Cell Biology)

Aug 2017

Copyright 2017 Xiao Lei

EPIGRAPH

故不积跬步，无以至千里；不积小流，无以成江海。骐骥一跃，不能十步
；驽马十驾，功在不舍。
- 荀子

English Translation:

“Unless steps and half steps are accumulated, no one can cover a thousand li,
unless little streams are accumulated, no rivers and seas can be formed. Even a
thorough-bred horse cannot cover ten paces by one leap; yet a worn-out old nag
can cover the distance of a ten-day journey, and it is all a matter of
perseverance.”
-Xun Zi (Translated by Yi-Pao Mei)

2

Dedication

To my family: love you Mom and Dad

3

Acknowledgement

I owe many thanks and gratitude to a lot people in my journey of graduate school.
First, I appreciate my advisor Dr. Lin Chen’s great effort in the mentorship during my
graduate life. Dr. Chen shows very good quality of an independent scientists in many aspects
which influence me deeply. Dr. Chen’s office door is open most of the time when he is at
school, and he is always willing to spend time with me to analyze experiment results and
provide his opinions and suggestions in my projects. He offers me freedom to try my own
thoughts and patiently watch me grow all these years. Under his guidance, I learn not only
how to solve a crystal structure, but also how to catch the main research point and think
critically in my research.
Second, I express my gratitude to my committee members Dr. Peter Qin and Dr. Matt
Pratt. Dr. Qin and Dr. Matt are always willing to offer help from their expertise and give me
valuable advices to in my annual research appraisal meetings (ARA) every year. They always
give positive words and views to me after my ARA meeting; these words are a great
motivation to me in a lot of desperate times when everything in research does not work out for
me.
In addition, I would thank a lot of friends and colleagues: Dr. Jiang Xu, Dr. Yi Kou ,
Dr. Yongqing Wu and Dr. Kaori Noridomi from Dr. Lin Chen’s lab; Dr. Yang Fu, Dr. Xiao
Xiao and Dr. Hanjing Yang from Dr. Xiaojiang Chen’s lab; Dr. Xiaojun Zhang, Dr. Yuan
Ding and Wei Jiang from Dr. Peter Qin lab; Dr. Carolina Dantas, Dr. Tsu-pei Chiu, and
Beibei Xing from Dr. Remo Rohs lab ；Dr. Varuzhan Balasanyan, Dr. Garrett Gross from Dr.
Don Arnold’s lab, Dr. Dai Chao from Dr. Xianghong Jasmine Zhou’s lab. They are very nice
4

person to talk with when I feel depressed in research, and we helped each other throughout the
years in graduate school. I would also like to thank Dr. Xiaojiang Chen and Dr. John Petruska
in Molecular and Computational Biology department (MCB) for their willing to share their
scientific visions and ideas to me. I would like to thank Dr. Shuxing-Li from USC
Nanophysics Core Facility Center to help when I need to use facility instruments. I would like
to thank Kathleen Boeck, Laura Cajero and Rokas Oginskis from MCB office to help me
when I need to ship my crystal dewars, order research supplies and call for lab repairs.
I would like to thank my housemate Dr. Chao Dai. We are very good friends and talk
about everything in life. We explore yummy food in Los Angeles and drink alcohol a lot, this
often takes me out of research depression temporarily. I would like to thank my girlfriend
Diana Dai, who always believe in me and encourage me to stay the course in graduate school.
I appreciate my homestay landlord, Mr. Bernard Steppes and Mrs. Brenda Steppes, who live
with me for almost 6 years for cooking nice food every diner and keeping my room clean. Mr.
Bernard and Mrs. Brenda’s optimistic view of life during their retired years and their art of
maintaining a healthy and great marriage for 40 years influence me a lot.
Last but not least, I would like to thank my parents and my grandparents for their
unconditional love. A lot of times my father asks if I make any research progress in our phone
calls; this often makes me nervous because I know he is a quick temper man and I gave him
“so so” answer every time. Unexpectedly, the tone from his side never sounds angry or
impatient. My father and mother show great understanding and support in my graduate life,
and I would always appreciate their love for me.

5

Table of Contents

EPIGRAPH 2
Dedication 3
Acknowledgement 4
Table of Contents 6
List of Figures 8
List of Tables 11
Abstract 12
Chapter 1: Introduction 13
1.1 MEF2: The MADS box family of transcription factors 13
Chapter 2: Crystal Structure of MEF2 D83V Mutant 16
2.1 Introduction 16
MEF2 family members in human 16
Crystal structure of MEF2 protein bound to DNA 17
Crystal structures of MEF2 protein bound to cofactors 20
The role of MEF2 transcription factors in lymphocyte development and cancer 23
2.2 Results 25
Chim WT and Chim D83V crystals and their X-ray diffraction 25
D83V mutation switches helix H3 conformation to strand conformation. 27
Comparison of the cofactor binding groove between Chim D83V and MEF2B WT 29
The C-terminal of Chim D83V interacts with its counterpart in crystal packing through
beta twist interaction 30
Structural mechanisms leading to the deformation of C-terminal helix H3 by the D83V
mutation 34
2.2 Discussion 35
D83V may create a new protein-protein interaction interface 35
Other mutations in DLBCL which are likely to disrupt MEF2B helix H3 36
The possibility of MEF2B D83V protein aggregation 37
Possible mechanisms for the weakened DNA binding affinity by the MEF2B D83V
mutant 37
2.3 Methods 38
Protein purification 38
DNA Purification 39
6

Crystallization and Structure Determination 40
Dynamic Light Scattering Study 45
Multiangle light scattering study 45
Electrophoresis mobility shift assay (EMSA) 45
Chapter 3: Structure of MEF2 and NKX2.5 complex reveals novel cofactor interaction
interfaces from MEF2 46
3.1 Introduction 46
The interaction between MEF2 and NKX2.5 plays important roles in lymphoma 46
Domain structure of NKX 2.5 transcription factors 47
NKX2.5’s role in cardiac muscle development 49
NKX2.5’s role in cancer 51
NKX2.5’s repressor role 52
3.2 Results 52
MEF2 and NKX2.5 binds DNA cooperatively 52
Crystallization and Diffraction of MEF2/NKX2.5/DNA crystals 53
Overall Structure of the MEF2/NKX2.5/DNA ternary complex 54
Protein and DNA interactions 59
Potential Protein-Protein Interfaces from Crystal Packing Analyses 62
3.2 Discussion 69
Speculative chromosome looping model mediated by MEF2 and NKX2.5 69
Minor groove DNA recognition by the MEF2 C terminal helix 70
Cancer mutations in protein-protein interaction interface 71
3.3 Methods 72
Protein expression and purification 72
DNA Purification 72
Ternary complex Crystallization and structure determination 73
Electrophoresis mobility shift assay (EMSA) 74
Reference 76

7

List of Figures

Figure 2.1 Domain organization and sequence comparison of human MEF2 proteins............11
Figure 2.2 Structure of MEF2A MADS box domain bound to DNA.......................................18
Figure 2.3 MEF2 specific domain is necessary for Cabin1 binding.........................................19
Figure 2.4 MEF2A structure including MADS box and MEF2 specific domain.....................20
Figure 2.5 Structures of MEF2 with various cofactors.............................................................21
Figure 2.6 Structures of MEF2 with cofactors..........................................................................22
Figure 2.7 Regulation from MEF2 transcription through phosphorylation with HDACs....... 23
Figure 2.8 MEF2B mutation pattern in DLBCL...................................................................... 24
Figure 2.9 MEF2B mutation hotspots and their Co-IP test for Cabin1 binding...................... 25
Figure 2.10 MEF2 Chim WT and Chim D83V crystals and diffractions................................ 26
Figure 2.11 Sequence alignment of MEF2B D83V constructs to MEF2A and MEF2B......... 27
Figure 2.12 MEF2 Chim WT align with MEF2B WT and EMSA tests on the binding of
MEF2 Chim WT and MEF2 Chim D83V to HDAC4 .............................................................28
Figure 2.13 Electron density of the C-terminal region of MEF2 Chim D83V and structural
alignment of MEF2 Chim D83V to MEF2 Chim WT and MEF2B WT................................. 29
Figure 2.14 Comparison of the cofactor binding groove of MEF2B Chim D83V to Chim WT
and MEF2B WT........................................................................................................................30
Figure 2.15 Chim D83V strand S4 beta twist interaction in crystal packing............................31
Figure 2.16 DLS analysis of MEF2B WT, MEF2 Chim WT, and MEF2 Chim
D83V.............32
8

Figure 2.17 MALS measurements results.................................................................................33
Figure 2.18 Mutation and PTM hotspots on MEF2B and D83 local environment.................. 34
Figure 3.1 Domain scheme of NKX 2.5 and structure and homeodomain.............................. 48
Figure 3.2 NKX2.5 disease related mutations map.................................................................. 49
Figure 3.3 EMSA assay of MEF2B and NKX2.5 on myocardin gene enhancer..................... 53
Figure 3.4 MEF2/NKX2.5/DNA crystals and X-ray diffraction.............................................. 54
Figure 3.5 Asymmetric unit of the MEF2/NKX/DNA crystal................................................. 55
Figure 3.6 Overall structure of the MEF2/NKX2.5/DNA complex......................................... 56
Figure 3.7 Movement of H2-S3 loop in NCS related copies in ASU...................................... 57
Figure 3.8 Shifts of DNA and NKX2.5 in the copy 2 and copy 3 after MEF2 alignment...... 58
Figure 3.9 Shifts of MEF2 and DNA in three NCS related copies after NKX2.5 alignment...59
Figure 3.10 Interaction of the N termini of both MEF2 and NKX2.5 to DNA minor
groove...................................................................................................................................... 60
Figure 3.11 Cooperative DNA minor groove recognition by arginine residues from both N
terminals of MEF2 and NKX2.5..............................................................................................61
Figure 3.12 The C terminal of MEF2 interacts with the minor groove of DNA.....................62
Figure 3.13 MEF2 and NKX2.5 interaction interface I from crystal packing.........................63
Figure 3.14 Detailed interactions between MEF2 and NKX in interface I............................. 64
Figure 3.15 Potential MEF2 and NKX interaction interface II............................................... 65
Figure 3.16 Detailed interaction in MEF2 and NKX2.5 interface II...................................... 66
Figure 3.17 Detailed interaction in MEF2 and NKX2.5 interface II...................................... 67
Figure 3.18 MEF2 and NKX2.5 potential interaction interface and disease or PTM affected
9

residues mapped in the interface..............................................................................................68

