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Impacts of post-translational modifications on interactions between G9a and its N-terminus binding partners

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Impacts of post-translational modifications on interactions between G9a and its N-terminus binding partners

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Impacts
of
Post-‐translational

Modifications
on
Interactions

Between
G9a
and
its
N-‐terminus

Binding
Partners

by

Yixin
Hu

A
Thesis
Presented
to
the

FACULTY
OF
THE
GRADUATE
SCHOOL

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

In
Partial
Fulfillment
of
the

Requirements
for
the
Degree

MASTER
OF
SCIENCE

(Biochemistry
and
Molecular
Biology)

August
2015

Copyright
2015

Yixin
Hu

Impact
of
Post-‐translational

Modifications
on
Interactions

Between
G9a
and
its
N-‐terminus

Binding
Partners

Yixin
Hu

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

August
2015

i

Acknowledgement

First
of
all,
I
would
like
to
thank
my
mentor
Dr.
Michael
Stallcup
in

particular
for
giving
me
chance
to
do
research
in
his
lab,
constantly
offering

invaluable
feedback,
guidance,
insight
and
encouragement
to
me,
and
being

such
an
excellent
advisor,
professor,
PI,
and
friend.
I
want
to
sincerely
thank

Dr.
Stallcup
with
my
whole
heart
for
his
effort
and
valuable
time.
I
would
also

like
to
sincerely
thank
other
thesis
committee
members
Dr.
Peggy
Farnham,

Dr.
Judd
Rice
and
Dr.
Zoltan
Tokes
for
their
comments
and
suggestions.

I
want
to
express
my
special
appreciation
and
thanks
to
Dr.
Chenyin

Ou
and
Dr.
Coralie
Poulard;
both
of
them
my
postdocs,
who
taught
me

research
techniques,
introduced
me
to
the
field
and
gave
me
lots
of
feedback

and
guidance.
I
highly
appreciate
their
assistance
and
involvement
in
my

growth
in
the
field
of
science.
I
also
offer
my
appreciation
to
my
colleagues,

Daniel
Gerke,
Brian
Lee,
Kiran
Sriram
and
others
for
discussions,
friendship,

and
being
a
wonderful
supportive
team.

Last
but
not
least,
I
would
like
to
thank
my
family
and
friends
who

encouraged
and
supported
me
as
I
worked
towards
my
graduate
degree.

Especially,
my
deepest
appreciation
goes
to
my
mother
(Shuqiong
Hu),
who

has
faith
in
me,
always
listens
to
me
and
supports
me.
That
means
a
lot
to
me!

I
want
to
give
her
a
big
thank!

Thank
you
very
much!

ii

Table
of
Content

Acknowledgement
.......................................................................................................
i

Table
of
Content
...........................................................................................................
ii

List
of
Figures
..............................................................................................................
iv

Abstract
.........................................................................................................................
vi

Introduction
..................................................................................................................
1

I.
Steroid
Hormone
Action:
Nuclear
Receptors
and
Nuclear
Receptor

coregulators
...................................................................................................................................
1

i.
Steroid
Hormones
......................................................................................................
1

ii.
Steroid
Hormone
Receptors
.................................................................................
2

iii.
Signaling
Pathway
–
Classic
genomic
pathway
...........................................
4

iv.
Nuclear
Receptor
Coregulators
..........................................................................
6

v.
Post-‐translational
Modifications
(PTMs)
........................................................
7

II.
Coregulator
G9a
...................................................................................................................
9

i.
G9a
Structure
and
isoforms
................................................................................
10

ii.
G9a
Corepressor
Function
..................................................................................
12

iii.
G9a
Coactivator
Function
..................................................................................
13

iv.
Post-‐translational
Modifications
(PTMs)
on
G9a
....................................
15

Results
..........................................................................................................................
20

Automethylation
on
G9a
K185
is
important
for
G9a
coactivator
function
.
20

Automethylation
on
G9a
K185
is
not
required
for
the
interaction
between

G9a
and
GR
or
ER
.................................................................................................................
22

Automethylation
on
G9a
K185
is
essential
for
its
interaction
with
HP1γ
..
24

iii

Aurora
kinase
B
interacts
with
G9a
and
the
kinase
activity
of
aurora
kinase

B
reduces
the
interaction
between
G9a
and
HP1
γ
...............................................
27

HP1
α
and
HP1
β
can
also
interact
with
G9a
...........................................................
29

Discussion
...................................................................................................................
31

[HP1
γ/Aurora
kinase
B
-‐
G9a
-‐
SHR]
Model
............................................................
31

Recognitions
and
Modifications
of
G9a
ARKT
Sequence
....................................
35

Differences
between
HP1
protein
isoforms
.............................................................
37

G9a
Coactivator
Function
.................................................................................................
39

Materials
and
Methods
...........................................................................................
42

Reference
....................................................................................................................
45

iv

List
of
Figures

Figure1.

Steroid
Hormone
Receptor
Structure

.
.........................................................................................................................................................
3

Figure
2.
Genomic
pathway
of
SHR
function

.
.........................................................................................................................................................
5

Figure
3.
Full
length
human
G9a
Structure

.
......................................................................................................................................................
11

Figure
4.
Post-‐translational
modifications
on
G9a
in
highly

conserved
sequence
mimic
histone
H3
met-‐H3K9/pho-‐H3S10
switch

.
......................................................................................................................................................
17

Figure
5.
Automethylation
on
G9a
K185
site
is
important
for
its

coactivator
function

.
......................................................................................................................................................
21

Figure
6.
Automethylation
on
G9a
K185
is
not
required
for
the

interaction
between
G9a
and
GR
or
ER

.
......................................................................................................................................................
23

Figure
7.
Automethylation
on
G9a
K185
is
essential
for
the

interaction
between
HP1
γ
and
G9a
and
the
interaction
between

HP1
γ
and
GR
or
ER
is
dependent
upon
this
automethylation
on
G9a

.
......................................................................................................................................................
26

Figure
8.
Aurora
kinase
B
interacts
with
G9a,
and
the
kinase

activity
of
aurora
kinase
B
reduces
the
interaction
between
G9a

and
HP1
γ

v

.
......................................................................................................................................................
28

Figure
9.
HP1
α
and
HP1
β
can
also
interact
with
G9a
in
cells

.
......................................................................................................................................................
30

Figure
10.
[HP1
γ/Aurora
kinase
B
-‐
G9a
-‐
NR]
Model

.
......................................................................................................................................................
34

vi

Abstract

Nuclear
receptors
regulate
transcription
of
target
genes
with
the
help
of

numerous
coregulators,
including
G9a.
G9a
is
well
known
as
a
histone

methyltransferase
and
a
corepressor,
but
our
lab
previously
found
that
G9a

also
acts
as
a
coactivator.
Moreover,
the
N-‐terminal
region
of
G9a
is

sufficient
and
required
for
coactivator
function.
Interestingly,
the
N-‐
terminal
region
of
G9a
undergoes
two
post-‐translational
modifications

(PTMs)
–
methylation
and
phosphorylation.
To
understand
how
these
two

PTMs
affect
the
mechanism
of
G9a
coactivator
function,
I
investigated
their

impact
on
the
interaction
between
G9a
and
a
few
binding
partners
of
G9a

that
interact
or
potentially
interact
with
the
G9a
N
terminal
domain
–

glucocorticoid
receptor
(GR),
estrogen
receptor
(ER)
α,
heterochromatin

protein
1
(HP1)
γ,
Aurora
kinase
B,
HP1
α
and
HP1
β.
By
performing

mutagenesis
on
two
post-‐translational
modification
sites
and
co-‐
immunoprecipitation,
I
studied
whether
the
G9a
PTMs
are
important
for

these
interactions
and
the
underlying
molecular
mechanism
of
G9a

coactivator
function.
The
results
show
that
the
automethylation
site
may

not
be
important
for
the
interaction
of
G9a
with
ERα
or
GR.
However,
HP1γ

interacts
only
with
the
methylated
form
of
G9a
concomitant
with
the

interaction
of
HP1γ
with
ERα
or
GR.
Nevertheless,
phosphorylation
of
G9a

by
aurora
kinase
B
seems
to
prevent
the
interaction
between
HP1γ
and
G9a.

The
results
suggest
that
these
PTMs
regulate
G9a
coactivator
activity
by

regulating
the
interaction
of
G9a
with
HP1γ.

1

Introduction

I.
Steroid
Hormone
Action:
Nuclear
Receptors
and
Nuclear
Receptor

coregulators

i. Steroid
Hormones

Steroid
hormones
(SHs)
are
lipophilic
molecules
synthesized
in
the

adrenal
gland
(e.g.
glucocorticoids
and
adrenal
androgens),
testes
(e.g.

testicular
androgens
and
estrogen),
and
ovary
and
placenta
(e.g.
estrogens).

SHs
bind
to
carrier
proteins
and
are
transported
through
blood
circulation

to
the
target
cells
(Beato
and
Klug
2000).

Steroid
hormones
have
been
known
to
play
important
roles
in
many

different
physiological
responses,
such
as
regulation
of
the
nervous
system,

various
types
of
metabolism,
immune
system
regulation,
conception,
and

intrauterine
fetal
development,
all
through
genomic
action
as
well
as
via

non-‐genomic
effects
(Falkenstein
et
al.
2000).
For
example,
glucocorticoids

regulate
transcription
of
many
genes
that
govern
important
physiological

pathways
such
as
metabolism
of
glucose,
fat
and
bone,
as
well
as
the

immune,
central
nervous
and
reproductive
systems.
Estrogen
plays
crucial

roles
in
mammary
gland
and
the
reproductive
tract,
as
well
as
the
central

nervous
system.
Androgens
regulate
reproduction
in
both
males
and

females
(Treviño
and
Weigel
2013).

