DBurow Thesis FULL DRAFTv3x

richessewoozyΒιοτεχνολογία

1 Οκτ 2013 (πριν από 4 χρόνια και 11 μέρες)

195 εμφανίσεις






THE MYC
-
INTERACTING ZINC FIN
GER PROTEIN
-
1:

DNA AND PROTEIN INTE
RACTIONS IN HUMAN EM
BRYONIC STEM CELLS







A Project




Presented to the faculty of the Department of Biological
Sciences

California State University, Sacramento




Submitted in partial satisfaction of


the requirements for the degree of




MASTER OF ARTS



in



Biological Sciences

(Stem Cell)


by


Dana Anne Burow


SPRING

2012





ii


THE
MYC
-
INT
ERACTING ZINC FINGER

PROTEIN
-
1:

DNA AND PROTEIN INTE
RACTIONS IN HUMAN EM
BRYONIC STEM CELLS




A
Project



by



Dana Burow













Approved by:


__________________________________, Committee Chair

Thomas Landerholm



__________________________________, Second Reader

Christine Kirvan



__________________________________, Third Reader

Jan Nolta



________________________

Date


iii



Student:
Dana Burow



I certify that this student has met the requirements for format containe
d in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.





_________________________, Graduate Coordinator

_________________

Ronald Coleman

Date


Department of Biological Sciences


iv


Abstract


of


THE MYC
-
INT
ERACTING ZINC FINGER

PROTEIN
-
1:

DNA AND PROTEIN INTE
RACTIONS IN HUMAN EM
BRYONIC STEM CELLS

by


Dana Burow


Stem cells can
divide indefinitely and maintain their capability to diff
erentiate
into many cell types, the key features of self
-
renewal and pluripotency,

which explains
their importance to regenerative medicine. Embryonic stem cells (ESCs), the most highly
pluripotent
of all stem cells, have the
added potential

of tumorigenesis. This is thought to
be driven
in part

through a shared gene expression program regulated by the transcription
factor, Myc.

Myc, first characterized as a potent oncogene, is shown to maintain plu
ripotency
and self
-
renewal in mouse ESCs. Myc regulation of pluripotency and self
-
renewal is
evident by
its
role

in the generation of induced pluripotent stem (iPS) cells. Myc is
thought to regulate target gene expression both locally
through classical m
echanisms,
and
globally through euchromatin remodeling. In this way, Myc can affect gene expression
on a large enough scale to reprogram differentiated cells into iPS cells. Miz
-
1, a
transcription factor named for its interaction with Myc, is thought to
form a co
-
repressor
complex with Myc
, silencing Miz
-
1 target genes including those associated with
di
fferentiation and proliferation.

Miz
-
1

contains

BTB/POZ and
13 C2H2 zinc
finger
s


v


and
is thought to bind initiator sequences

(INR)

in the
core
promoters of

target genes
thereby modulating their expression. Still, relatively little is known about the function of
Miz
-
1 as a transcriptional regulator and recent
epigenetics

analysis in h
ESCs suggest
Miz
-
1 binds alternative sequences, not associated with the
INR

of target gene promoters
.

Using a Miz
-
1 maltose binding protein (MBP) fusion protein tag system, t
his
study
implemented

an
in vitro
, high
-
throughput DNA binding assay and
Multiple em for
Motif Elicitation (
MEME
)

analysis to

identify putative Miz
-
1 DNA
biding motifs

de
novo
. The
consensus

motifs, ATCGAT and

G
A
TTACCGA

were then
confirmed

by
electrophoretic mobility shift analysis

(EMSA)

and
further
bioinformatics

analysis

revealed motif occurrences in functionally relevant gene ontology clusters including
:
transcription

regulation, growth, chromatin, and developmental genes
. MBP pull
-
down
mass spectrometry analysis also
identified interesting
Miz
-
1 protein cofactors
from hESC
nuclear extracts

that are associated with
reported

Miz
-
1 functions
.

Miz
-
1 DNA and protein interactions highlighted in this study confirm
its role

as a
master transcriptional regulator, cofactor and antagonist of

Myc in hESCs.

Though
,

the
findings

also
underline

the importa
nce of further
characterization of

pluripotency and

self
-
renewal
in

hESC
s so that potential

therapies may be safe and effective
.