10

List of Tables

Table 2.1 Data collection and refinement statistics for MEF2 Chim WT and DNA crystal.....42
Table 2.2 Data collection and refinement statistics for MEF2 Chim WT and DNA
crystal........................................................................................................................................44
Table 3.1 Detailed information on disease or PTM affected residues on MEF2 and NKX2.5
interaction interface ..................................................................................................................69
Table 3.2 Data processing and Refinement statistics of MEF2/NKX2.5/DNA complex.........74

11

Abstract
MEF2B gene is frequently mutated in lymphoma. Part I of my research focus on the
structural study of the MEF2B D83V, which is the most frequent MEF2B mutant in
lymphoma. The structure reveals a complete deformation of the C terminal helix in MEF2
specific domain by the D83V mutation. The deformation of the C terminal helix disrupts
cofactor binding to MEF2B and is likely to create a novel interface for unknown cofactors to
interact in the lymphomagenesis.
NKX2.5 is a potential MEF2 interaction partner in lymphomagenesis. Part II of my
research focus on the structural studies of two transcription cofactors: MEF2 and NKX2.5.
The structure reveals a novel MEF2 cofactor interaction interface, where many diseases
related mutations on MEF2 and NKX2.5 are located in the interface. The structure also
suggests the interaction between MEF2 and NKX2.5 is likely to be in a multi-protein
transcriptional machinery mediating long range chromosome interaction. As the synergy
between MEF2 and NKX2.5 is also well known to play a role in heart muscle regeneration,
the structure may help future studies to design therapeutic compounds to regeneration process.
treat lymphoma and cardiac disease.

12

Chapter 1: Introduction
1.1 MEF2: The MADS box family of transcription factors

The MADS box family of transcription factors are found in higher eukaryotic
taxonomy species, such as yeast, plant, insect and human [1] . They have a conserved MADS
box motif, which is responsible for protein dimerization and DNA binding. Myocyte enhancer
factor 2 is a member (MEF2) in the MADS box superfamily. In human, there are four genes
( Mef2a , Mef2b , Mef2c , Mef2d ) encoding four members of MEF2 protein. MEF2 members in
human have nearly identical MADS box and MEF2 specific domain at the N termini (residues
1-91) but have divergent transcription activation domain (TAD) at the C termini. MEF2
protein was initially found in skeletal and cardiac muscle cell, hence its name [2] , they bind to
(A/T) rich sequences at promoter or enhancer regions with consensus motif 5’ C/T TA (A/T) 4

TA G/A 3’ [3] . Early knockout studies indicate that MEF2A and MEF2C are needed for
cardiac morphogenesis, whereas MEF2B and MEF2D are redundant to some extent because
either null MEF2B or null MEF2D mice is viable with no obvious abnormalities [4] . Besides
the importance in skeletal and cardiac muscle development, MEF2 has been found to play
important roles in T cell apoptosis [5] and synapse remodeling [6,7] . MEF2’s roles in cancer
has also been reported. For instance, MEF2C is found to be over activated in T-acute
lymphoblastic leukemia cell lines (T-ALL) [8] . In another case, chromosome rearrangement
which makes reciprocal fusion protein DAZAP1/MEF2D and MEF2D/DAZAP1 is found in
TS-2 acute lymphoblastic leukemia [9] . Recent investigations show that MEF2B, which is
highly expressed in germinal center in lymph nodes, is frequently mutated in Diffuse Large
B-cell Lymphoma (DLBCL) patient [10–12] . The MEF2B mutation pattern shows that the
13

majority of mutations are non-synonymous substitutions rather than truncations and
frameshift mutations, which suggests that the MEF2B mutants may be positively selected
during cancer development. To date, all four members of MEF2 gene found in various cancers
show mostly amplification or non-synonymous substitution pattern than deletion or
truncations [13] . This fact further supports the hypothesis that MEF2 is positively selected as
a cancer driver mutation rather than passenger mutation.
Crystal structures show that MEF2 binds DNA as dimer; each monomer consists an N
terminal loop that inserts into the minor groove of DNA, helix H1, which sits above DNA,
strands S1 and S2, helix H2, strand S3 and C terminal helix H3. The strand S1, S2, S3 and
helix H2 from each monomer makes up a groove that is responsible for cofactor interaction
[14] . These MEF2 cofactors include co-repressors such as Cabin1, HDAC4 (Histone
deacetylase 4) and co-activators such as EP300 (Histone acetyltransferase p300) or FAK
(Focal adhesion kinase 1). The common interaction mode between MEF2 and these cofactors
is mediated by one helix from cofactors to the MEF2 hydrophobic binding groove through
hydrophobic and Van der Waals interactions [15] . Previous studies found that MEF2B D83V
was a frequent mutation in DLBCL. The MEF2B D83V mutant fails to bind to corepressor
Cabin1, leading to the upregulation of oncogene Bcl6 and proliferation of germinal center B
cells [16] . Bcl6 are expressed highly in germinal center (GC) B cells and it has been shown
that disruption of the Bcl6 gene results in an impaired germinal center formation and
abnormal B cells [17] . The deregulation of Bcl6 were reported in up to 40% of diffuse large
cell lymphomas and 10% of follicular lymphomas [18,19] . The critical role of BCL6 in
lymphomagenesis has been confirmed in a mouse model in which constitutive and aberrant
14

expression of Bcl6 promotes the development of human-like DLBCL [20] . In contrast with
the study that supports a general activator role of MEF2B D83V, another study found that
MEF2B D83V is in general decreasing target gene expression, including Bcl6 oncogene and
TGF-beta tumor suppressor gene expression. The authors propose that MEF2B D83V may
cause deficient recruitment of coactivator EP300, thus leading to global decrease transcription
of MEF2B target genes. Despite a general repressor role of MEF2B D83V from expression
array studies, increased expression target genes includes Myc oncogene [21] . The
rearrangement and deregulation of both Bcl6 and Myc in lymphoma has been reported before
[22] . Besides inconsistencies in the abovementioned two reports, one conclusion seems
consistent: MEF2B D83V could not interact with Cabin1. The main question my research
aims to address is how a surface mutation D83V on the helix H3 of MEF2 specific domain
could abrogate its ability to interact with Cabin1, as the affected region has not been shown to
involve in direct cofactor recruitment from all previous structural based studies.
In chapter 2, We report a crystal structure of MEF2B D83V mutant bound to MEF2
consensus DNA. The structure revealed a complete deformation of helix H3, and the
deformed helix H3 adopt a largely beta twist conformation.

15

Chapter 2: Crystal Structure of MEF2 D83V Mutant

Contributions: I performed all of the work presented in this dissertation with the
exceptions detailed here. Niroop Rajashekar did the EMSA assay. Niroop
Rajashekar and Haoran Shi helped crystallization condition optimization.
Dr. Yi Kou did the DLS experiment. Dr. Fu Yang did the luciferase assay.
Reused figures are under creative common license. Crystal structure figures
were generated by Pymol [23] . My advisor Dr. Lin Chen contributed part of
thesis writing. Dr. Lin Chen supervised the project.

2.1 Introduction

MEF2 family members in human
In human, there are four genes ( Mef2a , Mef2b , Mef2c , Mef2d ) encoding four members of the
MEF2 family of transcription factor. MEF2 members in human have nearly identical MADS
box domain at N-termini (residues 1-57) but have divergent transcription activation domain
(TAD) at C-termini. MEF2 Specific domain (residues 58-86) is located between MADS box
domain and TAD, which is responsible for cofactor binding (Figure 2.1) [14,24] .
16

Figure 2.1 Domain organization and sequence comparison of human MEF2 proteins
The amino acid numbering shown is of MEF2A and the percent sequence identities are all
relative to MEF2A. MADS box, MEF2 specific, and transactivation domain are each
highlighted in red, green and cyan respectively.

Crystal structure of MEF2 protein bound to DNA
In 2000, Eugenio Santelli and Timothy J. Richmond reported the first crystal structure
of a MEF2 transcription factor (Figure 2.2) [25] . The authors show that MEF2A bound to
DNA as a homodimer, with N terminal loop insert into the minor groove of DNA. The overall
protein shape is like a sandwich: two alpha helix H1 from each monomer sitting above DNA
backbone making up “bottom layer”, two beta sheet strand S1 and S2 from each layer making
up the middle part, and two alpha helix H2 on the top layer.
17

Figure 2.2 Structure of MEF2A MADS box domain bound to DNA
Figure adapted from (Santelli and Richmond, 2000).
Each MEF2 monomer is shown in green and blue respectively. DNA is shown in Orange.
Structure is in cartoon mode.