2

ii. Steroid
Hormone
Receptors

SHs
bind
to
steroid
hormone
receptors
(SHRs),
which
are
key

mediators
of
SH
action.
SHRs
are
transcription
factors
that
belong
to
the

nuclear
receptor
(NR)
superfamily.
There
are
several
members
of
the
SHR

family,
such
as
the
estrogen
receptor
(ER)
and
the
glucocorticoid
receptor

(GR).
Many
SHRs,
including
ER
and
GR,
have
different
isoforms
derived

from
distinct
genes
or
the
same
gene
(Treviño
and
Weigel
2013).

Generally,
steroid
hormone
receptors
are
composed
of
a
ligand-‐
binding
domain
(LBD),
a
DNA-‐binding
domain
(DBD),
a
hinge
region,
and

two
activation
domains
(AFs)
distributed
along
the
molecule
(Beato,

Herrlich,
and
Schütz
1995)
(Fig.1).
The
ligand-‐biding
domain
(LBD)
has
a

ligand-‐binding
cavity
that
can
recognize
and
bind
to
a
ligand.
The
DNA

binding
domain
(DBD)
has
2
zinc
finger
motifs,
which
allows
steroid

hormone
receptors
to
homo-‐
or
hetero-‐
dimerize
and
make
base-‐specific

contacts
with
palindromic
DNA
sequences.
Between
the
LBD
and
the
DBD,

there
is
a
hinge
region
that
connects
the
domains.
The
activation
functional

domain
2
(AF-‐2)
is
located
in
the
C-‐terminus
of
the
LBD
and
has
ligand-‐
dependent
transcriptional
activation
function.
The
activation
functional

domain
1
(AF-‐1)
is
located
in
the
N
terminal
region
of
SHR
and
is
involved

in
ligand-‐independent
transcriptional
activation
(Beato,
Chávez,
and
Truss

1996;
Rosenfeld,
Lunyak,
and
Glass
2006).

3

Figure1.

Steroid
Hormone
Receptor
Structure.

N:
N-‐terminus;
AF-‐1:
activation
function
domain
1;
DBD:
DNA-‐binding

domain;
LBD:
ligand-‐binding
domain;
AF-‐2:
activation
function
domain
2;
C:

C-‐terminus.

4

iii. Signaling
Pathway
–
Classic
genomic
pathway

Steroid
hormone
receptors
(SHRs)
have
been
characterized
to

function
in
two
major
ways:
(1)
a
genomic
pathway
(Fig.
2),
which
is

characterized
by
a
specific
delay
between
hormone
administration
and

biological
responses,
and
a
sensitivity
toward
inhibitors
of
transcription

and
translation,
and
(2)
a
non-‐genomic
pathway,
which
is
characterized
by

rapid
effects
that
involve
signaling
pathways
such
as
MAPK
and
Akt
(Davis

et
al.
2011).

In
the
genomic
pathway,
SHs
pass
through
the
cell
membrane
by

simple
diffusion
due
to
their
lipophilic
nature.
SHs
bind
to
specific
SHRs,

which
are
bound
by
a
complex
of
molecular
chaperones
in
the
absence
of

ligands.
These
molecular
chaperones,
e.g.
heat
shock
protein
90
(Hsp90),

help
folding
and
prevent
aggregation
of
SHRs.
Upon
binding
of
steroid

hormones,
SHRs
undergo
allosteric
conformational
changes,
disassociate

from
the
chaperone
complex
(Fig.
2,
Step
2b),
homo-‐
or
hetero-‐
dimerize,

localize
to
the
nucleus,
bind
to
hormone
responsive
elements
(HRE)
on
DNA

or
to
other
transcription
factors
(TFs),
recruit
coregulator
complexes,
and

interact
with
components
of
the
basal
transcriptional
machinery
to
exert

positive
or
negative
effects
on
the
regulation
of
target
genes
expression

(Giguère
et
al.
1988;
Glass
and
Rosenfeld
2000).

5

Figure
2.
Genomic
pathway
of
SHR
function.

(1)
SHs
pass
the
cell
membrane
by
simple
diffusion;
(2)
SHs
bind
to
a

specific
steroid
hormone
receptor
(SHR);
(2a&b)
SHR
disassociate
from
the

chaperone
complex
(Hsp
90);
(3)
SHRs
homo-‐
or
hetero-‐
dimerize;
(4)
SHRs

localize
to
nucleus
and
(a)
bind
to
a
hormone
response
element
(HRE)
or
(b)

bind
to
other
transcription
factors
(TF)
bound
to
their
DNA
response

element
(RE),
then
recruit
a
coregulator
complex;
(5)
the
coregulator

complex
interacts
with
the
transcriptional
machinery
and
regulates

transcription;
(6)
mRNA
transcription
is
affected
accordingly;
(7)
the

translation
process
is
influenced
subsequently;
(8)
physiological
response

to
specific
SHs.

6

iv. Nuclear
Receptor
Coregulators

In
the
past
two
decades,
a
large
number
of
studies
have
demonstrated

that
SHRs
mediate
the
action
of
steroid
hormones
in
close
collaboration

with
coregulators.

SHRs,
upon
recognizing
and
binding
HREs
on
DNA,
initiate
the

recruitment
of
numerous
coregulators,
which
include
coactivators
and

corepressors.
Coactivators
are
molecules
recruited
primarily
in
a
ligand

dependent
manner
by
nuclear
receptors
(NRs)
or
other
transcription

factors
(TFs)
to
enhance
NR-‐mediated
gene
expression.
Corepressors,
on

the
other
hand,
are
molecules
recruited
by
NRs
or
TFs
to
repress
gene

expression
(Lonard,
Lanz,
and
O’Malley
2007).
Many
coregulators
have

been
reported
to
exhibit
both
coactivator
function
and
corepressor
function

and
function
differently
in
different
circumstances
(Lonard
et
al.
2007).

Both
of
these
two
functions
are
important
in
the
delicate
balance
of

transcription
in
many
physiological
processes
(Feige
and
Auwerx
2007).

Coregulators
are
responsible
for
remodeling
the
chromatin
structure

and
promoting/inhibiting
the
assembly/disassembly
of
transcription

complexes.
It
is
assumed
that
coregulators
mediate
these
functions
through

two
major
mechanisms:
protein-‐protein
interactions
and/or
executing
a

series
of
enzymatic
modifications
required
for
regulated
gene
expression

(Glass
and
Rosenfeld
2000;
Rosenfeld
et
al.
2006).
Protein-‐protein

interactions
can
be
controlled
at
several
levels,
including
cofactor
activity,

stability,
intracellular
localization,
and
conformational
changes
that
alter

7

the
availability
of
binding
recognition
motifs.
Many
coactivators
have
been

observed
to
contain
one
or
more
copies
of
the
LXXLL
motif,
where
L

represents
leucine
and
X
any
amino
acid,
that
represents
a
general
structure

for
NR
recognition
(Glass
and
Rosenfeld
2000).
Coregulators
that
exhibit

enzymatic
activities
can
be
classified
into
two
classes.
(a)
Enzymes
that
are

capable
of
conducting
post-‐translational
modifications
(PTMs)
such
as

methylation
or
demethylation,
acetylation
or
deacetylation,

phosphorylation
or
dephosphorylation,
ubiquitination,
SUMOylation,
and

poly(ADP-‐ribosyl)ation.
(b)
Enzymes
that
contain
ATPase
activity
and

remodel
chromatin
structure
(and
often
cause
nucleosome
sliding)
in
ATP-‐
dependent
manner
(e.g.
the
SWI/SNF
complex)
(Rosenfeld
et
al.
2006).

Therefore,
understanding
the
fine-‐tuned
regulation
of
coregulators
is

critical
for
comprehending
the
regulation
of
transcription.
However,
the

mechanisms
of
the
switch
and
balance
between
coactivator
and
corepressor

function
of
a
specific
coregulator
are
unknown
and
remain
a
fundamental

question.

v. Post-‐translational
Modifications
(PTMs)

As
mentioned
above,
besides
the
cross
talk
between
nuclear
receptors

and
coregulators,
post-‐translational
modifications
(PTMs)
add
another

layer
to
the
regulatory
complexity.
PTMs
on
NRs
and
coregulators
are

involved
in
NR
and
coregulator
stability,
activity,
function,
intracellular

localization,
and
structures
that
influence
specific
bindings
in
the
cross-‐
talking
network
of
NRs,
coregulators
and
histone
marks
(Anbalagan
et
al.

8

2012;
Han,
Lonard,
and
O’Malley
2009).
In
order
to
understand
the
roles
of

PTMs
in
transcriptional
regulation,
intensive
studies
have
been
conducted

to
decipher
the
“PTM
code”
constructed
by
acetylation,
methylation,

phosphorylation,
ubiquitylation,
sumoylation
and
even
a
combination
of

such
modifications.

1) Post-‐translational
modifications
on
Nuclear
Receptors

PTMs
on
nuclear
receptors
have
been
reported
to
play
critical
roles
in

many
different
functional
mechanisms
in
physiological
processes
and

dysfunctional
effects
in
various
diseases
(Anbalagan
et
al.
2012).
For

example,
many
PTMs
have
been
found
on
estrogen
receptor
(ER)
α,
and

these
PTMs
have
been
reported
to
play
crucial
roles
in
modifying
ERα

expression
and
stability,
intracellular
localization,
and
sensitivity
to

hormonal
response.
To
name
one,
phosphorylations
on
serines
in
the
AF-‐1

domain
of
ERα
are
involved
in
ligand-‐independent
regulation
of
ERα-‐
mediated
transactivation
(Le
Romancer
et
al.
2011).

2) Post-‐translational
modifications
on
Coregulators

PTMs
on
many
coregulators
also
have
been
found
to
play
crucial
roles

in
transcription
regulation
control
(e.g.
the
switch
between
coactivator
and

corepressor
function
of
a
particular
coregulator)
and
can
influence

physiological
processes.
For
example,
the
methylation
of
steroid
receptor

coactivator
3
(SRC-‐3),
which
belongs
to
the
p160
coregulator
family,
has

been
found
to
induce
the
disassociation
between
SRC-‐3
and
CARM1
on
ERα

9

target
gene
promoters,
which
contributes
to
the
coactivator
complex

disassociation
in
promoter
clearance
during
multiple-‐rounds
of

transcription
(Han
et
al.
2009).