___________________________, Committee Chair

Thomas Landerholm


___________________________

Date


vi


ACKNOWLEDGEMENTS



I would like to acknowl
edge the
support and
dedication
of the
faculty mentors in the
Department of Biological Sciences at Sacramento State

University. I would also like to
recognize

the

Knoepfler lab at UC Davis for their guidance and the opportunity to be a

part of the lab for the past year
.
Thanks to the Segal lab at the UC Davis Genome Center
for their advice and collaboration on Bind
-
n
-
Seq.
Finally, t
hanks
goes out
to my
wonderful family
and friends who’s

love and support

has helped me accomplish my
goals
.


vii


TABLE OF CONTENTS


Page

Acknowled
gements

................................
................................
................................
............

vi

List of Tables

................................
................................
................................
...................

viii

List of Figures

................................
................................
................................
....................

ix

INTRODUCTION

................................
................................
................................
...............
1

METHODS

................................
................................
................................
..........................
7


Cloning, Recombinant Protein Expression and P
urification



































7


Bind
-
n
-
Seq:
in vitro
DNA Binding A
ssay

a
nd
de novo

Motif F
inding



















8


Electrophoretic Mobility S
hift
A
ssay






































































9


Bioinformatics Analysis of Motifs Identified by Bind
-
n
-
Seq.

































9


MBP
-
Miz
-
1
Pull
-
down Mass S
pectrometry
A
nalysis













































9

RESULTS

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


Miz
-
1 E
x
pression and P
urification by MBP


























































11


De n
ovo

Motif F
inding by Bind
-
n
-
Seq


































































11


Bioinformatics A
naly
sis of Motifs I
dentified by Bind
-
n
-
Seq
































13


EMSA

S
u
pports Miz
-
1 B
inding
ATCGAT and GATTACCGA



























16


MBP
-
Miz
-
1 Pull
-
down Mass Spectrometry A
nalysis











































16

DISCUSSION

................................
................................
................................
....................
25

Literature

Cited

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


viii


LIST OF TABLES


Table

Page


1.
Full
-
length Miz
-
1 M
otif

C
onsensus

Sequences I
dentified by Bind
-
n
-
Seq



















14


2.
Zinc finger Miz
-
1
M
otif

C
onsensus

Sequences I
dentified by Bind
-
n
-
Seq



















15


3
.
Putative Miz
-
1 DNA Binding M
otifs











































































17


4
.
Gene Ontology Clusters Identified by DAVID A
nalysis













































18


5
.

MBP
-
Miz
-
1 Mass S
p
ectrometry A
nalysis by Scaffold
3













































23



ix


LIST OF FIGURES


Figure

Page


1
.
SDS
-
PAGE
Detection of P
rotein from
P
urification by MBP
.

................................
......
12


2.
DAVID Analysis of Motif
-
Containing Miz
-
1 Bound G
enes
.

................................
.......
19


3.
EMSA
Indicates Miz
-
1 Binding Motif
-
containing Oligonucleotide P
robes
















20


4.
MBP
-
Miz
-
1
P
ull
-
down of c
-
Myc


















































































22


5.
Current
M
odel
P
roposed for Miz
-
1
Target

G
ene
R
egulation







































24



























1


INTRODUCTION


Stem cells are the focus of regenerative medicine and may hold the key to
unlocking cures for diseases ranging from neurological disorders to cancer and HIV
infection. In the early 1980s, embryonic stem cells (ESCs) were first isolated from
mouse embryos
and shown to grow indefinitely while maintaining pluripotency, the
ability to differentiate into many different cell types
(
Evans, 1981
)
. Since then there have
been significant gains in our understanding of these key features of stem cells
: self
-
renewal and pluripotency
. A complex network of transcription factors mediate s
tem cell
differentiation. In 2006, scientists were able to reverse this process and generate induced
pluripotent
stem
cells, termed
iPS

cells, from differentiated fibroblast epithelial cells in
mice
(
Takahashi & Yamanaka, 2006
)

and later in humans
(
Takahashi et al., 2007
)
. The
induction of pluripotent stem cells (iPS cells) demonstrated by
Takahashi and Yamanaka

requires the forced expression

of a set of transcription factors, including the proto
-
oncogene, c
-
Myc

(
Takahashi & Yamanaka, 2006
)
.