Eugenio Santelli and Timothy J. Richmond propose that the two antiparallel alpha
helices H2 on the top may form a cleft for cofactor interaction, however, experimental
evidence from our research group shows that MEF2 MADS box domain alone is not sufficient
for cofactor binding and MEF2 specific domain is needed for cofactor binding (Figure 2.3)
[14] .
18

Figure 2.3 MEF2 specific domain is necessary for Cabin1 binding
Figure reused from (Han et al., 2003)
EMSA assay testing the interaction between MEF2 and Cabin1 using both MEF2B L
(residues 1-93) and MEF2B S (residues 1-78) truncates.

Further structural study shows that the MEF2 specific domain folds with MADS box
domain in dimerization and DNA binding and the two domains could be considered as a
single domain (Figure 2.4) [24] . The helix H3 from one monomer is anchored to helix H1
from the other monomer by hydrophobic interactions: I84, V83 and L88 residues from helix
H3 interact with the hydrophobic pocket composed by F26, M29, Y33 and aliphatic chain of
K30. In the MEF2A structure, and the strand S3 from one monomer interacts with strand S2
from the other monomer. This structure shows a well-defined strand S3 and helix H3, which
contradicts to a previous proposition that S3 and H3 of MEF2 is unstructured based on NMR
studies [26] . Wu et al. also proposed that DNA may help helix H3 stabilization, as the
trajectory of H3 runs toward DNA.
19

Figure 2.4 MEF2A structure including MADS box and MEF2 specific domain
Figure adapted from [24]
( a) MEF2A structure. Each monomer chain is colored by green and cyan. ( b ) Detailed
hydrophobic interaction between H3(green) from one monomer to helix H1(blue) from the
other monomer in MEF2 dimer.

Crystal structures of MEF2 protein bound to cofactors
To date, a number of structures of MEF2 bound with cofactors show a conserved
binding mode: alpha helix from cofactors bound to the top hydrophobic cofactor binding
groove of MEF2 (Figure 2.5) [14,15,27,28] . These cofactors include co-repressors such as
Cabin1 and HDAC4 and co-activators such as EP300 and FAK.
20

Figure 2.5 Structures of MEF2 with various cofactors
Figure Adapted from (Han et al., 2005; Han et al., 2003; He et al., 2011;Cardoso et al., 2016)
(a) MEF2 structure with Cabin1 (PDB code: 1n6j). (b)MEF2 structure with HDAC9. (c)
MEF2 structure with EP300. (d) MEF2 structure with FAK
Each MEF2 monomer is in green and cyan color. Cabin1, HDAC9 and EP300 is in red. FAK
dimer is in grey and magenta.

The fact that these cofactors bind to the same hydrophobic groove on MEF2 (Figure
2.6) suggests that these factors may compete each other under certain physiological contexts.
Alternatively, within the same cell but in different genomic contexts, MEF2 could recruit
different cofactors to distinct genomic loci to repress or activate resective target genes. For
example, a model in muscle gene activation proposes that MEF2’s association with HDAC
causes chromosome deacetylation and gene repression in the resting state; under stimulated
conditions with activated kinase signaling pathways, HDAC could be phosphorylated by
21

calcium/calmodulin-dependent protein kinase (CaMK) or yet to be identified kinases and
released from MEF2; MEF2 then recruits EP300 and causes chromosome acetylation and
gene activation (Figure 2.7) [29,30] .

Figure 2.6 Structures of MEF2 with cofactors
Figure reused from (Cardoso et al., 2016)
Structure superposition by C α
of one MEF2 monomer. Cabin1 (blue), HDAC9 (red), p300
(purple) and FAK (grey) have common interaction interface with MEF2 hydrophobic groove.
Structure is shown in cartoon mode, each MEF2 monomer is in green and cyan.

22

Figure 2.7 Regulation from MEF2 transcription through phosphorylation with HDACs
Figure reused from (McKinsey et al., 2002)
MEF2 could recruit class II HDACs (HDAC4/5/7/9) or EP300 in muscle development
The role of MEF2 transcription factors in lymphocyte development and cancer
MEF2 has been known to play a role in T cell apoptosis and B cell proliferation.
During negative selection of T cells in thymus, MEF2 is released from Cabin 1's repression by
calcium signal and drives Nur77 expression, which induces T cell apoptosis [5] . MEF2 binds
the IgJ chain promoter and drives IgJ chain gene expression during early B cell differentiation
[31–33] . Even though MEF2C is known to function in muscle development, there is high
expression of MEF2C in mature B cells relative to that of other tissues, including heart and
skeletal tissues [34] . MEF2C promotes germinal center formation and B-cell proliferation in
response to BCR stimulation [34–36] . Dysregulations of MEF2C has been reported in many
leukemia cases [8,37–39] . MEF2B expression is high in germinal center and frequent MEF2B
mutations are found in lymphoma patient samples, especially in large B cell diffuse large
B-cell lymphoma (DLBCL) [10,11,40] . The mutation pattern of MEF2B in DLBCL features
frequent non-synonymous substitutions (Figure 2.8), rather than deletions and frameshift
23

mutations as in MLL2 and TP53 in DLBCL. This implies that MEF2B mutant is likely a
gain-of-function mutant and positively selected during DLBCL development.

Figure 2.8 MEF2B mutation pattern in DLBCL
Figure reused from (Morin et al., 2011)
Non-synonymous somatic mutations (green circles), frameshift-inducing indel mutations
(orange triangles; inverted triangles for insertions and upright triangles for deletions),
nonsense mutation (red circles).

Structural mapping of the three most frequent non-synonymous substitutions (K4,
Y69, D83) mutation on MEF2B suggests that K4E mutation may affect DNA binding,
whereas Y69H or Y69C mutation may affect cofactor binding and this implication is
supported by experimental evidence (Figure 2.9) [16,21] .
24

Figure 2.9 MEF2B mutation hotspots and their Co-IP test for Cabin1 binding
Figure (a) adapted from (Han et al. 2003); Figure (b) reused from (Ying et al. 2013)
(a) mutation hotspots mapped on MEF2B (K4, Y69, D83) in DLBCL patients, structure is
shown in cartoon mode. Hotspots residues are shown in sphere mode (b) Co-IP experiments
results on MEF2B mutations on Cabin1 binding.

As D83V is the most frequent mutation in MEF2B , and this mutation is on the protein
surface and has not been shown to be involved in direct contact with ligand in previous
structures. We are motivated to solve the MEF2B D83V structures in the hope of finding a
structural based mechanisms to explain its potential functions in cancer cells.
2.2 Results
Chim WT and Chim D83V crystals and their X-ray diffraction
To overcome the low solubility of MEF2B WT D83V and prepare large amount of
protein for biochemical and structural studies, we made a chimera protein (MEF2 Chim WT)
containing amino acid residues of 1 to 64 from MEF2A and residues 65 to 92 residues from
MEF2B, and introduced D83V mutation into the MEF2 chimera WT construct (MEF2 Chim
25

D83V). The chimera proteins displayed similar DNA binding properties as the native MEF2
and are expressed and purified using our established systems for native MEF2 proteins. Chim
WT crystals appeared within a week after crystal tray set up; best crystals diffracted to 2.2Å.
Chim D83V crystals appeared between two to three weeks after crystal tray set up; best
crystals diffracted to 3Å (Figure 2.10) (Table 2.1-2.2).

Figure 2.10 MEF2 Chim WT and Chim D83V crystals and diffractions
(a) MEF2 Chim WT crystals. (b) MEF2 Chim WT crystal diffraction. (c) MEF2 Chim D83V
crystals. (d) MEF2 Chim D83V crystal diffraction.

26

D83V mutation switches helix H3 conformation to strand conformation.
The MEF2 Chim D83V construct has the same MEF2B specific domain as wild type
and overall 90% amino acid identity as MEF2B WT (Figure 2.11).

Figure 2.11 Sequence alignment of MEF2B Chim D83V constructs to MEF2A and
MEF2B

EMSA assay showed MEF2 Chim D83V could not bind to HDAC4 whereas MEF2B
WT and MEF2 Chim WT binds to HDAC4 as expected (Figure 2.12). The EMSA also
showed that the D83V mutant binds DNA well although the shifted band has a lower
mobility, which is consistent with the fact the Asp to Valine substitution would reduce the
negative charge on the protein (Figure 2.12b). We also determined the structure of MEF2
Chim WT and the structure shows nearly identical structure as that of the WT MEF2B
determined previously (RMS 0.307 and 0.412 by C a
alignment to the MEF2B WT in

MEF2B/Cabin1 complex and MEF2B/HDAC complex respectively) (Figure 2.12). This
supports that MEF2 Chim WT is a good structural control as wild type MEF2B.

27

Figure 2.12 MEF2 Chim WT align with MEF2B WT and EMSA tests on the binding of
MEF2 Chim WT and MEF2 Chim D83V to HDAC4
(a) Structural alignment of MEF2 Chim WT (orange) to MEF2B WT in MEF2B/Cabin1
complex (green) and in MEF2B/HDAC4 complex (blue). (b) EMSA assay testing HDAC4
binding to MEF2B WT, MEF2 Chim WT and MEF2 Chim D83V, HDAC to MEF2 ratio is
increasing from 1:1, 2:1, 4:1 in the assay.