II.
Coregulator
G9a

G9a
is
a
histone
methyltransferase,
which
preferentially
mono-‐
or
di-‐
methylates
histone
H3
at
Lys-‐9
(H3K9),
and
to
a
lesser
extent,
Lys-‐27

(H3K27)
(Peters
et
al.
2003;
Rice
et
al.
2003;
Tachibana
et
al.
2001,
2002,

2005).
G9a
is
associated
with
transcriptional
repression
events
in

euchromatin
(Hwang,
Eissenberg,
and
Worman
2001;
Tachibana
et
al.
2002,

2005).
However,
it
has
also
been
shown
to
activate
transcription
in

collaborate
with
several
nuclear
receptors
(Bittencourt
et
al.
2012;
Lee
et
al.

2006;
Purcell
et
al.
2011),
transcription
factor
RUNX2
(Purcell
et
al.
2012)

and
hematopoietic
activator
NF-‐E2
(Chaturvedi
et
al.
2009).

G9a
plays
a
critical
role
in
a
wide
range
of
physiological
pathways

(Shankar
et
al.
2013).
For
example,
G9a
has
been
shown
to
be
important
for

the
regulation
of
genes
required
for
normal
embryonic
development

(Tachibana
et
al.
2002,
2005)
and
immune
responses
are
affected
when
G9a

is
knocked
out
in
lymphocytes
(Thomas
et
al.
2008).
G9a
has
also
been

shown
to
repress
PPARγ
expression
and
adipogenesis
(Li
et
al.
2013;
Wang

et
al.
2013).
In
addition,
both
G9a
and
GLP
(G9a-‐like
methyltransferase)

have
been
found
to
methylate
and
inactive
the
tumor
suppressor
protein

p53
(Huang
et
al.
2010).
Consistent
with
this
finding,
down-‐regulation
of

10

G9a
has
been
shown
to
suppress
cancer
cell
growth,
by
inducing

centrosome
disruption
and
chromosome
instability
(Kondo
et
al.
2008),
and

invasion
and
metastasis
of
lung
cancer
in
mice,
by
silencing
the
cell

adhesion
molecule
Ep-‐CAM
(Chen
et
al.
2010).

i. G9a
Structure
and
isoforms

G9a
belongs
to
the
Su(var)3-‐9
family
of
methyltransferases
and
is

composed
a
SET
domain,
six
ankyrin
repeats,
a
cysteine-‐rich
region,
a

polyglutamate
region,
nuclear
localization
signals,
and
an
activation
domain

(Fig.
3).
The
SET
domain
is
the
catalytic
domain
for
methyltransferase

activity
(Shankar
et
al.
2013;
Tachibana
et
al.
2001).
The
ankyrin
repeat

domain
has
been
identified
to
be
a
mono-‐
and
dimethyllysine
binding

module,
important
for
protein-‐protein
interactions
(Collins
et
al.
2008;

Milner
and
Campbell
1993),
and
has
been
reported
to
play
roles
in
DNA

methyltransferase
recruitment
(Epsztejn-‐Litman
et
al.
2008).
In
addition,
a

specific
residue
of
the
ankyrin
repeat
domain
(Asp905)
is
essential
for

binding
of
an
unknown
protein
that
is
involved
in
embryonic
stem
cell
self-‐
renewal
and
differentiation
(Bittencourt
et
al.
2014).
A
nuclear
localization

signal
has
been
identified
in
the
N
terminal
region
of
human
G9a
(Estève
et

al.
2005)
and
amino
acids
1-‐280
of
human
G9a
have
been
previously
shown

to
be
important
and
sufficient
for
coactivator
function
in
transient
reporter

gene
assays
(Purcell
et
al.
2011).
Moreover,
the
N-‐terminal
region
of
G9a

contains
an
autonomous
activation
domain
(Lee
et
al.
2006).
In
addition,
Dr.

11

Stallcup’s
group
has
previously
shown
that
G9a
binds
to
ERα
(Purcell
et
al.

2011)
and
GR
(Bittencourt
et
al.
2012)
via
its
N
terminal
region.

Figure
3.
Full
length
human
G9a
Structure.

The
numbering
shows
the
location
of
amino
acid
residues.
N:
N-‐terminus;
1-‐
280
residues
that
are
critical
for
G9a
coactivator
function
are
shown
in
red;

E:
polyglutamate;
NLS:
nuclear
localization
signal;
Cys:
cysteine-‐rich
region;

ANK:
six
ankyrin
repeats;
Pre
and
Post:
pre-‐
and
post-‐SET
domains;
SET:
H3

Lys-‐9
methyltrasferase
domain.
C:
C-‐terminus.

12

ii. G9a
Corepressor
Function

As
mentioned
above,
G9a
is
a
coregulator
that
has
been
shown
to
play

roles
in
both
repression
and
activation
of
gene
transcription.
Intensive

studies
have
shown
that
G9a
has
been
recruited
to
the
transcription

complexes
of
some
genes
by
transcription
factors,
such
as
CCAAT

displacement
protein/cut
(CDP/cut)
(Nishio
and
Walsh
2004),
growth

factor
independent
1
(Gfi1)
(Duan
et
al.
2005),
positive
regulatory
domain
I-‐
binding
factor
1
(PRDI-‐BF1)
(Gyory
et
al.
2004),
neuron
restrictive
silencing

factor
(NRSF)
(also
known
as
REST)
(Roopra
et
al.
2004),
multi-‐domain

protein
UHRF1
(Kim
et
al.
2009),
and
the
noncoding
RNA
Air
(Nagano
et
al.

2008)
to
remodel
chromatin
structure
or
to
recruit
other
cofactors
and

repress
active
gene
transcription.

The
primary
mechanism
of
G9a
corepressor
function
is
via

methylating
the
histone
H3
N
terminal
tail
in
euchromatin
and
associating

with
corepressor
complexes.
G9a
preferentially
executes
methylation
on

histone
H3
at
Lys-‐9
(H3K9),
and
Lys-‐27
(H3K27)
to
a
lesser
extent
(Peters

et
al.
2003;
Rice
et
al.
2003;
Tachibana
et
al.
2001,
2002,
2005),
which
is

associated
with
transcriptional
repression
(Hwang
et
al.
2001;
Tachibana
et

al.
2002,
2005).
Methylation
of
H3K9
leads
to
the
recruitment
of
proteins

such
as
heterochromatin
protein
1
(HP1),
which
can
bind
to
the
Lys-‐9

methylated
histone
3
tail
(H3K9)
via
a
chromo
domain.
This
recruitment

starts
silencing
mechanisms
by
either
assembling
heterochromatin
(mainly

caused
by
H3K9me3)
or
by
silencing
gene
transcription
by
different

13

mechanisms
(Bannister
et
al.
2001;
Jacobs
and
Khorasanizadeh
2002;

Lachner
et
al.
2001;
Lomberk,
Wallrath,
and
Urrutia
2006).
There
are
also

other
mechanisms
of
G9a
corepressor
function.
For
example,
the
ankyrin

repeat
domain
of
G9a
has
been
reported
to
contribute
to
DNA
methylation-‐
mediated
repression
of
transcription
by
recruiting
DNA
methyltranferases

(Dnmt3a
and
Dnmt3b),
and
by
recognizing
the
H3K9me2
histone
mark

(Collins
et
al.
2008;
Epsztejn-‐Litman
et
al.
2008).
The
ankyrin
repeats

domain
has
also
been
reported
to
play
roles
in
G9a
corepressor
function

through
mechanisms
involving
a
specific
amino
acid
in
the
ankyrin
repeat

(Bittencourt
et
al.
2014).
Furthermore,
G9a
has
been
shown
to
repress
the

transcription
of
IFN-‐γ
without
its
known
catalytic
activity,
indicating
that

some
other
mechanism
might
be
responsible
for
its
corepressor
function

(Lehnertz
et
al.
2010).

iii. G9a
Coactivator
Function

Even
though
the
corepressor
function
of
G9a
has
been
well
studied

and
established,
there
are
reports
showing
that
G9a
can
function
as
a

coactivator,
contributing
to
the
activation
of
gene
expression.

The
G9a
coactivator
function
was
first
observed
by
Dr.
Stallcup’s

group
using
a
transient
report
gene
assay;
these
studies
showed
that
the

mechanism
of
G9a
coactivator
function
is
methyltransferase
activity

independent
(Lee
et
al.
2006).
Subsequently,
the
coactivator
function
of
G9a

on
endogenous
genes
was
also
observed
(Bittencourt
et
al.
2012;

14

Chaturvedi
et
al.
2009,
2012;
Kondo
et
al.
2008;
Lehnertz
et
al.
2010;
Oh
et

al.
2014;
Purcell
et
al.
2011,
2012).

Some
studies
have
been
conducted
to
understand
the
coactivator

function
of
G9a.
Dr.
Stallcup’s
group
successfully
identified
that
the
N

terminal
region
of
G9a
(1-‐280
in
human
G9a)
is
sufficient
and
required
for

its
coactivator
function
in
transient
reporter
gene
assays
(Purcell
et
al.
2011)

and
contains
an
autonomous
activation
domain
(Lee
et
al.
2006).
G9a
has

been
reported
to
cooperate
with
other
coactivators
such
as
Grip1,
CARM1

and
p300
to
activate
transcription
in
the
context
of
nuclear
receptors
in
a

methyltransferase
activity-‐independent
manner
(Lee
et
al.
2006).