Stem and cancer cells have many of the same properties, postulated to be driven
in part through a shared gene expression program regulated by the transcription factor,
c
-
Myc. Myc was first characterized through its association as a potent oncogene, though

the mechanisms of oncogenic activation were initially vague
(
Eisenman, 2001
)
.
Members of the Myc f
amily of transcription factors are well documented as being
deregulated in many cancers. This deregulation of Myc through translocation and
duplication events results in increased Myc expression and allows for oncogenic
activation
(
Meyer, 2008
)
, whereas the normal, high levels of
endogenous expression in
2


ESCs plays a key role in mediating pluripotency and self
-
renewal
(
N. V. Va
rlakhanova,
Cotterman, R.F., deVries, W.N., Morgan, J., Donahue, L.R., Murray, S., Knowles, B.B.,
Knoepfler, P.S., 2010
)
. Pluripotency and self
-
renewal properties, the foundation of stem
cells, are implicated in their cancerous counterpart, teratoma, a
s early as 1960
(
Pierce,
1960
)
. Teratoma tumors contain all three
germ

layers and continue to be challenging to
overcome with current cancer therapies. Despite the promise of stem cell therapies,
there
still remains the problem of ESC
-
related tumorigenesis.

Myc is a key player in the generation of iPS cells and though alternate routes to
iPS cell formation have been presented in recent years, the endogenous role of c
-
Myc is
implicated in the shift
to pluripotency and self
-
renewal in each of these cases. A 2008
study by
Nakagawa

was able to generate iPS cells without the forced expression of c
-
Myc
(
Nakagawa M., 2008
)
. Though they generally have significantly reduced
reprogramming efficiency, it is suggested that e
ndogenous c
-
Myc expression in these iPS
cells is able to mediate the shift to pluripotency and self
-
renewal through its activation by
the other exogenous fact
ors used in reprogramming, including Oct4, Sox2, and Klf4

(
Stadtfeld, 2010
)
. This notion is supported by a study demonstrating that neural stem
cells can be converted to iPS cells through exogenous Oct4 expression alone since the
other defined factors are endogenously expressed in neural stem cells
(
Kim, 2009
)
.
In
2010,
Stadtfeld

have also linked the original set of iPS cell fo
rming factors to another
proven set (Oct4, Sox2, Nanog, and Lin28) through the deregulation

of

c
-
Myc by Lin28

(
Stadtfeld, 2010
)
.

The importance of Myc in the mediation of pluripotency and self
-
renewal in stem cells cannot be overlooked.

3


The Myc protein family is a group of transcription factors containing the basic
helix loop helix leucine zipper (bHLHZ) dom
ain. Myc, like other members of the
bHLHZ superfamily of transcription factors, can heterodimerize with another bHLHZ
protein, Max, and bind the enhancer box (E
-
box) sequence, CACGTG, activating target
genes
(
Chaudhary, 1999
)
. Myc can also have repressive action on gene expression
through association with and inhibition of the activating function of the Miz
-
1
transcription factor
(
Peukert,
1997
)
. In this way, Myc modulates gene expression to in
turn regulate diverse cellular processes including: metabolism, cell cycle, differentiation,
apoptosis, senescence, and DNA replication
(
Grandori, 2000
)
.

I
n more recent studies, Myc
is

shown to regulate global euchromatin structure and
is tightly associated with certain histone modifications
(
Knoepfler et al., 2006
)
. Myc and
the cofactor
transformation/transcription domain
-
associated protein (
TRRAP
)

are known
to recruit histone acetyl transferases (HAT)
(
McMahon, 2000
)
. HATs are well
-
characterized proteins that function in the acetylation of amino
-
terminal lysine residues
of histone

proteins, especially those located near transcriptional start sites
(
Knoepfler,
20
07
)
. Myc is shown to regulate euchromatin on a global scale and to some extent
independent of its role as a classical transcription factor
(
Cotterman et al., 2008
)
.

In a
r
ecent review, a model

is proposed that defines Myc’s role in the self
-
renewal of stem
cells and tu
morigenesis through both local
transcriptional activation
, by classical

mechanisms,
and global euchromatin structure, whereby overexpression results in

tumorigenesis
(
Knoepfler, 2007
)
. In iPS cell formation, c
-
Myc is hypothesized
to allow
Oct4 and Sox2, two other defined factors in iPS cell formation, to bind to genomic targets
4


through its mediation of global histone acetylation
(
Takahashi & Yamanaka, 2006
)
.
While Myc is most well known for its transcriptional activating function, interest in its
repressive functions is growing and is implicated in its regulation of stem cell
pluripotency

and self
-
renewal
.