We see unambiguous electron densities of the c terminal region (residues 80 to 88) in MEF2
Chim D83V structure (Figure 2.13). The structural alignment between MEF2 Chim D83V and
Chim WT or MEF2B WT shows complete deformation of helix H3, though the rest part of the
structure is nearly identical (C a align RMS 0.434 between MEF2 Chim D83V to MEF2 Chim

WT). The deformed helix 3 in Chim D83V adopts a beta strand conformation, and this beta
strand (strand S4) moves away from DNA and helix H1 (Figure 2.13).
28

Figure 2.13 Electron density of the C-terminal region of MEF2 Chim D83V and
structural alignment of MEF2 Chim D83V to MEF2 Chim WT and MEF2B WT
(a) Electron density of the C terminal residues 78-87 of MEF2 Chim D83V. Density is shown
at 1 sigma level at 2Fo-Fc map. Structure is shown in stick mode. (b) Structural alignment of
MEF2 Chim D83V (red) to Chim WT (orange) and MEF2B WT in MEF2B/Cabin1 complex
(green) and MEF2B/HDAC4 complex (blue). Structure is shown in ribbon mode.

Comparison of the cofactor binding groove between Chim D83V and MEF2B WT
The MEF2 cofactor binding groove is composed of the central six strand (S1, S2 and
S3 from each monomer) on bottom as floor and two helix H2 above as rim. The structural
alignment between MEF2 Chim D83V to MEF2 Chim WT or MEF2B WT shows the H2 of
cofactor-binding groove is shifted outward along the helix axis (Figure 2.14). Previous
observation shows similar shift of helix H2 in cofactor free MEF2A structure compared with
29

MEF2/cofactor complex structure [24] .

Figure 2.14 Comparison of the cofactor binding groove of MEF2B Chim D83V to Chim
WT and MEF2B WT
(a) MEF2B cofactor binding groove is shown in ribbon model. Each monomer is in green and
blue. (b) Comparison of the cofactor binding groove of MEF2 Chim D83V to Chim WT and
MEF2B WT. MEF2 Chim D83V is in red, MEF2 Chim WT in orange. MEF2B WT in
MEF2B/Cabin1 complex and MEF2B/HDAC4 complex is in green and blue. Red arrow
showing the shift of helix H2 in cofactor free structures as to cofactor bound MEF2 structures.

The fact that MEF2 Chim D83V and MEF2 Chim WT has very similar cofactor
binding groove, but one fails to bind to Cabin1 while the other can bind, suggesting factors
beyond the hydrophobic groove conformation contribute to the different Cabin1 binding
properties.
The C-terminal of Chim D83V interacts with its counterpart in crystal packing through
beta twist interaction
Crystal packing analysis shows that the MEF2 Chim D83V C terminal S4 from one
molecule interacts with the same region from another molecule. The detailed interaction is
30

largely antiparallel beta twist interaction by extensive main chain backbone hydrogen bonding
between the two strands (Figure 2.15).

Figure 2.15 Chim D83V strand S4 beta twist interaction in crystal packing
(a) Chim D83V strand S4 from one MEF2 Chim D83V molecule (green) interacts with strand
S4 from another MEF2 Chim D83V molecule (blue). Structure is shown in both cartoon and
line model. (b) Stick model showing mainchain hydrogen bond interactions (dashed grey
lines)

The beta twist interaction through C terminal strands between two molecules may be a
factor that causes MEF2 D83V mutant aggregation. To test if Chim D83V aggregates in vitro,
we performed dynamic light scattering test (DLS) of MEF2 Chim D83V with or without
DNA; DLS results show the protein is in dimer form as MEF2B WT (Figure 2.16).

31

Figure 2.16 DLS analysis of MEF2B WT, MEF2 Chim WT, and MEF2 Chim D83V
Peaks of the complex is between 1 nm and 10 nm, suggesting complex is a dimer with DNA,
which is consistent with crystal structure ( ~ 40 nm).
32

In addition to DLS, multi-angle light scattering (MALS) measurements also show
MEF2 Chim D83V protein is a dimer and not aggregated (Figure 2.17). However, these
evidence from in-vitro assays could not rule out the possibility that MEF2B D83V may
aggregate in the cellular context. Alternatively, the observed interactions mediated by S4
could represent new interaction mode MEF2B D83V could engage with other MEF2
molecules of different cofactors in the context of cancer cells.

Figure 2.17 MALS measurements results
(a) MEF2B WT. (b) MEF2 Chim WT. (c) MEF2 Chim D83V. Red line is the light scattering
signal.

33

Structural mechanisms leading to the deformation of C-terminal helix H3 by the D83V
mutation
The strand S3 and helix H3 at the C terminal of the MEF2 specific region are
susceptible for disease mutations and post translational modifications (PTM). In this region,
the strand S3 makes a sharp turn to the helix H3, which is almost perpendicular to strand S3.
Residues in and close to this turn are in a crowded local environment. D83 is located in the
turn, the side chain of which interacts with T80 amine through hydrogen bonding and with
R79 through charge-charge interaction (Figure 2.18).

Figure 2.18 Mutation and PTM hotspots on MEF2B and D83 local environment
Figure adapted from (Han et al. 2003)
(a) mutation hotspots in MEF2B strand S3 and H3 region. (b) MEF2 Chim D83 local
environment showing hydrogen bonding distance (2.9Å) from D83 to main chain T80 amine.

The D83V mutation introduced a beta-branched valine residue, which increases the
clash possibility in local environment, and disrupts hydrogen bonding with T80 and a
34

charge-charge interaction with R79. In addition, the beta branched residue V83 is introduced
into two flanking beta branched residues T80 and I84. Beta branched residues favor strand
conformation more than helix conformation [41,42] , this tandem beta branched residues TVI
could break the helix H3 conformation as shown by our experiment observation in crystal.
The disruption of helix H3 conformation destabilizes its anchor to helix H1, and the result
strand S4 has more freedom to move away from its intra-molecular interaction with helix H1
and DNA to inter-molecular interaction with another MEF2 in the nearby unit cell in the
crystals.
2.2 Discussion

D83V may create a new protein-protein interaction interface
In this chapter, we show that D83V mutation completely disrupt the conformation of
helix H3 in the MEF2B specific domain. A previous molecular dynamic study proposed that
the D83V mutation disrupts the strand S3 folding and partial helix H3 deformation [43] . In
contrast with the molecular dynamics study, our work shows that the fold of strand S3 is well
maintained as MEF2B WT, but the helix H3 is totally deformed. As the MEF2B specific
domain was shown to be important for cofactor binding, the helix to strand conformational
switch is likely affecting MEF2 cofactors binding through allosteric mechanisms. Our study
shows that the stability of helix H3 is important for Cabin1 and HDAC4 recruitment, as D83V
mutant with deformed helix H3 fails to bind to Cabin1 and HDAC4. However, the D83V
mutant may interact with other unknown partners in DLBCL development through interaction
mediated by strand S4. There are reports that MADS box family members SRF and MCM1,
which have similar tertiary structure as MEF2 core domain, interacting with their partners
35

through beta strand interaction in a manner similar to the interactions mediated by S4 in the
D83V mutant [44,45] . There are reports that regions besides the hydrophobic groove in MEF2
may be also a potential protein-protein interaction interface. For example, mutations in
MEF2C strand S3 and helix H3 region (N73I/E74A/H76L or E77V/S78N/R79Q/T80A)
abrogate its interaction to MyoD [30] . As revealed by our crystal structure, cancer mutations
like D83V could potentially disrupt or change these protein-protein interaction interfaces.
Future studies needs to be done to identify new partners of MEF2 mutant and reveal their
functions.
Other mutations in DLBCL which are likely to disrupt MEF2B helix H3
The helix H3 region in MEF2 seems to be predisposed for conformational switch, it is
in a alpha helix conformation but rich in beta branched residues, including T82 and I84 in this
region, which disfavor alpha helix as compared with beta strand. This region is frequently
targeted in cancer and post-translational modification (PTM). For instance, mutations such as
E77K, S78R, N81K, N81Y, D83A and D83G are also identified in DLBCL patient samples,
albeit with lower frequency than D83V. These mutations may also affect helix H3 stability.
For example, N81K or N81Y may disrupt the H3 stability because N81 is a much more
favored N-capping residue of helix than K or Y [46] [47] . Besides mutations, there are reports
showing that S3 and H3 region is also a target of PTM. For instance, MEF2C T80
phosphorylation causes deficiency in co-activator P300 recruitment to skeletal muscle
development promoters [48] . There are case reports that MEF2C S82 is targeted by
phosphorylation and K89 is targeted by ubiquitination though the functional consequence is
not yet clear [49,50] .
36