Consistent
with
that
finding,
G9a
has
been
reported
to
function
as
a
scaffold

protein
to
recruit
p300
and
CARM1
on
a
subset
of
GR
target
genes

(Bittencourt
et
al.
2012).
G9a
has
also
been
reported
to
be
recruited
by

RUNX2
to
the
promoters
of
target
genes
to
activate
their
transcription
in
a

methyltransferase
activity-‐independent
manner
(Purcell
et
al.
2012).
In

addition,
it
is
reported
that
G9a
stabilizes
Mediator
occupancy
on
the

promoter
of
the
adult
β
globin
gene
to
exert
its
coactivator
function,
while
it

recruits
the
H3K4
demethylase
Jarid1a
to
the
promoter
of
the
embryonic
β

globin
gene
and
results
in
corepressing
transcription
(Chaturvedi
et
al.

2009,
2012).
It
is
suggested
that
different
binding
partners
may
play
critical

roles
in
the
switch
between
G9a
coactivator
and
corepressor
function.
It
has

also
been
shown
that
G9a
is
recruited
to
the
promoter
or
enhancer
regions

of
its
positively
regulated
target
genes,
indicating
that
G9a
may
act
directly

15

on
their
expression
(Bittencourt
et
al.
2012;
Chaturvedi
et
al.
2009,
2012;

Lee
et
al.
2006;
Oh
et
al.
2014;
Purcell
et
al.
2011,
2012).
In
addition,
G9a
is

shown
to
bind
to
RNA
polymerase
II,
indicating
that
G9a
may
play
a
role
in

the
establishment
of
a
preinitiation
or
reinitiation
complex
during

transcription
(Chaturvedi
et
al.
2009).

iv. Post-‐translational
Modifications
(PTMs)
on
G9a

As
mentioned
above,
the
N
terminal
region
of
G9a
is
sufficient
and

required
for
its
coactivator
function
in
transient
reporter
gene
assays

(Purcell
et
al.
2011).
Interestingly,
two
PTMs
occur
in
the
G9a
N
terminal

region.
G9a
catalyses
its
own
automethylation
on
Lys-‐239
in
mouse
G9a

(Chin
et
al.
2007),
which
is
Lys-‐185
in
human
G9a
(hG9a).
Moreover,
aurora

kinase
B
catalyses
in
vitro
phosphorylation
of
Thr-‐186
of
human
G9a

adjacent
to
Lys-‐185
(Sampath
et
al.
2007).

1) [H3K9
methylation
-‐
H3S10
phosphorylation
switch]
histone
mimic

The
post-‐translational
modifications
of
the
G9a
N-‐terminal
region
are

in
an
ARKT
sequence,
which
closely
resembles
the
G9a
consensus
sequence

ARKS
found
on
histone
H3K9.
The
lysine
methylation
and
threonine

phosphorylation
on
this
ARKT
sequence
in
G9a
mimics
the
[H3K9

methylation
-‐
H3S10
phosphorylation
switch]
on
histone
3
ARKS
sequence

(Sampath
et
al.
2007).

MetH3K9-‐phoH3S10
is
a
switch
motif
controlling
the
chromatin

structure
during
mitosis
(Fischle,
Wang,
and
Allis
2003;
Fischle
et
al.
2005;

16

Hirota
et
al.
2005)
(Fig.
4b)
and
some
other
nuclear
functions.
The

methylation
of
histone
H3
lysine
9
is
performed
by
SET-‐domain
histone

methyltransferases
such
as
G9a
and
Su(Var)3-‐9
(Peters
et
al.
2003;
Rice
et

al.
2003;
Tachibana
et
al.
2001,
2002,
2005),
which
create
a
binding
site
for

proteins
such
as
HP1
(Jacobs
et
al.
2001;
Nielsen
et
al.
2002)
and
Polycomb

(Fischle,
Wang,
Jacobs,
et
al.
2003).
The
phosphorylation
of
H3
serine
10
is

performed
by
kinases
such
as
aurora
kinase
B
(Cheung,
Allis,
and
Sassone-‐
Corsi
2000;
Fischle
et
al.
2005;
Hsu
et
al.
2000).
The
phosphorylation
on

H3S10
by
aurora
kinase
B
displaces
the
HP1
protein
that
was
bound
to
the

methylation
on
H3K9
and
contributes
to
the
chromatin
structure

remodeling.
Interestingly,
besides
the
role
this
mechanism
plays
in
forming

heterochromatin,
this
mechanism
has
been
reported
to
be
involved
in
gene

activation,
in
response
to
thyroid
hormone
(Tardáguila,
González-‐Gugel,

and
Sánchez-‐Pacheco
2011).

Besides
histone
H3
and
G9a,
this
sequence
is
found
to
be
highly

conserved
in
several
other
non-‐histone
protein,
such
as
GLP
(ARKT),
and

mAM
(ARKT)
(Chin
et
al.
2007;
Sampath
et
al.
2007)
(Fig.
4a).

17

Figure
4.
Post-‐translational
modifications
on
G9a
in
highly
conserved

sequence
mimic
histone
H3
met-‐H3K9/pho-‐H3S10
switch.

(A)
Partial
protein
sequence
alignment
of
histone
H3,
human
G9a
(hG9a),

mouse
G9a
(mG9a),
human
GLP
(hGLP),
mouse
GLP
(mGLP),
mAM:
a

SetDB1
methyltransferase
activator
protein.
Highly
conserved
motifs
are

underlined.
Putative
methylacceptor
sites
are
shown
in
red,
putative

phosphoacceptor
sites
are
shown
in
blue.
(B)
A
model
of
the
met-‐
H3K9/pho-‐H3S10
switch
mechanism.
HP1
proteins
recognize
tri-‐
methylation
on
H3K9.
Phosphorylation
by
aurora
kinase
B
is
sufficient
to

prevent
the
binding
between
HP1
proteins
and
H3K9me3
histone
mark.

18

2) Putative
binding
partners
on
G9a
PTMs

Besides
recognizing
methylated
H3K9,
it
has
been
reported
that
HP1γ

can
interact
with
G9a,
and
the
automethylation
on
G9a
K185
is
important

for
this
interaction.
Interestingly,
two
other
members
in
the
mammalian

HP1
family,
HP1
α
and
β,
can
also
interact
with
G9a
in
vitro
(Chin
et
al.
2007;

Sampath
et
al.
2007).
The
three
HP1
proteins
are
encoded
by
different

genes
that
belong
to
the
chromobox
(CBX)
genes
class
(Lomberk
et
al.

2006).
Since
these
genes
encode
proteins
with
distinct
localization
patterns,

despite
similar
structures,
these
three
members
of
HP1
family
may
exert

different
functions
by
different
mechanisms
(Vermaak,
Henikoff,
and
Malik

2005).
While
HP1
proteins
have
been
primarily
associated
with

heterochromatin,
HP1γ
has
also
been
found
to
associate
with
functions
in

euchromatin
and
even
associate
with
positive
regulation
of
gene
expression

(Piacentini
and
Pimpinelli
2010;
Vakoc
et
al.
2005).

Furthermore,
while
aurora
kinase
B
is
responsible
for
the

phosphorylation
of
H3
serine
10
(Cheung
et
al.
2000;
Fischle
et
al.
2005;

Hirota
et
al.
2005;
Hsu
et
al.
2000),
it
has
also
been
reported
to

phosphorylate
G9a
on
the
threonine
in
ARKT
consensus
sequence,
and
this

phosphorylation
has
been
reported
to
prevent
the
interaction
between
G9a

and
HP1γ
(Sampath
et
al.
2007).

19

In
summary,
two
PTMs,
methylation
and
phosphorylation,
are
found

in
the
highly
conserved
sequence
ARKT
motif
in
the
G9a
N
terminal
domain,

which
is
required
and
sufficient
for
G9a
coactivator
function.
More

interestingly,
these
two
PTMs
mimic
the
histone
H3
metH3K9-‐phoH3S10

switch,
indicating
a
possible
underlying
mechanism
of
these
two
PTMs
for

the
switch
of
G9a
coactivator
and
corepressor
function.

To
understand
how
these
PTMs
affect
the
coactivator
function
of
G9a,

I
investigated
the
impact
of
these
two
PTMs
on
the
interaction
between
G9a

and
its
N-‐terminus
binding
partners,
including
estrogen
receptor
(ER)
α,

glucocorticoid
receptor
(GR),
Heterochromatin
protein
1
(HP1)
γ,
Aurora

kinase
B,
HP1
α
and
HP1
β.

20

Results

Automethylation
on
G9a
K185
is
important
for
G9a
coactivator
function

Before
I
started
working
on
this
project,
Coralie
Poulard
performed

transient
luciferase
reporter
gene
assays
using
G9a
having
various

mutations
at
Lys-‐185.
The
level
of
luciferase
reporter
gene
expression
is

dependent
upon
the
activation
of
the
steroid
receptor
and
its
coactivators.

When
dexamethasone
(dex)
or
estradiol
(E2)
was
added
to
cells,
along
with

plasmids
encoding
the
glucocorticoid
receptor
or
estrogen
receptor,

luciferase
activity
was
increased
accordingly
(Fig.
5b
and
5c,
compare
bars

1
and
2).
When
Grip1,
which
is
a
well-‐known
coactivator,
was
added
to
the

system,
luciferase
activity
was
higher
than
when
only
adding
nuclear

receptors
(Fig.
5b
and
c,
bars
2-‐3).
When
hG9a
full
length
wild
type
(hG9a

FL
WT)
was
added
to
the
system,
it
cooperated
with
Grip1
to
activate
the

transcription
of
luciferase
reporter
gene.
The
transcription
activity
of

luciferase
reporter
gene
was
even
higher
when
the
concentration
of
G9a

was
higher
(Fig.
5b
and
c,
bars
2-‐5).
When
the
automethylation
site
K185
on

G9a
was
mutated
from
K
to
A
(hG9aFL
K185A)
or
R
(hG9aFL
K185R),
the

methylation
cannot
be
made
on
that
site
anymore
(Fig.
5a)
and
at
the
same

time
luciferase
activity
was
less
(Fig.
5b
and
c,
bars
4-‐9),
indicating
the

importance
of
the
automethylation
on
G9a
K185
for
the
G9a
coactivator

function.
The
level
of
G9a
expression
was
examined
by
western
blot
using

the
cell
extracts.