Miz
-
1, na
med for its interaction with Myc (
M
yc
-
i
nteracting
z
inc finger protein
-
1),
was first characterized in 1997 and found to function strongly in growth arrest
(
Peukert,
1997
)
. Miz
-
1 is a BTB/POZ (
BR
-
C, ttk and bab/pox virus and zinc
-
finger
) domain
-
containing transcription factor that
is thought to directly bind

core promo
ter initiator
(INR) sequences and recruit

the co
-
activator protein, p300,
in order to activate target
genes, such as negative regulators
of cell cycle control and growth
an
d

positive regulators
of differentiation
(
Kime & Wright, 2003
;
Seoane et al., 2
001
;
Staller, 2001
;
M. Wanzel,
Herold, S., Eilers, M., 2003
;
Wu, 2003
)
. Myc
can bind

Miz
-
1 through its HLH domain
and is thought to repress Miz
-
1 gene activation through competition with p300
(
Staller,
2001
)
.


Recent
epigenetic
studies
also
support

the hypothesis that the mechanism by
which Myc represses expre
ssion of differentiation genes, thereby maintaining

pluripotency an
d self
-
renewal,

is related to Miz
-
1. Co
-
immunoprecipitation using anti
-
c
-
Myc antibody suggests recruitment of
Histone Deacetylase 2

(
HDAC2
)

and
DNA
(cytosine
-
5)
-
methyltransferase 3a (
D
NMT
3a
)

to form a repressor complex (
Varlakhanova

unpublished data).
The DNA CpG methyltransferase, D
NMT
3a, is known to interact
with Myc by means of Miz
-
1, forming a corepressor complex that functions at the
promoter of target genes like p21Cip1, a cyclin
-
dependent kinase inhibitor
(
Brenner et
al., 2005
)

and Mad4, a
nother

transcriptional
regulator of proliferation and differentiation
5


(
Ki
me & Wright, 2003
)
. This
confirms

that the mechanism by which Myc represses
differentiation gene expression in
hESCs

is through Miz
-
1.

Biological

and epigenetic characterization of Miz
-
1
in hESCs
demonstrates that
Myc and Miz
-
1 function coordinately in

the regulation of
pluripotency and self
-
renewal
through their repression of differentiation associate
d

genes
.

Myc and Miz
-
1 also display
antagonistic roles in the regulation of
genes important to stem cell pluripotency, self
-
renewal and differentiation
.
Genome
-
wide chromatin immunoprecipitation
-
microarray
(ChIP
-
chip) analysis demonstrate
s

that

Myc also
occupies nearly 30% of Miz
-
1 targets
,

and
that
these are predominantly differentiation associated genes
,

including many
members of the
Hox
gene
family
(
N. V. Varlakhanova, et al, 2011
)
. Additionally, parallel
ChIP
-
chip analysis o
f activating
euchromatin

marks, including acetylation of lysine 9 on
histone 3 (
AcH3K9
) and
trimethylation of lysine 4 on histone 3

(
H3K4me3
)
,

and

Miz
-
1
DNA
binding
show a
significant

overlap

between

genes involved in
cellular metabolism
and growth
.

Conversely,

g
enes not associated with active euchr
o
matin

marks
and those
associated with the

inactivation mark
trimethylation of lysine 27 on histone 3
(H3K27me3)

were predominately diff
erentiation associated g
enes, including
many
Hox
genes
(
N. V. Varlakh
anova, et al, 2011
)
.

Myc knockdown in hESCs results in an
upregulation of
differentiation associated genes and
a
downregualtion

of

pluripotency and
growth associated genes
,
while
conversely Miz
-
1 k
nockdown in hESCs results in a

downregulation

of differentiation associated genes and
an
upregualtion pluripotency and
growth associated genes
(
N. V. Varlakhanova, et al, 2011
)
.