The possibility of MEF2B D83V protein aggregation
D83V mutation introduces a hydrophobic valine residue on the protein surface; the
surface exposed hydrophobic valine may lead to aggregation of the mutant proteins by
hydrophobic interactions. This is reminiscent of the mechanism of sickle cell disease. Crystal
structures shows that the sickle cell disease mutation E6V in the β2 subunit of hemoglobin
(HbS) interacts with hydrophobic surface residues F85 and L88 from β1 subunit, cause
aggregation of the mutant HbS protein to fibril structure, which leads to disease phenotype
[51–53] . Our in-vitro study shows that MEF2 Chim D83V is not aggregated, however, we
could not rule out the possibility that MEF2B D83V may aggregate in cell. But this is a less
likely mechanism for how D83V favors cancer cells. If cancers merely want to eliminate
MEF2 via aggregation, one could expect see many deletion of frameshift mutations. One of
our future study to to study if the MEF2B D83V aggregation in cell context.
Possible mechanisms for the weakened DNA binding affinity by the MEF2B D83V
mutant
Previous studies show that the MEF2B D83V mutant has lower affinity to DNA than
wild type in cell base EMSA assays [21] . Our study did not test the DNA binding affinity of
the native MEF2 mutant because its DNA binding domain (MADS box domain) is from
MEF2A. Our structure shows that the deformed helix H3 in MEF2B D83V moves away from
helix H1 and DNA compared with MEF2B wild type. This implies that the helix H3 region
needs to be ordered for stronger DNA binding, due to its stabilization effect on DNA binding
helix H1.
MEF2C S59 phosphorylation has been shown to enhance DNA binding and MEF2
37

target gene transcription [54] , as S59 is close to MEF2 specific region, there is also a
possibility that S59 phosphorylation may modulate the MEF2 specific region conformation
and exerts the effect.
In short, our MEF2 D83V mutant crystal structure shows a naturally occurring cancer
mutation drastically changing secondary structure of the affected region. The affected region
is likely to be a novel interface between MEF2 and cofactor interaction. MEF2 and yet
unknown cofactor interacts through the novel interface and the consequence is likely to lead
to a gain-of-functional role for mutant protein to be selected in cancer development. This may
help the development of target-specific therapeutics design using our structure as a guide. Our
studies also provide a natural example of how a single point mutation could alter the folding
of a protein, providing important insights into the mechanisms of protein folding.
2.3 Methods
Protein purification
MEF2B WT, MEF2 Chim WT and MEF2 Chim D83V are cloned in pET30b bacterial
expression vectors with T7 promoter and transformed into E.Coli BL21(DE3) strain. Protein
is induced in 2XYT medium with 0.5 mM IPTG at 22 °C overnight (16 to 20 hours). Each
purification is from 1L cell pellet; pellet is lysed by sonication. MEF2B WT and MEF2 Chim
WT are purified as previously indicated for MEF2A purification (Wu et al. 2010). MEF2
Chim D83V is purified by heparin column (GE Healthcare) with buffer A containing 20 mM
HEPES pH 7.0, 0.5 mM EDTA, 0.5 mM TCEP and buffer B containing all the component in
buffer A with 1.5 M NaCl. Protein peak comes out at around 1 M NaCl from heparin column,
add Polyethyleneimine (PEI) at a final concentration to 0.1% (W/V) to protein peak to strip
38

DNA contamination (Burgess 1991). Add saturated (NH 4 ) 2 SO 4
solution to final 80% (V/V) to
precipitation the protein and remove PEI; dissolve protein pellet in 20 mM HEPES pH 7.0,
250 mM NaCl, 0.5 mM EDTA, 0.5 mM TCEP for Mono S (GE Healthcare) purification.
Sample fractions from Mono S are combined and concentrated through Amicon 3K
centrifugal filter (EMD Millipore) and further purified by size exclusion Superdex 75 (GE
Healthcare).The final storage buffer for both MEF2 Chim WT, MEF2 Chim D83V and
MEF2B WT is: 10 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM EDTA, and 0.5 mM TCEP.
DNA Purification
The DNA used in crystallization for MEF2 Chim D83V is 5’ AACTATTTATAAGA 3’ and
its complementary strand 5’ TTCTTATAAATAGT 3’ with one base overhang at end. DNA
used in crystallization for MEF2 Chim WT is 5’ AAACTATTTATAAGA 3’ and its
complementary strand 5’ TTCTTATAAATAGTT 3’. The DNA were purchased from
Integrated DNA Technologies (Coralville, IA) at 1- μmol scale in the crude and desalted form.
The crude DNA was dissolved in a buffer (100 mM NaCl, 10 mM NaOH, pH 12.0) and
purified by a Mono Q cation-exchange column (GE Healthcare) on FPLC (GE Healthcare).
The peak fractions were pooled and neutralized to pH 7.0 by HEPES prior to overnight
dialysis against water. The desalted DNA sample was lyophilized to powder, resuspended in
water, and quantified at 260 nm. Complementary DNA strands were annealed at 95 °C in the
annealing buffer (100 mM NaCl, 5 mM HEPES, pH 7.6) in PCR machine (Eppendorf
Mastercycler Personal 5332 Thermal Cycler) for 2 min and cool naturally to room
temperature for 1 hour.
39

Crystallization and Structure Determination
Protein and DNA were mixed at a molar ration of 1:1.2, the final protein concentration
in the mixture is around 8mg/ml to 10mg/ml. Hanging drop crystal trays were set up at room
temperature, 1 µL protein complex and 1 µL mother liquor was mixed . MEF2 Chim WT
with DNA crystals appear within a week; crystals are orthorhombic shapes with dimensions of
~100x100x100 microns in crystallization buffer (0.1 M HAc pH 4.7, 150 mM NaCl, 10 mM
CaCl 2 , 5 mM MgCl 2 and 28% PEG 400). Crystals were harvested, cryoprotected in 0.1 M

HAc pH 4.7, 150 mM NaCl, 10 mM CaCl 2 , 5 mM MgCl 2 , 10% Glycerol and 30% PEG 400
and flash frozen in liquid nitrogen. Data was collected at Advanced Light Source (ALS
Berkeley) beamline 8.2.2 with the following settings: detector distance 300 mm, oscillation
angle 0.5 o
, exposure time for each frame 1 s, overall rotation 270 o . MEF2 Chim D83V with
DNA crystals appear between 2 to 3 weeks; crystals are hexagonal bars shapes with
dimensions of ~100x100x50 microns in crystallization buffer (0.1M MES pH 5.94, 0.2 M
NaCl and 18% PEG2000MME). Crystals are harvested, cryoprotected in 0.1M MES pH 5.94,
0.2 M NaCl, 20% Glycerol and 25% PEG2000MME and flash frozen in liquid nitrogen. Data
was collected at Advanced Photon Source (APS Chicago) beamline 23 ID-B with the
following settings: detector distance 400 mm, oscillation angle 1 o , exposure time for each

frame 0.5 s, overall rotation 210 o .
The crystal diffraction data of MEF2 Chim WT with DNA complex was processed
with iMosflm and space group determination by pointless [55–57] . The structure was
determined by molecular replacement using the MEF2 and DNA complex part from PDB ID
3P57 as search model using the Molrep program in the CCP4 program Suite [58–61] . Model
40

building was done in Coot and refinement was done in Refmac5 [62,63] . Crystallographic and
refinement statistics table (Table 2.1) is generated by “Generation of Table 1 for Journal” tool
in Phenix Suite [64] .
41

Table 2.1 Data collection and refinement statistics for MEF2 Chim WT and DNA crystal
42

The crystal diffraction data of MEF2 Chim D83V with DNA complex was processed
with iMosflm and space group determination by pointless [55–57] . The structure was
determined by molecular replacement using part of MEF2 Chim WT (residues 1-80 with
DNA) and DNA complex as search model using the Phaser program in the CCP4 program
Suite [60,61,65] . Model building is in Coot and refinement is in Refmac5 [62,63] . Refmac5
intensity based twin refinement is used, after twin refinement the Rfree dropped from 31% to
21%, however, the Rfree after twin refinement could not compared directly with Rfree
without twin refinement. The electron density map is very similar after twin refinement
compared to the density map without twin refine. Space group P32 is verified by Zanuda to
be the best solution space group [66] . Crystallographic and refinement statistics table (Table
2.2) is generated by “Generation of Table 1 for Journal” tool in Phenix Suite [64] .
43

Table 2.2 Data collection and refinement statistics for MEF2 Chim WT and DNA crystal

44

Dynamic Light Scattering Study
Experiments were conducted at the University of Southern California NanoBiophysics
Core Facility. Solutions of the protein or protein-DNA complex were made between
5-8.0mg/ml in PBS buffer and filtered by 0.45 μm filter. Triplicate samples were examined in
low volume plastic cuvettes using Wyatt DLS system. The measurements were conducted at
20 ± 0.1 °C. Data were accumulated for 60 s, and the autocorrelation functions were
obtained. Particle diameters were recorded as a frequency distribution curve, and the average
diameter and range were plotted.
Multiangle light scattering study
Experiments were conducted at the University of Southern California NanoBiophysics
Core Facility. Purified MEF2B, Chim WT and Chim D83V protein were subjected to HPLC
chromatography (Shodex KW 803) in buffer C (250 mM Na2SO4, 50 mM HEPES pH 7.5,
0.5 mM TCEP). The column effluent was passed directly on-line into Dawn Heleos MALS
detector (Wyatt Technology) and Optilab rEX refractometer (Wyatt Technology). Data was
analyzed by ASTRA 6 software.
Electrophoresis mobility shift assay (EMSA)
EMSA was performed in 20 mM Hepes (pH 7.6), 250 mM NaCl, 1 mM DTT, 12%
glycerol. The concentration of DNA was kept at 50 μM; approximately 80 μM MEF2B, Chim
WT and Chim D83V was used in all binding reactions except the DNA control. In the MEF2
with HDAC4 lanes, HDAC4 ratio to MEF2 is increasing from 1:1, 2:1 and 4:1. The binding
reactions were analyzed on a 4%–20% (w/v) acrylamide gradient native gel in TBE and
stained with Sybr Safe DNA Dye (Thermo Fisher Scientific).

45

Chapter 3: Structure of MEF2 and NKX2.5 complex reveals novel
cofactor interaction interfaces from MEF2

I performed all of the work presented in this dissertation with the exceptions
detailed here. Reused figures are under creative common license. Crystal
structure figures are generated by Pymol [23] . My advisor Dr. Lin Chen
contributed partly to writing on this Chapter. Dr. Lin Chen supervised this
project.
3.1 Introduction

The interaction between MEF2 and NKX2.5 plays important roles in lymphoma
In chapter 2, we hypothesize that the Sheet S3 and helix H3 region in MEF2 specific
domain is likely to be an interface for cofactor recruitment in lymphomagenesis, and the
mutation hotspots in this region in lymphoma patients may affect partner interaction or partner
switching. Early study suggests one of the cofactor of MEF2 in lymphomagenesis is likely to
be NKX2.5. The upregulation of NKX2.5 due to chromosome aberration drives MEF2C
expression in lymphomagenesis [8] . Besides roles in lymphomagenesis, the synergy between
MEF2 and NKX2.5 was reported multiple times from early studies of heart development
[67–69] . This motivates us to pursue the complex structure of MEF2 and NKX2.5 as to
understand the molecular mechanism between these two factors in lymphomagenesis and
heart development.