21

Figure
5.
Automethylation
on
G9a
K185
site
is
important
for
its

coactivator
function.

(Data
from
Coralie
E.
Poulard,
PhD.)
(A):
in
vitro
methlytion
assay.
GST

hG9a
N
wild
type
or
mutant
proteins
were
methylated
by
GST
mG9a
deltaN

(containing
SET
domain)
in
presence
of
radioactive
methyl
donor
SAM.

Reaction
products
were
analysed
by
SDS
PAGE
followed
by
flurography.

The
amount
of
GST
protein
was
checked
by
coomassie
staining.
(B)
and
(C):

CV-‐1
cells
were
transfected
with
MMTV(GRE)-‐LUC
(B)
or
MMTV(ERE)-‐LUC

(C)
reporter
plasmids
(200
ng)
and
expression
vectors
encoding
GR
(1
ng),

ER
α
(2
ng),
Grip1
(50
ng),
hG9aFL
WT
(150
ng
and
400
ng),
hG9aFL
K185A

(150
ng
and
400
ng),
and
hG9aFL
K185R
(150
ng
and
400
ng)
as
indicated.

Cells
were
grown
with
or
without
100
nM
of
dexamethasone
(dex)
or
20
nM

of
estradiol
(E2)
as
indicated
and
assayed
for
luciferase
activity.
Whole-‐cell

extracts
were
analyzed
for
G9a
expression
by
immunoblotting
with
anti-‐HA

antibody.

22

Automethylation
on
G9a
K185
is
not
required
for
the
interaction

between
G9a
and
GR
or
ER

To
further
investigate
the
importance
of
G9a
K185
automethylation
and
the

underlying
mechanism
of
G9a
interactions,
I
studied
the
impact
of
this

automethylation
on
the
interaction
between
G9a
and
the
glucocorticoid

receptor
(GR)
or
the
estrogen
receptor
(ER)
α
by
co-‐immunoprecipitation

(Co-‐IP)
experiments.
Dr.
Stallcup’s
group
previously
observed
that
G9a

binds
to
ER
α
(Purcell
et
al.
2011)
and
GR
(Bittencourt
et
al.
2012)
using
its

N
terminal
region.
Immunoprecipitation
(IP)
of
flag-‐tagged
GR
(Fig.
6a)
or

ERα
(Fig.
6b)
resulted
in
the
co-‐immunoprecipitation
of
HA-‐tagged
hG9a.

When
the
automethylation
site
on
G9a
K185
is
mutated
from
K
(HA
hG9a
FL

WT)
to
R
(HA
hG9a
FL
K185R),
the
interaction
between
G9a
and
GR
(Fig.
6a)

or
ERα
(Fig.
6b)
was
not
affected,
indicating
that
the
automethylation
on

G9a
K185
is
not
required
for
the
interaction
between
G9a
and
GR
or
ERα.

23

Figure
6.
Automethylation
on
G9a
K185
is
not
required
for
the

interaction
between
G9a
and
GR
or
ER.

(A)
and
(B):
Cos7
cells
were
transfected
with
pcdna3-‐hGR
(full-‐length)
or

pSG5-‐hERα
(full-‐length)
and
pSG5-‐HA-‐hG9a
WT
(full-‐length)
or
pSG5-‐HA-‐
hG9a
K185R
(full-‐length).
GR
or
ERα
were
immunoprecipitated
from
cell

extracts
with
an
anti-‐GR
antibody
or
anti-‐ERα
antibody,
as
indicated.

Proteins
bound
were
analyzed
by
immunoblot
with
anti-‐GR
or
anti-‐ERα

antibody
to
control
for
levels
of
immunoprecipitated
GR
and
ERα,
or
with

anti-‐HA
antibody
to
detect
G9a.
A
3%
input
cell
extract
sample
was
directly

loaded
for
immunoblot
as
control
of
G9a
(anti-‐HA
antibody)
and
GR
(anti-‐
GR
antibody)
or
ERα
(anti-‐ERα
antibody)
and
actin
(anti-‐actin
antibody).

Results
shown
are
from
a
single
experiment,
which
is
representative
of
two

independent
experiments.

24

Automethylation
on
G9a
K185
is
essential
for
its
interaction
with
HP1γ

Two
groups
previously
described
that
the
automethylation
site
is
essential

for
the
interaction
between
G9a
and
HP1γ
protein
(Chin
et
al.
2007;

Sampath
et
al.
2007).
In
addition,
it
appears
that
the
phosphorylation
on

G9a
T186
prevents
the
interaction
between
G9a
and
HP1γ
(Sampath
et
al.

2007).
Thus,
to
further
investigate
the
importance
of
the
automethylation

on
G9a
K185
for
G9a
coactivator
function,
I
studied
the
interaction
of
HP1γ

with
G9a
or
nuclear
receptors
(GR
or
ER).
Co-‐immunoprecipitation

experiments
were
conducted.
Immunoprecipitation
(IP)
of
HP1γ
causes
the

co-‐immunoprecipitation
of
HA-‐tagged
G9a,
GR
(Fig.
7a)
and
ER
(Fig.
7b).

When
the
automethylation
site
on
G9a
K185
is
mutated
from
K
(HA
hG9a
FL

WT)
to
R
(HA
hG9a
FL
K185R)
or
A
(HA
hG9a
FL
K185A),
HP1γ
failed
to

interact
with
G9a
(Fig.
7a
and
7b),
confirming
that
the
automethylation
on

G9a
K185
is
required
for
the
interaction
between
HP1γ
and
G9a.
And

surprisingly,
we
found
that
the
interaction
between
HP1γ
and
GR
or
ER
was

also
affected
when
the
automethylation
site
on
G9a
K185
is
mutated,

indicating
that
the
interaction
between
HP1γ
and
GR
or
ER
is
dependent
on

the
methylation
status
of
G9a
(Fig.
7a
and
7b).
In
addition,
when
T186
is

mutated
from
T
(HA
hG9a
FL
WT)
to
E
(HA
hG9a
FL
T186E),
the
charge
of

glutamic
acids
(E)
mimics
the
phosphorylation
of
threonine
(T)
and
the

interaction
between
HP1γ
and
G9a
is
inhibited.
However,
when
T186
is

mutated
from
T
(HA
hG9a
FL
WT)
to
A
(HA
hG9a
FL
T186A),
HP1γ
interacts

less
with
G9a
(Fig.
7a
and
7b),
indicating
that,
since
the
mutation
from
T
to

25

E
or
A
doesn’t
really
impair
the
stability
of
G9a,
the
interaction
between

HP1
γ
and
G9a
seems
to
require
the
methylation
on
G9a
K185
and
the

specific
sequence
of
ARKT
on
G9a.

26

Figure
7.
Automethylation
on
G9a
K185
is
essential
for
the
interaction

between
HP1
γ
and
G9a
and
the
interaction
between
HP1
γ
and
GR
or

ER
is
dependent
upon
this
automethylation
on
G9a.

(A)
and
(B):
Cos7
cell
were
transfected
with
pcdna3-‐hGR
(full-‐length)
or

pSG5-‐hERα
(full-‐length)
and
pSG5-‐HA-‐hG9a
WT
(full-‐length)
or
pSG5-‐HA-‐
hG9a
K185R
(full-‐length)
or
pSG5-‐HA-‐hG9a
K185A
(full-‐length)
or
pSG5-‐
HA-‐hG9a
T185E
(full-‐length)
or
pSG5-‐HA-‐hG9a
T185A
(full-‐length).
HP1γ

was
immunoprecipitated
from
cell
extracts
with
an
anti-‐HP1γ
antibody,
as

indicated.
Proteins
bound
were
analyzed
by
immunoblot
with
anti-‐HP1γ

antibody
to
control
for
levels
of
immunoprecipitated
HP1γ,
or
with
anti-‐HA

antibody
to
detect
G9a
or
anti-‐GR
antibody
to
detect
GR
or
anti-‐ERα

antibody
to
detect
ERα.
A
3%
input
cell
extract
sample
was
directly
loaded

for
immunoblot
as
control
of
G9a
(anti-‐HA
antibody),
GR
(anti-‐GR
antibody),

ERα
(anti-‐ERα
antibody)
and
actin
(anti-‐actin
antibody).

27

Aurora
kinase
B
interacts
with
G9a
and
the
kinase
activity
of
aurora

kinase
B
reduces
the
interaction
between
G9a
and
HP1
γ

Aurora
kinase
B
has
been
shown
in
a
previous
report
to
phosphorylate
G9a

on
T186
in
vitro
and
the
phosphorylation
of
G9a
on
T186
seems
to
disrupt

the
interaction
between
G9a
and
HP1
γ
in
peptide
pull
down
assay

(Sampath
et
al.
2007).
To
further
explore
whether
aurora
kinase
B
may
also

be
present
in
the
complex
and
involved
in
the
underlying
mechanism
of

G9a-‐HP1γ
interaction,
I
studied
whether
aurora
kinase
B
interacts
with
G9a,

and
Coralie
Poulard
tested
the
importance
of
the
kinase
activity
for
the

interaction
between
HP1γ
and
G9a.
In
co-‐immunoprecipitation
experiments,

both
the
immunoprecipitation
of
flag
tagged
aurora
kinase
B
(Fig.
8a)
and

immunoprecipitation
of
HP1γ
(Fig.
8b)
led
to
the
co-‐immunoprecipitation
of

HA
tagged
hG9a,
indicating
both
aurora
kinase
B
and
HP1γ
interact
with

hG9a.
And
when
the
cells
are
transfected
with
plasmid
encoding
aurora

kinase
B
leading
to
its
over-‐expression,
HP1γ
interacted
less
with
G9a
as

well
(Fig.
8b),
indicating
that
the
kinase
activity
of
aurora
kinase
B
may

prevent
the
interaction
between
HP1
γ
and
G9a.
This
result
needs
to
be

reproduced.