Interestingly and c
ontrary

to
the current literature

(
Kime & Wright, 2003
;
Seoane, et al., 2001
;
Staller, 2001
;
M.
6


Wanzel, Herold, S., Eilers, M., 2003
;
Wu, 2003
)
,

which describes Miz
-
1 binding
localized to core promoter INR sequences,
the

recent

work of
Varlakhanova

demonstrates

that the global distribution of Miz
-
1 binding is predominantly localized to regions more
than
1000 bases

upstream of the transcriptional

start sites
of target genes

(
N. V.
Varlakhanova, et al, 2011
)
. It

is important to note that

unlike the
Varlakhanova

study,
previous studies
only analyzed

Miz
-
1
regulation

of few candidate genes and did not
assess global genomic binding.
Cis
-
Regulatory Element Annotation System (CEAS)

analysi
s

(
Ji X, 2006
)

of Miz
-
1 ChIP
-
chip data

from the Varlakhanova

study failed
to
identify

potenti
al

DNA binding motifs

for Miz
-
1
, however,
Miz
-
1 INR sequence
-
independent DNA binding is of

clear

significant to the
global
function of Miz
-
1
.


INR
-
independent Miz
-
1 binding

represents more than half of

total

Miz
-
1 genomic binding
sites,
and identification of
novel

Miz
-
1 DNA binding motif
s

is central to furthering our
understanding of this important Myc antagonist.

Understanding

the interworking of the complex Myc network

of transcription
factors
, including Miz
-
1,

that m
ediates stem cell s
elf
-
renewal,
pluripotency

and
differentiation

will help to further our
knowledge

of both stem and cancer cell biology
.
Teasing out the subtle differences between Myc
-
mediated
pluripotency and
self
-
renewal
in stem cells and that in cancer cells is of vital
importance in furthering stem cell based
therapies so that they are both safe and effective, and may also lead to novel cancer
treatments.

The present work identifies putative Miz
-
1 DNA binding motifs and potential
protein cofactors and serves as a platfor
m for further investigation into specific Miz
-
1
DNA and protein interactions in hESCs.


7


METHODS

Cloning, Recombinant Protein Expression and Purification.

A
plasmid
vector coding for
a N
-
termal fusion of
E. coli

maltose binding protein (MBP) to
full
-
length
human Miz
-
1
was
cloned

by

restriction ligation

using

pMAL
-
c5G (New England Biolabs

Ipswich, MA
)
and Miz
-
1 cDNA
generated

from H9
hESC

mRNA.
T
he
sequence encoding the
13 C2H2
zinc fingers (
nucleotides

805
-
2379
)

of Miz
-
1
cDNA
were cloned
by Gateway (Invitrog
en
Life Technologies,
Carlsbad, CA
)

into a
plasmid
vector coding for an N
-
terminal GST
-
MBP
tag
(Segal Lab, UC Davis Genome Center, Davis, CA).

Transformed
E. coli

BL21
STAR

(Invitrogen Life Technologies) were grown at 37°C and 225rpm.
Expression
of the MBP
-
hMiz
-
1 fusion

constructs was

induced at 2.5 hours by

sopropyl

-

-

-
thio alactopyranoside


(
IPTG
)
. Cells were harvested 5 hours post
-
induction by
centrifugation (3500rpm, 20 min, 4°C) and lysed in Zinc Buffer A [ZBA; 10mM Tris (pH
7.5), 90mM KCl, 1mM
MgCl
2
, 90μM ZnCl
2
, 5mM DTT] by sonication (6 rounds: 30 sec
(high), 30 sec rest). Protein lysate was isolated by centrifugation (20,000rpm, 30 min,
4°C) then incubated at 4°C with amylose

linked agarose

beads (New England Biolabs)
for 20 min. Protein lysat
e was cleared by gravity flow and beads subsequently washed
with 10 column volumes of ZBA. MBP
-
hMiz was eluted in 3

mL ZBA and 10

mM
maltose then dialyzed in 2

L ZBA

overnight

to deplete free maltose.

MBP
-
hMiz
-
1 protein
was concentrated using Amicon Ultra
Filter units (Millipore
, Billerica, MA
). Purity and
quantity of the MBP
-
hMiz fusion protein was assessed by SDS
-
PAGE and Bradford
Assay

(Thermo Fisher Scientific,
Waltham
, MA)
.