46

Domain structure of NKX 2.5 transcription factors
The Nk homeobox gene (NKX) was first identified in the Drosophila genome by
screen with degenerated homeobox oligonucleotides [70] . NKX2.5 gene encodes a 324-amino
acid protein with three domains: a TN domain, a homeodomain (HD) and a NK2 specific
domain [71] . The NKX2.5 homeobox domain (HD) binds consensus motif 5’ AAGTG 3’
[72] . The homeobox domain contains three alpha helices and the helix 3 is the main DNA
recognition helix (Figure 3.1).

47

Figure 3.1 Domain scheme of NKX 2.5 and structure and homeodomain
Figure b and c reused from (Pradhan et al. 2012)
(a) Domain structure of NKX 2.5. (b) Sequence alignment of NK family HD domains.
NKX2.5 138−197 corresponds to HD residue 1−60. (c) structure of NKX2.5 binds to DNA.

Even though all the reported NKX2.5 crystal structures show that NKX2.5 is a
monomer, there are reports suggesting NKX2.5 could exist as a homo- or hetero-dimer. There
is no consensus on which region mediates NKX2.5 dimerization. Early report shows residues
K193 and R194 inside the HD domain are important for NKX 2.5 dimerization, as double
mutant of K193I and R194D abolish NXK2.5 HD dimerization, however, this double mutant
still dimerizes with full length wild type NKX2.5 albeit with lower affinity, suggesting
regions outside HD domain may also be involved in dimerization [73] . Later studies found
that the tyrosine rich domain (YRD), which contains nine tyrosine residues in C terminal
region of NKX2.5 (residues 236-275), is important for NKX2.5 homodimerization and
heterodimerization with other partners like ETS1 [74] .
48

The NKX2.5 C terminal region has been shown to have an inhibitory effect on its own
transcription activity. For instance, an atrial natriuretic factor (ANF, which is a target gene of
NKX) luciferase reporter assay showed that NKX2.5 truncations ended in P236 and Q200
residues activate the atrial natriuretic factor (ANF) promoter 5-fold more than wild type
NKX2.5 [75] . In another case, NKX2.5 truncation ended in Q198 is as similarly active as wild
type whereas NKX2.5 truncation ended in Y259 has much lower activity than wild type (less
than half the activity as wild type) [76] . Interestingly tyrosine rich domain (YRD) is between
residue P236 and residue Y259 in NKX2.5; as YRD is involved in homo- or
hetero-dimerization of NKX2.5. This implies that the oligomerization state of the protein is
related to transcription activation or inhibition.
NKX2.5’s role in cardiac muscle development

NKX 2.5 plays important roles in cardiac development and maturation. It shows high
expression in early heart progenitor cells [77] . NKX2.5-/- knockout mouse shows embryonic
lethality at embryonic stage 10.5 [78] . NKX2.5 is heavily mutated in congenital heart diseases
(Figure 3.2) [79,80] .

Figure 3.2 NKX2.5 disease related mutations map
49

Figure reused from [80]
Truncating insertions and deletions and nonsense mutations are shown above. Red indicating
variants in congenital heart disease patients. Bold and colored indicates variants reported in
inbred consanguineous population. Green indicating variants in the control group. Underlined
variants refer to familial segregation, and italicized variants indicate a sporadic report of the
variant in a case. Tin: Tinman Domain. YRD: tyrosine rich domain.

The evidence of direct interaction between MEF2 and NKX2.5 is very limited. In one
study [67] , the authors show that NKX2.5 and MEF2C are co-expressed in embryonic mouse
heart. They also show that NKX2.5 and MEF2C can pull down each other in
co-immunoprecipitation (Co-IP) assay. However, Gal4-VP16 based luciferase assay using full
length MEF2C and NKX2.5 did not show direct physical interaction between the two factor.
Even though evidence of direct physical interaction between MEF2 and NKX2.5 is very
limited, the functional synergy between MEF2 and NKX2.5 in heart development is
intensively studied and broadly observed. NKX 2.5, MEF2C and GATA4 are involved in the
early step of cardiac development because they appear in the precardiac mesoderm earlier
than any other known transcription factors implicated in cardiogenesis [2,81] . NKX2.5 and
MEF2 activate the expression of each other, and drive the differentiation of cardiomyoblasts
to cardiomyocytes [69,82] . Early studies in Nppa gene (a gene coding atrial natriuretic factor,
which is a vasodilator hormone secreted by atrial myocytes) promoter regions find direct
interactions and synergy between MEF2 and GATA4 [83] , and between GATA4 and NKX2.5
[84] . The lack of evidence of the synergy between MEF2 and NKX2.5 in these studies is
likely due to the luciferase constructs used in these studies only include 700bp upstream of the
Nppa gene. Later studies found that the 700 bp region upstream the transcription start site
(TSS) alone could not recapitulate the endogenous Nppa gene activity in the heart. The
50

authors identified three additional NKX2.5 binding region at 5′ regulatory elements ( −34 kb,
−31 kb, and −21 kb to TSS) that are essential to recapitulate the endogenous Nppa gene
activity [85] . The putative MEF2 region identified within the 700 bp region upstream of Nppa
gene only shows very weak affinity of MEF2 binding [83] . Chromatin immunoprecipitation
(ChIP) with massively parallel DNA sequencing (Chip-seq) studies shows MEF2 is found
mainly in the intergenic and enhancer regions of skeletal and cardiac muscle cells with -50 kb
away from TSS. Because chromosome looping could bring long distance elements together to
drive target gene expression [86] , we hypothesized that MEF2 and NKX2.5 could mediate
chromosome long range interaction.
NKX2.5’s role in cancer
NKX family members are also involved in in cancer. Overexpression of NKX2.2,
NKX2.5, NKX3.1 and NKX6.1 has been shown to be involved in Ewing's sarcoma, ovarian
yolk sac tumors, lobular breast carcinomas and clear cell sarcoma respectively [87] . Aberrant
expression of NKX family members is common in T-cell acute lymphoblastic leukemia
(T-ALL) [87] . Six NKX members (NKX2.1, NKX2.2, NKX2.3, NKX2.5, NKX3.1, NKX6.1)
have been reported to be deregulated in specific T-ALL subgroups. Most of NKX members
are normally not expressed in T-cell development, in T-ALL, aberrant high expression of
NKX is usually caused by chromosomal rearrangement of [87,88] .
The interplay between MEF2 and NKX has been reported in T-ALL. For instance, in
one case of T-ALL, overexpression of NKX2.5 leads to overexpression of MEF2C, which
repress the expression of NR4A1, inhibit BCL2-regulated apoptosis and promotes survival of
leukemic cells [8] .
51

NKX2.5’s repressor role
The repressor role of NKX members has been reported in many cases. The Nkx2.2
repressor complex regulates islet β-cell specification and prevents β-to-α-cell reprogramming
[89] [87] . The Nkx2.5/Bmp2/Smad1 negative feedback loop controls heart progenitor
specification and proliferation [90] . NKX2.5 has been shown to act as an inhibitor of
reprogramming of cardiac fibroblasts to cardiomyocytes [91] . In short, NKX family members
could act as a repressor or an activator depending on cellular context.
3.2 Results
MEF2 and NKX2.5 binds DNA cooperatively
EMSA assay was performed to test if MEF2 and NKX2.5 could bind with DNA
cooperatively. Myocardin gene enhancer DNA containing both MEF2 and NKX2.5 consensus
binding motif was used in EMSA and results show both MEF2 and NKX2.5 could bind DNA
cooperatively. EMSA also shows that NKX2.5 alone binds DNA much weaker compared with
MEF2/NKX2.5 complex (Figure 3.3).
52

Figure 3.3 EMSA assay of MEF2B and NKX2.5 on myocardin gene enhancer
(a) SYBR Safe DNA dye staining the gel. (b) Coomassie Brilliant Blue R-250 protein dye
staining the gel. In panel (a), weak staining of DNA in lanes 4 to 6 for the
MEF2B/NKX2.5/DNA ternary complex is likely due to the protein occupying of the DNA
and limiting the access of the SYBR Safe DNA dye. In panel (b) the protein staining shows
very well formed the MEF2B/NKX2.5/DNA ternary complex (lanes 4 to 6) but weaker
NKX2.5/DNA complex (Lane 3). This suggests the binding of MEF2 increases the binding of
NKX2.5 and their interaction is likely cooperative.

Crystallization and Diffraction of MEF2/NKX2.5/DNA crystals
The MEF2 Chim WT yields high quality MEF2/NKX2.5/DNA ternary complex
crystals within a week after crystallization tray setup. THE Crystal diffracts to 2.1 Å at
synchrotron beam light (Figure 3.4).
53

Figure 3.4 MEF2/NKX2.5/DNA crystals and X-ray diffraction
(a ）Crystals of MEF2/NKX2.5/DNA ternary complex (b) X ray diffractions of a
MEF2/NKX2.5/DNA ternary complex crystal

Overall Structure of the MEF2/NKX2.5/DNA ternary complex
There are three non-crystallographic symmetry related (NCS) copies of
MEF/NKX2.5/DNA complex in the asymmetric unit (ASU) (Figure 3.5).
54

Figure 3.5 Asymmetric unit of the MEF2/NKX/DNA crystal
(a ）Three NCS related copies MEF2/NKX/DNA complexe in ASU. Structure shown in
ribbon mode. NCS related copies are show in green, red and blue. NKX is shown in yellow.