28

Figure
8.
Aurora
kinase
B
interacts
with
G9a,
and
the
kinase
activity
of

aurora
kinase
B
reduces
the
interaction
between
G9a
and
HP1
γ.

(A)
and
(B):
Cos7
cell
were
transfected
with
pSG5-‐Flag-‐aurora
kinase
B

(full-‐length)
or
pSG5-‐HA-‐hG9a
WT
(full-‐length).
(A):
Aurora
kinase
B
was

immunoprecipitated
from
cell
extracts
with
an
anti-‐Flag
antibody,
as

indicated.
Proteins
bound
were
analyzed
by
immunoblot
with
anti-‐Flag

antibody
to
control
for
levels
of
immunoprecipitated
aurora
kinase
B,
or

with
anti-‐HA
antibody
to
detect
G9a.
A
3%
input
cell
extract
sample
was

directly
loaded
for
immunoblot
as
control
of
G9a
(anti-‐HA
antibody),
aurora

kinase
B
(anti-‐Flag
antibody)
and
actin
(anti-‐actin
antibody).
(B):
(Data

from
Coralie
E.
Poulard,
PhD.)
HP1γ
was
immunoprecipitated
from
cell

extracts
with
an
anti-‐HP1γ
antibody,
as
indicated.
Proteins
bound
were

analyzed
by
immunoblot
with
anti-‐HP1γ
antibody
to
control
for
levels
of

immunoprecipitated
HP1γ,
or
with
anti-‐HA
antibody
to
detect
G9a.
A
3%

input
cell
extract
sample
was
directly
loaded
for
immunoblot
as
control
of

G9a
(anti-‐HA
antibody),
aurora
kinase
B
(anti-‐Flag
antibody)
and
HP1γ

(anti-‐HP1γ
antibody).

29

HP1
α
and
HP1
β
can
also
interact
with
G9a

It
was
previously
shown,
by
doing
GST
pull
down
and
peptide
pull
down

experiments,
that
G9a
interacts
with
HP1
α
and
HP1
β
in
vitro
(Chin
et
al.

2007;
Sampath
et
al.
2007).
Hence,
I
tested
whether
the
HP1
α
and
HP1
β

interact
with
G9a
in
cells
and
compared
the
difference
of
interaction
level

with
G9a
between
HP1
α,
HP1
β
and
HP1
γ.
Co-‐immunoprecipitation

experiment
was
performed.
The
preliminary
data
shows
that

immunoprecipitation
of
myc
tagged
HP1
proteins
leads
to
the
co-‐
immunoprecipitation
of
HA
tagged
G9a
(Fig.
9a),
indicating
besides
HP1
γ,

HP1
α
and
HP1
β
can
also
interact
with
G9a
in
cells.
Also,
by
quantifying
and

normalizing
hG9a
bands
to
HP1
proteins
bands,
interaction
between
HP1
γ

and
G9a
is
2.5-‐fold
more
than
HP1
α
and
2.3-‐fold
more
than
HP1
β
(Fig.
9b).

It
suggests
that
HP1
γ
may
bind
tighter
to
G9a
than
do
HP1
α
or
HP1
β.
I

need
to
reproduce
this
data
to
validate
the
result.

30

Figure
9.
HP1
α
and
HP1
β
can
also
interact
with
G9a
in
cells.

Cos7
cells
were
transfected
with
pcdna3-‐myc-‐HP1
α
(full-‐length),
pcdna3-‐
myc-‐HP1
β
(full-‐length),
or
pcdna3-‐myc-‐HP1
γ
(full-‐length)
along
with

pSG5-‐HA-‐hG9a
WT
(full-‐length).
Myc
tagged
HP1
proteins
were

immunoprecipitated
from
cell
extracts
with
an
anti-‐myc
antibody.
Proteins

bound
were
analyzed
by
immunoblot
with
anti-‐myc
antibody
to
control
for

levels
of
immunoprecipitated
HP1
proteins,
or
with
anti-‐HA
antibody
to

detect
G9a.
A
3%
input
cell
extract
sample
was
directly
loaded
for

immunoblot
as
control
of
G9a
(anti-‐HA
antibody),
HP1
proteins
(anti-‐myc

antibody)
and
actin
(anti-‐actin
antibody).
(B)
The
intensity
of
IP
and
Co-‐IP

bands
were
quantified
and
normalized
by
dividing
the
intensity
of
HA
bands

by
myc
bands.

31

Discussion

[HP1
γ/Aurora
kinase
B
-‐
G9a
-‐
SHR]
Model

Based
on
the
results
in
this
study,
we
have
shown
that:

(1) Automethylation
of
G9a
K185
is
important
for
G9a
coactivator

function.

(2) HP1γ
interacts
with
G9a
and
automethylation
on
G9a
K185
is

important
for
the
interaction,
indicating
a
possible
mechanism
of
why

the
automethylation
on
G9a
K185
is
important
for
the
G9a
coactivator

function.
Therefore,
I
propose
the
possible
model
of
[HP1γ-‐metG9a]

interaction
to
be
important
for
G9a
coactivator
function.

(3) Overexpressed
aurora
kinase
B
interacts
with
G9a,
and
kinase
activity

of
Aurora
kinase
B
seems
to
prevent
the
interaction
between
HP1γ

and
G9a.
This
indicates
that
aurora
kinase
B
could
catalyze
the

phosphorylation
of
G9a
T186
that
prevents
the
interaction
between

G9a
and
HP1γ.

(4) G9a
interacts
with
ERα
and
GR.
Previous
studies
showed
the

recruitment
of
G9a
to
ERα
or
GR
responsive
elements
on
G9a

positively-‐regulated
ERα
or
GR
target
genes
(Bittencourt
et
al.
2012;

Lee
et
al.
2006;
Purcell
et
al.
2011),
indicating
a
model
of
[G9a-‐SHR]
to

be
important
for
G9a
coactivator
function.

However,
automethylation
on
G9a
K185
was
not
important
for
the

interaction
between
G9a
and
GR
or
ER.
Later,
further
GST
pull-‐down

experiments
conducted
by
Coralie
Poulard
showed
consistent
results.

32

(5) Interaction
between
HP1γ
and
GR
or
ERα
is
dependent
on
the

methylation
status
of
G9a,
indicating
the
possible
model
that
there
is
a

complex
between
HP1γ
and
these
SHRs,
and
G9a
is
an
important
part

of
the
complex.

Therefore,
putting
all
of
the
new
findings
together,
a
[HP1γ-‐metG9a-‐
SHR]
model
is
proposed
to
be
important
for
G9a
coactivator
function.
And

aurora
kinase
B,
by
catalyzing
phosphorylation
on
G9a,
could
reverse
G9a

coactivator
function.
All
together,
we
propose
a
[HP1γ/Aurora
kinase
B-‐
G9a-‐SHR]
model
to
be
a
possible
mechanism
for
G9a
coactivator
function

and
regulation
(Fig.
10).

Subsequently,
data
in
our
lab
from
Coralie
Poulard
showed
that
HP1γ

is
recruited
to
the
GR
binding
sites
of
the
genes
that
are
positively
regulated

by
G9a
after
dex
treatment.
This
indicates
that
HP1γ
may
be
involved
in

activating
transcription
of
genes
that
are
positively
regulated
by
G9a
and

SHR.
Furthermore,
knock
down
of
HP1γ
down-‐regulated
the
expression
of

these
same
genes,
while
inhibition
of
aurora
kinase
B
up-‐regulated
their

expression.
These
consistent
results
support
the
proposed
model
and

strongly
suggest
that
HP1γ
and
Aurora
kinase
B
are
primary
candidates
that

play
important
roles
in
G9a
coactivator
function
and
regulation.

Certainly,
some
questions
in
this
model
are
still
poorly
understood.

Such
as,
which
part
of
G9a
is
interacting
with
aurora
kinase
B?
Is
it
G9a
N

terminal
region?
And
if
aurora
kinase
B
interacts
with
G9a
N
terminal

region,
what
is
the
impact
of
the
two
PMTs
on
the
interaction
between
G9a

33

and
aurora
kinase
B?
What
is
the
impact
of
the
activity
of
Aurora
kinase
B

on
interaction
between
G9a
and
Aurora
kinase
B?
These
questions
could
be

answered
by
co-‐immunoprecipitation
(Co-‐IP),
GST-‐pull
down,
mammalian

double
hybrid
experiments
or
mass
spectrometry.

In
addition,
even
though
the
Co-‐IP
experiments
in
our
study
used

plasmids
to
overexpress
the
proteins
of
interest,
they
may
give
us
insight
as

to
what
could
be
happening
in
the
transcription
regulation
of
endogenous

genes.
Endogenous
Co-‐IP
or
more
chromatin
immunoprecipitation
(ChIP)

experiments
are
needed
to
be
performed
to
further
prove
our
model.

34

Figure
10.
[HP1
γ/Aurora
kinase
B
-‐
G9a
-‐
NR]
Model.

(A)
Upon
binding
of
steroid
hormone,
steroid
hormone
receptors
recognize

hormone
response
element
(HRE)
on
DNA
and
recruit
numerous

coregulators,
including
G9a.
G9a
K185
is
methylated
by
its
SET
domain
and

this
methylation
helps
recruit
HP1γ
to
the
site.
Synergistically
with
some

other
coactivators,
G9a
enhances
the
transcription
of
SHR
target
genes.
(B)

Upon
phosphorylation
of
G9a
T186
by
aurora
kinase
B,
the
interaction

between
G9a
and
HP1γ
is
disrupted
and
G9a
coactivator
function
is
reduced

along
with
transcription
of
the
target
gene.