8


Bind
-
n
-
Seq in vitro DNA Binding
Assay

and
de novo Motif

Finding
.
MBP
-
hMiz
-
1
protein
s

at various concentrations
(Table
s

1
-
2)
were bound to random
oligonucleotides
with barcodes
in Bind
-
n
-
Seq

(BnS)

bindin buffer [BnSBB; 0. 2μ /μL Herrin Sperm
NA, 00μM ZnCl
2
, 5mM DTT, 5% BSA] for 30 minutes with
agitation at

room temp
.
Binding reactions were then washed 6X for 10 min each with BnS wash buffer [BnSWB;
0mM Tris (pH 8.5), 00μM ZnCl
2
, 1mM MgCl
2
, 5mM DTT] under various K
Cl salt
concentrations
. Bound oligonucleotides were eluted for 10 min in EB buffer (Qiagen
,
Hilden, Germany
) and 10mM maltose. Quantitative PCR was performed by the Opticon
Monitor system and SYBR green detection (Program: 94°C for 4 min in
i
tial denaturation,
26 cycles of 94°C for 30 sec, 63°C for 30 sec and 72°C for 1 min) to determine optimal
a
mplification cycles for each set of oligonucleotides. Oligonucleotides were amplified
using iProof DNA Polymerase (Bio Rad
, Hercules, CA
), cleaned by PCR Purification Kit
(Qiagen), quantified by NanoDrop

(Thermo Fisher Scientific
)
, and 100ng of each
sample
d pooled for sequencing.

Amplified, pooled oligonucleotides with barcodes were
sequenced on the MiSeq (Illumina
, San Diego, CA
) at the UCD Genome Center Core
facility and reads sorted and filtered for quality by
the
MiSeq platform software.

Bioinformatics

were performed by the Segal Lab at the UC Davis Genome center
.

Sorted,
filtered reads were analyzed in randomly sampled

clusters of 10,000 reads by MEME
.
Intermediate motifs were matched back to the original dataset and subsequent rounds of
MEME performed

to generate the most enriched motifs for each BnS condition (table 1).

De novo

motif finding

was performed at the UC Davis Genome Center by the Segal lab.

9


Electrophoretic Mobility Shift Assay
.

EMSA

was performed by binding MBP
-
hMiz

to
synthesized and hybridized probes (5’ CAAAAGTGCG
ATCGAT
GCTGCGTGGT 3’
and 5’ CAAAAGTGCG
GATTACCGA
GCTGCGTGGT 3’)
and poly(deoxyinosinic
-
deoxycytidylic) acid nonspecific competitor
in ZBA for 20 min at room temperature then
visualized on Novex 6% DNA Retard
ation polyacrylamide gels (Invitrogen Life
Technologies) by SYBR green
DNA stain
and SYPRO ruby
protein
stain

(
Invitrogen Life
Technologies
)

at 300nm UV transillumination.

Bioinformatics

A
nalysis of
M
otifs
I
dentified by B
ind
-
n
-
S
eq
. Gapped Local Alignment
of
Motifs (GLAM2)

(
Frith, 2008
)

analysis
was performed on motifs identified by BnS for
each full
-
length and zinc
-
finger Miz
-
1 constructs. Then, GLAM2

results were searched
against
Miz
-
1 ChIP
-
chip sequence
data

(
N. V. Varlakhanova, et al, 2011
)

for
motif
occurrences

by
Find Individual Motif Occurrences (FIMO)
(
Grant, 2011
)

a
nalysis with a
p
-
value c
ut
-
off of 0.0001. The subsequent
motif
-
containing
gene list was analyzed
using

the
D
atabase for
A
nnotation,
V
isualization and

I
ntegrated
D
iscovery (
DAVID)

(
Huang,
2009a
,
2009b
)

with a p
-
value cut
-
off of 0.001 for identification of enriched gene
ontology clusters.

MBP
-
Pull down Mass Spec
trometry Analysis.
MBP
-
hMiz
-
1 was bound to amylose linked
agarose beads (New England Biolabs) and
in vitro

transcribed and translated c
-
Myc (T
N
T
Translation/Transcription Kit, Promega) in ZBA for 20 minutes
then washed with 10
volumes ZBA,
eluted with LDS
sam
ple

loading buffer (Invitrogen Life Technologies)
and detected by Western Blot against human c
-
Myc. MBP
-
hMiz
-
1 was bound to amylose
linked
agarose beads (New England Biolabs) and H9 human embryonic stem cell nuclear
10


extracts in ZBA for 20 minutes then washed with 10 volumes ZBA and eluted in ½X
ZBA and 10mM maltose. Samples were submitted to the UC Davis Proteomics Core for
Mass Spectrometry
analysis.