The overall structure shows that MEF2 and NKX2.5 sitting on opposite side of the
double stranded DNA, with the N terminal loop of MEF2 interacting with the minor groove of
DNA and the C terminal alpha helix H3 of NKX2.5 interacting with the major groove of DNA
(Figure 3.6).
55

Figure 3.6 Overall structure of the MEF2/NKX2.5/DNA complex
(a ）NKX2.5 is in blue and MEF2 is in green and red. (b) 180 ο rotation view of (a).

The alignment of MEF2 structures in the three NCS related copies in ASU shows
noticeable movement of the loop (residues 73-77) between helix H2 and sheet S3 (loop
H2-S3) (Figure 3.7). The movement of H2-S3 loop was reported previously in MEF2 bound
with different ligand (HDAC or Cabin1) [15] , and in Apo MEF2 compared with ligand bound
MEF2 [24] . The movement of the H2-S3 loop in our structure is likely contributed by
different NKX2.5 binding mode to MEF2 in the crystal packing environment.

56

Figure 3.7 Movement of H2-S3 loop in NCS related copies in ASU
(a) NCS related molecule copy 1 in green, copy 2 in red and copy 3 in blue. Structure is
shown in ribbon mode.

The alignment of MEF2 between copy 2 and copy 3 shows a shift in the DNA end
where NKX interacts. The DNA region which is for NKX2.5 binding in NCS copy 3 was bent
upward and back into the plane compared to NCS copy 2 (Figure 3.8).

57

Figure 3.8 Shifts of DNA and NKX2.5 in the copy 2 and copy 3 after MEF2 alignment
(a) NCS related molecule copy 2 in red and copy 3 in blue. Model shown in ribbon mode.

The alignment of NKX2.5 in three NCS copies shows a significant shift in the DNA
ends where MEF2 interacts (Figure 3.9).
58

Figure 3.9 Shifts of MEF2 and DNA in three NCS related copies after NKX2.5
alignment
(a) NCS related molecule copy 1 is in green, copy 2 in red and copy 3 in blue. Structure is
shown in ribbon mode. (b) An inward rotation movement of MEF2/DNA into the plane from
viewer: outside copy 2 (red), middle copy 1 (green) and backside copy 3 (blue).

Protein and DNA interactions
The detailed interaction pattern between MEF2 and DNA and between NKX2.5 and
DNA respectively is similar to previous reports [24,92] . However, the ternary complex reveals
a cooperative DNA minor groove binding by both the N terminal loop of MEF2 and N
terminal loop of NKX2.5 (Figure 3.10).
59

Figure 3.10 Interaction of the N termini of both MEF2 and NKX2.5 to DNA minor
groove
(a) MEF2 and NKX is shown in ribbon mode and in green, DNA is shown in sphere mode.
(b) different view angle from (a).

Detailed analysis reveals that the residue R3 from the N terminal of MEF2 and residue
R142 from the N terminal of NKX 2.5 both insert into the same minor groove of DNA (5’
AAGAAA 3’). The Arg residues from both MEF2 and NKX2.5 are involved in hydrogen
bonding with DNA bases and backbone sugars (Figure 3.11). It has been proposed that the
interaction of arginine to DNA minor groove also involves shape recognition [93] . Based on
our structure, NKX2.5 congenital heart disease associated mutation R142C [94] is likely to
affect its cooperativity with MEF2 to recognize DNA.
60

Figure 3.11 Cooperative DNA minor groove recognition by arginine residues from both
N terminals of MEF2 and NKX2.5
(a) DNA is shown in sphere mode in orange. Two arginines from the N terminal of MEF2 and
NKX2.5 are shown in stick mode. (b) Detailed interaction of Arg 3 from MEF2 and Arg 142
from NKX 2.5 in DNA minor groove, both protein and DNA are shown as stick model,
colored by element (C: green; O: red; N: blue; P: yellow). Yellow dashed line shows polar
interaction between Arg residue and DNA bases.

We also notice the C terminal of one NCS copy of MEF2 protein interacting with
DNA minor groove (Figure 3.12), this observation has never been reported before. The C
terminal Arg 91 and Arg 94 contact to phosphate and sugar backbone of DNA through
charge-charge and hydrogen bonding interactions. Arg 94 inserts into the DNA minor groove,
stabilized by two flanking phosphates from T8 and G16.
61

Figure 3.12 The C terminal of MEF2 interacts with the minor groove of DNA
(a) Overall structure. MEF2 is colored by cyan and green, N terminal and C terminal residues
are shown as sticks. DNA is shown in sphere mode in orange. NKX is colored by magenta.
(b) Zoom view of the C terminal residues of MEF2A and DNA interaction. Featuring Arg 94
and Arg 91 as sticks. DNA is shown as cartoon and stick.

Potential Protein-Protein Interfaces from Crystal Packing Analyses
The analysis of crystal packing of three NCS copies reveal potential protein-protein
interaction interfaces which may contribute to chromosome long range interactions.
The first interface (interface I) identified is between sheet S3 and helix H3 from MEF2
to helix H1 from NKX2.5 (Figure 3.13). Buried surface area (BSA) is around 477.85 Å 2 .
62

Figure 3.13 MEF2 and NKX2.5 interaction interface I from crystal packing
(a) Structure is shown in ribbon mode, NKX in orange, MEF2 and DNA in green. (b)
Different view angle from (a).

Detailed interactions shows charge-charge interactions between D83 of MEF2 to R161
of NKX2.5, hydrogen bonding between main chain and side chain of S78 of MEF2 to R161
and K158 of NKX2.5, hydrogen bonding between main chain amine of M62 of MEF2 to main
chain carbonyl Q159, and hydrogen bonding between side chain carbonyl of S73 of MEF2 to
R155 from NKX2.5 (Figure 3.14). Based on this interface, MEF2 mutations such as D83V
and S78R in cancer may impair the interaction between MEF2 and NKX protein. Notably,
this is also the region implicated in the physical interaction and functional synergy between
MEF2 and MyoD.
63

Figure 3.14 Detailed interactions between MEF2 and NKX in interface I
Residues involved in interactions are shown as sticks, the rest residues are shown as ribbon,
MEF2 in green and NKX2.5 in orange. Polar interactions are shown as dashed gray lines.

The second interface (interface II) mainly involves the sheet S3 and helix H3 from
MEF2 and the loop between helix H1 and helix H2 (H1-H2 loop) from NKX2.5 (Figure 3.15).
Buried surface area (BSA) is around 359.6 Å 2 .

64

Figure 3.15 Potential MEF2 and NKX interaction interface II
(a) structure is shown in ribbon mode. MEF2 is in red, NKX in orange, DNA is in red and
blue. Arrow points to the interaction interface. (b) shape complementarity between NKX and
MEF2 interaction interface, both NKX and MEF2 in shown in surface mode.

The detailed interaction features extensive water mediated hydrogen bonding and Van
der Waals interactions (VdW) with shape complementary of the two protein (Figure 3.16)
(Figure 3.17). MEF2 R90 side chain involves Cation-π interaction with NKX2.5 Y162. MEF2
D83 involves water mediated interaction to NKX2.5 S164. MEF2 E77 side chain involves
water mediated interaction to NKX2.5 Q159 side chain. MEF2 E77 main chain involves
hydrogen bonding to NKX2.5 Q170. MEF2 E86 involves charge-charge interaction to
NKX2.5 K168.
65

Figure 3.16 Detailed interaction in MEF2 and NKX2.5 interface II
Structures is shown in stick mode. MEF2 is in green and NKX2.5 in orange. Polar interactions
are shown in yellow dashed line.

66

Figure 3.17 Detailed interaction in MEF2 and NKX2.5 interface II
Structures shown as sticks. MEF2 is in green and NKX2.5 is in orange. Polar interactions is
shown in yellow dashed line.

The potential MEF2 and NKX2.5 protein-protein interaction interfaces are at MEF2
specific domain, comprising sheet S3 and helix H3, this interface does not overlap with the
interface for Cabin1, HDAC4, EP300 interaction; this allows MEF2 bind multiple cofactors as
a center of different signal crosstalks. Previous studies show that MEF2C mutations on
(N73I/E74A/H76L or E77V/S78N/R79Q/T80A) disrupt its synergistically with the MyoD
transcription factor (Figure 3.18) [14,95] ; this implies MyoD and NKX2.5 may have the same
interaction interface to MEF2. NKX2.5 is heavily mutated in congenital heart disease (CHD).
Many of the disease affected mutations and post transcriptional modification (PTM) affected
residues on NKX2.5 can be mapped in the MEF2-NKX2.5 protein protein interaction
67

interface (Figure 3.18) (Table 3.1).

Figure 3.18 MEF2 and NKX2.5 potential interaction interface and disease or PTM
affected residues mapped in the interface
Panel a reused from [14] .
(a) Surface representation of MEF2 (grey), the hydrophobic groove for class II HDACs and
Cabin1 interaction (Yellow), and the interface mapped for MyoD (magenta) interaction based
on mutagenesis studies. (b) Disease or PTM affected residues (spheres) mapped on MEF2 and
NKX2.5 interface I. (c) Disease or PTM affected residues (spheres) mapped on MEF2 and
NKX2.5 interface II.