35

Recognition
and
Modifications
of
G9a
ARKT
Sequence

The
two
PTMs
on
G9a
that
play
important
roles
in
our
model
are

found
in
a
highly
conserved
ARKT(S)
sequence,
indicating
that
they
could

be
involved
in
many
distinct
recognition
mechanisms
of
regulation.

In
our
study,
we
have
also
shown
that
the
ARKT
sequence
can
be

recognized
and
modified
by
G9a
SET
domain
and
aurora
kinase
B.

Furthermore,
the
modifications
on
the
G9a
ARKT
motif
can
be
recognized

by
HP1γ
and
this
recognition
may
play
important
roles
in
G9a
coactivator

function.
One
of
our
results
showed
that
when
G9a
T186
is
mutated
to
E,
it

interacts
with
HP1γ
poorly
(Fig.
7a
and
7b).
It
was
previously
shown
that

the
presence
of
H3S10
phosphorylation
reduced
the
methylation
of
H3K9

by
G9a
(Chin
et
al.
2005).
And
consistent
to
this,
unpublished
data
from

Coralie
Poulard
has
shown
that
when
G9a
T186
is
mutated
from
T
to
E,
the

methylation
on
G9a
K185
is
strongly
decreased.
Moreover,
she
also
showed

that
the
methylation
level
on
G9a
K185
is
only
marginally
reduced,
even

though
the
interaction
between
HP1γ
and
G9a
is
greatly
reduced
when

T186
is
mutated
to
A.
This
indicates
that
G9a
methyltransferase
activity
can

be
performed
on
ARKA
sequence,
but
HP1γ
specifically
recognizes
the

ARKT
sequence
and
does
not
bind
well
to
ARKA
even
when
methylated
on
K.

Together,
it
indicates
that
interaction
between
HP1γ
and
G9a
needs
K185

methylated
G9a
and
the
specific
ARKT
sequence
on
G9a.

Besides
HP1γ,
automethylation
on
G9a
ARKT
sequence
has
also
been

reported
to
be
important
for
its
interaction
with
chromodomain-‐Y-‐like

36

(Cdyl)
in
vitro
(Sampath
et
al.
2007),
a
chromodomain-‐containing
protein

(Caron
et
al.
2003;
Escamilla-‐Del-‐Arenal
et
al.
2013)
that
has
been

previously
purified
in
a
G9a
containing
complex
in
vivo
(Shi
et
al.
2003).

While
cdyl
has
previously
been
reported
to
function
as
a
corepressor
(Caron

et
al.
2003),
it
was
also
reported
to
have
histone
acetyltransferase
activity

and
preferentially
acetylate
histone
H4
(Lahn
et
al.
2002),
which
is
usually

associated
with
gene
activation
(Vettese-‐Dadey
et
al.
1996).
In
addition,

cdyl
was
reported
to
be
methylated
by
G9a
(Rathert
et
al.
2008).
Therefore,

it
would
be
interesting
to
investigate
whether
cdyl
plays
any
role
in
G9a

coactivator
function.
Co-‐immunoprecipitation,
GST-‐pull
down,
or

mammalian
double
hybrid
methods
could
be
used
to
investigate
the

interactions
between
cdyl
and
G9a.
Transient
reporter
gene
assay,
RNAi,

qPCR,
and
micro
array
methods
could
be
used
for
investigating
gene

expression
regulation.
ChIP
could
be
used
to
investigate
the
recruitment
of

cdyl
to
the
responsive
elements
on
DNA.

Furthermore,
and
intriguingly,
it
seems
that
the
recognition
of
and

modifications
on
the
ARKT(S)
sequence
on
histone
and
non-‐histone

proteins
are
involved
in
dual
roles
or
cross
talk
in
transcription
activation

and
repression.
In
parallel
with
our
findings
that
the
methylation
on
the
G9a

N-‐terminal
ARKT
sequence
correlates
with
G9a
coactivator
function,

methylation
on
H3K9
has
been
found
to
play
crucial
roles
in
gene
silencing

in
hetero-‐
and
eu-‐chromatin
(Fischle
et
al.
2005;
Fischle,
Wang,
Jacobs,
et
al.

2003;
Hirota
et
al.
2005;
Hwang
et
al.
2001),
but
H3K9
methylation
within

37

gene
bodies
also
is
associated
with
gene
activation
(Piacentini
and

Pimpinelli
2010;
Vakoc
et
al.
2005).
These
add
an
extra
layer
of
regulation

complexity.

Therefore,
some
questions
would
be
interesting
to
be
addressed.
Such

as,
are
there
different
roles
of
the
modifications
on
ARKS(T)
motif
in
histone

and
non-‐histone
proteins?
What
cofactors
are
recognizing
these
ARKS(T)

motifs?
What
roles
do
they
play
in
transcriptional
regulation?
Methods
that

could
be
used
to
answer
these
questions
may
involve
investigating
protein-‐
protein
interactions,
gene
expression,
open
chromatin
regions,
and
histone

marks.
Resolving
these
questions
may
give
us
some
insight
of
what
is
going

on
in
this
dual
function.

Differences
between
HP1
protein
isoforms

Despite
structure
similarities,
proteins
in
the
HP1
family,
which
play

important
roles
in
recognizing
ARKT(S)
motifs,
seem
to
be
involved
in

different
roles
in
transcription
regulation.
While
HP1α
and
HP1β
have
been

associated
more
with
regulation
in
heterochromatin
(Bannister
et
al.
2001;

Jacobs
and
Khorasanizadeh
2002;
Jacobs
et
al.
2001;
Lachner
et
al.
2001;

Lomberk
et
al.
2006;
Nielsen
et
al.
2002),
HP1γ
has
been
associated
more

with
regulation
in
euchromatin
(Dialynas
et
al.
2007;
Minc,
Courvalin,
and

Buendia
2000;
Vakoc
et
al.
2005).
Furthermore,
it
has
also
been
reported

that
there
are
isoform-‐specific
interactions
and
recognitions
(Higo
et
al.

2010;
Machado,
Dans,
and
Pantano
2010;
Vassallo
and
Tanese
2002;
Yahi
et

38

al.
2008).
Interestingly,
besides
being
involved
in
gene
silencing,
all
three

isoforms
of
HP1
proteins
have
been
recently
reported
to
be
involved
in

gene
activation
(HP1α
(Sdek
et
al.
2013),
HP1β
(Shiota
et
al.
2010;
Zhang,

Wang,
and
Sun
2010),
and
HP1γ
(Piacentini
and
Pimpinelli
2010;
Vakoc
et

al.
2005)).

Therefore,
it’s
interesting
to
ask
if
HP1α
and
HP1β
can
play
similar

roles
in
G9a
coactivator
function
as
does
HP1γ?
The
K185
site
on
G9a
has

been
previously
reported
to
be
important
for
the
co-‐localization
between

HP1
α
and
G9a
in
a
GFP
overexpression
experiment
(Chin
et
al.
2007).
In

our
study,
we
have
shown
that
overexpressed
HP1α
and
HP1β
can
interact

with
G9a,
suggesting
the
possible
interaction
in
the
non-‐overexpressed

state.
These
results
confer
a
possibility
that
HP1α
and
HP1β
could
be

playing
similar
roles
as
HP1γ
in
G9a
coactivator
function.
But
further

investigations
are
needed.

To
compare
the
similarities
and
differences
in
the
role
of
HP1
proteins

played
in
G9a
coactivator
function,
some
further
questions
are
needed
to
be

answered.
Such
as,
what
is
the
impact
of
the
two
PMTs
of
G9a
on
the

interaction
between
G9a
and
these
proteins?
which
part
of
HP1
protein
is

interacting
with
G9a?
Is
it
the
chromodomain
(CD)
or

chromodomainshadow
(CDS)?
Do
the
three
isoforms
use
the
same
domain

to
interact
with
G9a?
What
are
the
differences
of
the
chromodomains
of

these
three
isoforms
in
recognizing
the
methylation
on
histone
versus
non-‐

39

histone
proteins?
These
questions
may
be
answered
by
structural
biology

and
protein-‐protein
interaction
investigations.

G9a
Coactivator
Function

Even
though
a
model
of
G9a
coactivator
function
is
proposed
above

(Fig.
10),
the
model
is
still
in
its
preliminary
stage
and
the
underlying

mechanism
is
still
poorly
understood.
Three
directions
can
be
suggested
for

further
study
of
G9a
coactivator
function:

(1)
Further
explore
the
roles
PTMs
and
N-‐terminus
binding
partners

may
play
in
G9a
coactivator
function

Since
the
N
terminal
region
of
G9a
is
required
and
sufficient
for
its

coactivator
function
in
transient
reporter
gene
assays
(Purcell
et
al.
2011)

and
automethylation
in
this
region
is
important
for
G9a
coactivator
function

(Poulard
et
al.
non-‐published),
additional
hypotheses
needed
to
be
tested
to

understand
the
role
this
automethylation
plays
in
coactivator
function
of

G9a.

Besides
post-‐translational
modifications,
protein-‐protein
interactions

could
also
be
important
for
G9a
coactivator
function.
Sometimes
these
two

features
are
intertwined
and
cannot
be
really
separated.
G9a
recruits

Mediator
to
the
promoter
of
adult
β
globin
gene
to
exert
its
coactivator

function,
while
it
recruits
H3K4
demethylase
Jarid1a
to
the
promoter
of

embryonic
β
globin
gene
and
results
in
corepressing
the
transcription

(Chaturvedi
et
al.
2012).
This
suggests
that
the
different
binding
partners

40

G9a
interacts
with
may
play
critical
roles
in
the
switch
between
G9a

coactivator
and
corepressor
function.

To
date,
which
binding
partners
G9a
may
interact
with
when
it
is

acting
as
a
coactivator
is
still
poorly
understood.
One
possible
direction

could
be,
as
mentioned
above,
investigating
whether
cdyl,
which
requires

automethylation
for
its
interaction
with
G9a,
plays
any
roles
in
G9a

coactivator
function.
Furthermore,
the
studies
of
finding
new
binding

partners
of
G9a
are
largely
based
on
candidate
approaches.
More

interaction
studies
must
be
performed
to
make
a
larger
map
to,
maybe

bioinformatically,
predict
the
true
model
of
transcription
complex
when

G9a
functions
as
a
coactivator
and
the
switch
between
its
coactivator
and

corepressor
function.

(2)
Finding
out
which
specific
sequences
on
G9a
serve
as
binding
sites

for
SHRs

Even
though
ERα
(Purcell
et
al.
2011)
and
GR
(Bittencourt
et
al.
2012)

have
been
shown
to
interact
with
the
G9a
N-‐terminus,
no
known
binding

motifs
of
nuclear
receptors
are
found
in
the
G9a
N-‐terminal
region,
such
as

LXXLL
motif
(Purcell
et
al.
2011).
Although,
our
study
showed
that
the

automethylation
on
G9a
K185
is
not
important
for
the
interaction
between

G9a
and
GR
or
ERα,
the
binding
sites
of
G9a
interaction
with
steroid

hormone
receptors
could
play
critical
roles
in
its
coactivator
function.

Therefore,
finding
out
which
specific
sequences
that
G9a
uses
to
bind
SHRs

will
give
us
an
insight
in
understanding
G9a
coactivator
function,
and

41

structural
biology
may
help
to
answer
this
question.
Further
protein-‐
protein
interaction
and
gene
expression
investigation
may
help
to
predict

the
underlying
regulation
mechanisms.

(3)
Physiological
relevance
of
G9a
coactivator
function

Even
though
G9a
has
been
found
to
play
dual
roles
in
some

physiological
pathways,
such
as
regulating
transcription
of
β
globin
gene

(Chaturvedi
et
al.
2009,
2012),
other
physiological
processes
where
G9a

coactivator
function
is
involved
are
still
poorly
understood.
Dr.
Stallcup’s

group
has
found
that
there
are
some
endogenous
genes
positively
regulated

by
G9a
after
dexamethasone
(Dex)
treatment
in
A549
cell
line
and
estradiol

(E2)
treatment
in
MCF7
cell
line,
and
some
other
genes
are
negatively

regulated
by
G9a
(Bittencourt
et
al.
2012;
Purcell
et
al.
2011,
2012).

To
understand
what
physiological
relevance
G9a
coactivator
function

may
be
involved
in,
further
physiological
tests
and
bioinformatics

approaches
must
be
performed.

42

Materials
and
Methods

Cell
Culture.
COS-‐7
cells
were
purchased
from
ATCC
and
maintained
in

Dulbecco's
modified
Eagle's
medium
(DMEM)
supplemented
with
10%

(vol/vol)
fetal
bovine
serum
(FBS)
at
37
°C
and
5%
CO2.

Plasmids.
Plasmids
encoding
the
hG9a
(full-‐length)
were
constructed
in

pSG5-‐HA.
hG9a
K185A,
K185R,
T186A
and
T186E
mutants
were
generated

with
the
QuickChange
II
XL
Site-‐Directed
Mutagenesis
Kit
(Agilent

Technologies),
using
pSG5-‐HA-‐hG9a
(full-‐length).
Plasmids
encoding
aurora

kinase
B
were
constructed
in
pSG5-‐flag.
Plasmids
encoding
HP1α,
HP1β
and

HP1γ
were
constructed
in
pcnda3-‐myc.
Plasmid
encoding
GR
was

constructed
in
pcnda3.
Plasmid
encoding
ERα
was
constructed
in
pSG5.

Antibodies.
The
following
antibodies
were
purchased
and
used
for
this

study:
anti-‐HA
from
Roche,
anti-‐flag
and
anti–β-‐actin
from
Sigma,
anti-‐myc

from
Millipore,
anti-‐GR
and
anti-‐ERα
from
Santa
Cruz
Biotechnology,
and

anti-‐HP1gamma
from
Abcam.

Transfection.
For
co-‐immunoprecipitation
assays,
COS-‐7
cells
(Gluzman

1981)
were
plated
in
medium
(4.5g/L
glucose
phenol
red
Dulbecco's

modified
Eagle's
medium
with
10%
fetal
bovine
serum)
at
1
×
10
6

cells/well

in
10-‐cm-‐diameter
plates
the
day
before
transfection.
Transfection
of

expression
plasmids
was
performed
using
lipofectamine
2000
transfection

43

agent
purchased
from
invitrogen
according
to
the
manufacturer's
protocol.

Molar
amount
of
total
DNA
mass
was
balanced
with
empty
vector.
After
6
h

of
transfection,
the
cells
were
grown
in
hormone-‐free
medium
(High

glucose
Dulbecco's
modified
Eagle's
medium
without
phenol
red
with
10%

charcoal-‐dextran
treated
fetal
bovine
serum)
for
48
h,
and
treated
with
100

nM
dexamethasone
(Dex)
or
100
nM
estradiol
(E2)
for
6
h.

Co-‐immunoprecipitation.
Cell
lysis
and
co-‐immunoprecipitation
assays
on

whole
cell
extracts
were
performed
with
radioimmune
precipitation
(RIPA)

buffer
(50
mM
Tris·HCl
(pH
8.0),
150
mM
NaCl,
1mM

Ethylenediaminetetraacetic
Acid,
1%
Nonidet
P-‐40,
0.25%
sodium

deoxycholate),
with
proteases
inhibitors.
600
ul
of
RIPA
buffer
is
used
to

lyse
cells
of
each
10-‐cm-‐diameter
plate.
Then
the
samples
were
incubated
at

-‐20°C
degree
for
20
mins
and
were
clarified
by
centrifugation
for
15
min
at

14000
rpm
(maximum
speed
of
a
microcentrifuge).
Protein
concentrations

were
determined
by
the
method
of
Bradford
Assay
(Bradford
1976)
using

Protein
Assay
Dye
Reagent
Concentrate
from
Bio-‐Rad
with
bovine
serum

albumin
(BSA)
as
standard.
A
portion
of
the
supernatant
(30
μg
of
proteins)

was
removed
for
direct
immunoblot
analysis
and
served
as
input
for
co-‐
immunoprecipitation
experiments.
The
remaining
supernatant
(1
mg
of

proteins)
was
used
for
immunoprecipitation.
Total
volume
is
balanced
with

RIPA
complete
buffer.
Samples
were
then
incubated
with
specific
antibodies

(2
μg)
as
indicated
for
16
h
at
4°C;
the
immunoconjugates
were
precipitated

44

by
incubation
with
Protein
A/G
PLUS-‐Agarose
beads
from
Santa
Cruz

Biotechnology
for
2
h,
followed
by
washing,
elution,
and
immunoblotting.

Immunoblotting.
Total
protein
and
Laemmli
loading
dye
were
heated
at

100°C
degree
for
5
mins.
Immunoprecipitates
were
resolved
by
sodium

dodecyl
sulfate-‐polyacrylamide
gel
electrophoresis
(SDS-‐PAGE).
Standard

molecular
weight
marker
proteins
used
were
Tri-‐color
Prestained
Protein

Marker
II
[10-‐245kDa]
from
Bioland
Scientific
LLC.
Then
proteins
were

transferred
from
the
gel
to
PVDF
membrane
using
TransBlot
Turbo
transfer

system
from
Bio-‐Rad.
All
incubations
for
blocking
and
immunostaining

were
performed
at
room
temperature.
Blots
were
incubated
30
mins
in
5%

Milk
blocking
solution
consisting
of
PBST
(137
mM
NaCl,
2.7
mM
KCl,
10

mM
Na2HPO47H2O,
2
mM
KH2PO4,
0.1%
Tween-‐20).
The
primary
antibody

as
indicated
was
diluted
in
blocking
solution
and
incubated
with
the
blot

overnight
at
4°C
degree.
The
blot
was
washed
three
times
for
10
mins
each

in
PBST,
and
incubated
for
60
mins
in
PBST
containing
the
secondary

antibody;
and
anti-‐mouse
and
anti-‐rabbit
antibody
from
Promega,
and
anti-‐
rat
from
Santa
Cruz
Biotechnology
were
used
as
secondary
antibodies.
After

three
more
10-‐min
washes
in
PBST,
the
SuperSignal
West
Pico

Chemiluminescent
Substrate
(an
enhanced
chemiluminescence
[ECL]

substrate)
from
Thermo
Scientific
was
used
to
visualize
the

immunostaining
pattern.

45

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Abstract (if available)

Abstract Nuclear receptors regulate transcription of target genes with the help of numerous coregulators, including G9a. G9a is well known as a histone methyltransferase and a corepressor, but our lab previously found that G9a also acts as a coactivator. Moreover, the N-terminal region of G9a is sufficient and required for coactivator function. Interestingly, the N-terminal region of G9a undergoes two post-translational modifications (PTMs)—methylation and phosphorylation. To understand how these two PTMs affect the mechanism of G9a coactivator function, I investigated their impact on the interaction between G9a and a few binding partners of G9a that interact or potentially interact with the G9a N terminal domain—glucocorticoid receptor (GR), estrogen receptor (ER) α, heterochromatin protein 1 (HP1) γ, Aurora kinase B, HP1 α and HP1 β. By performing mutagenesis on two post-translational modification sites and co-immunoprecipitation, I studied whether the G9a PTMs are important for these interactions and the underlying molecular mechanism of G9a coactivator function. The results show that the automethylation site may not be important for the interaction of G9a with ERα or GR. However, HP1γ interacts only with the methylated form of G9a concomitant with the interaction of HP1γ with ERα or GR. Nevertheless, phosphorylation of G9a by aurora kinase B seems to prevent the interaction between HP1γ and G9a. The results suggest that these PTMs regulate G9a coactivator activity by regulating the interaction of G9a with HP1γ.

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