68

Protein Location Disease/PTM Ref
MEF2B E77K DLBCL [40]
MEF2B S78R DLBCL [11]
MEF2B N81K/Y DLBCL [10]
MEF2B D83V/G/A DLBCL [10]
MEF2B K89Ub Ubiquitination [50]
MEF2C T80phos Phosphorylation [48]
NKX2.5 E154G CHD [80]
NKX2.5 Q170stop CHD [96]
NKX2.5 Q160P Atrial Septal Defect [97]
NKX2.5 L171P Atrioventricular
(AV) block
[98]
NKX2.5 Q149stop CHD [79]
NKX2.5 S164phos Phosphorylation [99]
NKX2.1 S187stop
NKX2.5 S164
Lung Cancer [100]
NKX2.2 Y152phos
NKX2.5 Y162
Phosphorylation [50]

Table 3.1 Detailed information on disease or PTM affected residues on MEF2 and
NKX2.5 interaction interface
3.2 Discussion
Speculative chromosome looping model mediated by MEF2 and NKX2.5
The MEF2/NKX2.5/DNA ternary complex is described in this chapter. The potential
interfaces between MEF2 and NKX2.5 suggest the two could function in mediating long
range chromosome looping together with other factors like GATA4. GATA4 has been
69

reported multiple times to directly interact with NKX2.5 [68,73,74,84,101] . In GATA family
members, GATA1 has been shown to mediated chromosome looping before [102] . Structures
of GATA3 shows that the two zinc finger from N terminal domain and C terminal domain
could bind to different DNA molecules [103] . Chip-seq studies found more than half of
MEF2A binding sites in cardiomyocytes and skeletal cell line are in the intergenic region (>50
kb) from transcription start site (TSS) [104] . It’s likely that MEF2 protein from intergenic
regions act as transcriptional enhancers with multiple other transcriptional factors and
mediators and form a big transcriptional machinery and drive target gene expression. It has
been reported that the chromosome looping mediated by a transcriptional complex including
NKX2.5 is responsible for driving atrial natriuretic factor (ANF) expression in heart
development [85] . The model that MEF2 and NKX2.5 are in a protein complex mediating
long range chromosome interaction is only speculative at the current stage. More experiments
are needed to test the potential interaction interfaces between MEF2 and NKX2.5 and study
chromosome looping between these two factors. We are planning to do Co-IP test to find if
certain disease related MEF2 and NKX2.5 mutations have an impact on their mutual
interactions. We are also planning to do chromosome capture studies (e.g. 3C, ChiAPET) to
find the role of MEF2/NKX2.5 complex in chromosome looping.
Minor groove DNA recognition by the MEF2 C terminal helix
The structure of the MEF2/NKX2.5/DNA complex also revealed an unexpected DNA
recognition mechanism of MEF2: the C terminal loop extension from the helix H3 could
interact to the minor groove of DNA by a arginine mediated, shape-based recognition
mechanism. Previous structure studies could not capture this feature, likely due to the DNA
70

used in crystallization that does not contain an intact minor groove on the flanking side of
core consensus DNA. It’s interesting to note that R94 is not conserved between MEF2
members, in MEF2B, MEF2C and MEF2D the corresponding residues are G94, N94 and
N94. This difference may contribute to different DNA binding, especially to the flanking
regions of the binding site, by the four members.
The synergy between MEF2 and NKX2.5 is intensively reported, however, the
evidence of direct interaction between the two factors is very limited, only one report showing
Co-IP of these two factors under transient expression of both factors with tags. It may be due
to the nature of the interaction of the two factors: it only happens in the right cellular context
in a big complex and DNA binding is needed for both factors to interact with each other. The
fortuitous capture of the MEF2 and NKX2.5 interaction interface in this study gives insights
into understanding the molecular interaction during heart or cancer development.
Cancer mutations in protein-protein interaction interface
The structure of MEF2 and NKX2.5 shows that many disease related mutations are
mapped in protein protein interaction interface. The mutations on each of the protein is likely
to have an impact on their interactions; the impact can be interaction disruption or interaction
enhancement, either of which is likely to play a role in carcinogenesis.

71

3.3 Methods
Protein expression and purification
MEF2 Chim WT is purified as described in Chapter 2. Human NKX2.5 homeobox
domain (residues 138-197) with Cys 193 substituted by Ser is cloned in pET28-Sumo vector
with a his tag before Sumo tag. Protein was first purified by Ni-NTA agarose resin
(QIAGEN). After Ni purification the elute target protein is cleaved by sumo protease Ulp1 to
remove the his and sumo tag, the protein is then purified by Heparin FF column (GE
Healthcare) on FPLC system (GE Healthcare) . The Heparin FF buffer A is: 20 mM HEPES
pH 7.0, 0.5 mM EDTA, 0.5 mM TCEP. Heparin FF buffer B has the same components as
buffer A with additional 1.5 M NaCl. The final storage buffer for both MEF2 Chim WT and
NKX2.5 is: 10 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM EDTA, and 0.5 mM TCEP.
DNA Purification
5’ ACTATTTTAAGAACGTGCT 3’ and its complemental strand were purchased
from Integrated DNA Technologies (Coralville, IA) at 1- μmol scale in the crude and desalted
form. The crude DNA was dissolved in a buffer (100 mM NaCl, 10 mM NaOH, pH 12.0) and
purified by a Mono Q cation-exchange column (GE Healthcare) on FPLC (GE Healthcare).
The peak fractions were pooled and neutralized to pH 7.0 by HEPES prior to overnight
dialysis against water. The desalted DNA sample was lyophilized to powder, resuspended in
water, and quantified at 260 nm. Complementary DNA strands were annealed at 95 °C in the
annealing buffer (100 mM NaCl, 5 mM HEPES, pH 7.6) for 2 min in PCR machine
(Eppendorf Mastercycler Personal 5332 Thermal Cycler) and cool naturally to room
temperature for 1 hour.
72

Ternary complex Crystallization and structure determination
MEF2 Chim WT, NKX2.5, and DNA was mixed at a molar ration of 1:1:1.2, the final
protein concentration in the mixture is around 8mg/ml to 10mg/ml. Crystal high-throughput
screening was performed by Crystal Gryphon robot (ARI instruments). One condition (0.15 M
DL-Malic Acid pH 7.0 , 20% PEG 3350) from NeXtal Tubes Protein Complex Suite (Qiagen)
yields good diffraction crystals. Sitting drop crystal trays were set up at 18 ℃, and incubated
at room temperature. 0.3 µL protein complex and 0.3 µL mother liquor was mixed. MEF2
Chim WT/NKX2.5/DNA ternary crystals appear within two weeks; crystals are long bar
shapes with dimensions of ~300x100x100 microns. Crystals were harvested, cryoprotected in
0.15 M DL-Malic Acid pH 7.0 , 30% PEG 3350 and flash frozen in liquid nitrogen. Data was
collected at Advanced Photon Source (APS Chicago) beamline 23 ID-B with the following
settings: detector distance 300 mm, oscillation angle 0.5 o
, exposure time for each frame 0.5 s,
overall rotation 180 o . Best crystal diffracted to 2.1 Å. The crystal diffraction data was
processed with iMosflm and space group determination by pointless [55–57] . The structure
was determined by molecular replacement in Phaser MR in CCP4 program suite using MEF2
Chim WT, NKX2.5 from PDB ID 3RKQ and DNA generated from Coot as search models
[60–62,65] . Model building was done in Coot and refinement was done in Refmac5 [62,63] .
Crystallographic and refinement statistics table (Table 3.2) is generated by “Generation of
Table 1 for Journal” tool in Phenix Suite [64] .
73

Table 3.2 Data processing and Refinement statistics of MEF2/NKX2.5/DNA complex

Electrophoresis mobility shift assay (EMSA)
EMSA was performed in 20 mM Hepes (pH 7.6), 250 mM NaCl, 1 mM DTT, 12%
74

glycerol. The concentration of DNA was kept at 120 μM; 120 μM MEF2 Chim WT was used
in all binding reactions except the DNA control. In the MEF2 Chim WT with NKX2.5 lanes,
NKX2.5 ratio to MEF2 Chim WT is increasing from 1:1, 2:1 and 4:1. The binding reactions
were analyzed on a 4%–20% (w/v) acrylamide gradient native gel in TBE and stained with
Sybr Safe DNA Dye (Thermo Fisher Scientific) and Coomassie Brilliant Blue R-250 Dye
(Bio-rad).

75

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84

Abstract (if available)

Abstract MEF2B gene is frequently mutated in lymphoma. Part I of my research focus on the structural study of the MEF2B D83V, which is the most frequent MEF2B mutant in lymphoma. The structure reveals a complete deformation of the C terminal helix in MEF2 specific domain by the D83V mutation. The deformation of the C terminal helix disrupts cofactor binding to MEF2B and is likely to create a novel interface for unknown cofactors to interact in the lymphomagenesis. ❧ NKX2.5 is a potential MEF2 interaction partner in lymphomagenesis. Part II of my research focus on the structural studies of two transcription cofactors: MEF2 and NKX2.5. The structure reveals a novel MEF2 cofactor interaction interface, where many diseases related mutations on MEF2 and NKX2.5 are located in the interface. The structure also suggests the interaction between MEF2 and NKX2.5 is likely to be in a multi-protein transcriptional machinery mediating long range chromosome interaction. As the synergy between MEF2 and NKX2.5 is also well known to play a role in heart muscle regeneration, the structure may help future studies to design therapeutic compounds to regeneration process. treat lymphoma and cardiac disease.

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