Data was analyzed by Scaffold 3 (Proteome Software, Inc.,
Portland, Oregon).



11


RESULTS

Miz
-
1
E
xpression and
P
urification
by MBP
.

Induction of recombinant
MBP
-
hMiz
-
1
protein expression
by IPTG under

the Tac promoter in
E. coli
is an efficient and effective
means of robust protein production
in vitro
. The MBP tag allows

for efficient purification
by amylose
-
linked agarose
beads and elution with maltose
.

SDS
-
PAGE and
Bradford
A
ssay confirm

MBP
-
Miz
-
1 purity
and
concentration
s of gr
eater than 2 μM, important for
subsequent implementation in the

in vitro

DNA binding assay
.

De Novo Motif Finding by Bind
-
n
-
Seq.
Bind
-
n
-
Seq is a high
-
throughput,
in vitro

DNA
biding assay that allows for the systematic and rapid detection of DNA binding motifs in
parallel. While other protein
-
DNA binding approaches have been identified and widely
implemented, including ChIP
-
chip, ChIP
-
Seq, protein
-
binding microarrays (PBM)
,
cyclical amplification and selection of targets (CAST), systematic evolution of ligands by
exponential enrichment (SELEX) and even one and two
-
hybrid systems, Bind
-
n
-
Seq has
distinct advantages over these other analyses.
In vivo

approaches including ChIP

technologies, and one and two
-
hybrid systems are powerful but experimentally complex
and limited in their application, by for example, the availability of ChIP quality
antibodies.
I
n vitro

analyses including, PBM, CAST and SELEX are limited in scope by
th
e size of the DNA library available for analysis and/or the labor required to successfully
execute the technique. Bind
-
n
-
Seq, through its simple design and implementation of
next
-
generation sequencing technology, overcomes challenges of experimental
compl
exity and scope. Short, randomly generated

12



Figure 1.
SDS
-
PAGE Detection of Protein
from
P
urification

by MBP
.
Significant
amounts of recombinant protein was obtained by purification by MBP, while little
carryover of bacterial proteins is evident.



13


oligo
nucleotides (21bp binding region) with barcodes are bound to MBP
-
protein
constructs and amylose
-
linked agarose beads,
washed and eluted with maltose and
identified by massively parallel sequencing to generate approximately 100,000 reads per
sample, while m
aintaining the ability to run up to 64 samples in parallel
(
Zykovich,
2009
)
. In this study, MBP fused to Full
-
length Miz
-
1 and MBP fused to Miz
-
1 zinc
fingers
(residues 269
-
793) constructs were each analyzed by Bind
-
n
-
Seq across 5
different binding buffer and wash buffer conditions. The 5 most highly enriched
consensus sequence motifs

identified for each the full
-
length and zinc
-
finger construct
and condition are presented in Tables 1 and 2 respectively. All motifs had significant
enrichment of greater than 5
-
fold and up to 25
-
fold over background. Interestingly,
conditions of higher st
ringency (higher salt concentration) did not see the lowest
enrichment, rather, conditions of low protein concentration produces the lowest
enrichment values. Further Gapped Local Alignment of Motifs (GLAM2) analysis
(
Frith,
2008
)

was performed on each set of consensus sequences identified for the respective
protein constructs and the results are presented in Table 3. The motifs GATTACCGA and
ATCGAT were identified as the most significant matches for the ful
l
-
length and zinc
finger constructs respectively.

Bioinformatics Analysis of Motifs Identified by Bind
-
n
-
Seq
.

Motifs retrieved from BnS
were analyzed by GLAM2 to generate a list of principal motifs for each full
-
length and
zinc finger constructs. The two t
op
-
scoring motifs are shown in Table 3. The MEME
-
formatted motifs from GLAM2 were subsequently used to search Miz
-
1 ChIP
-
chip data
by
Find Individual Motif Occurrences

(FIMO). FIMO analysis revealed 3052 and 2411

14


Table 1.
Full
-
length Miz
-
1 Motif

Consensus

Sequences

Identified by Bind
-
n
-
Seq
.
The five
most highly enriched consensus sequences for each binding condition of the BnS assay
for the full
-
length Miz
-
1 construct is shown along with the enrichment score.


Binding Condition

Consensus Sequence

Fold
-
Enri
chment

50nM [protein]

1mM [salt]

ATAATCGAT

17.417

GATTACCGA

16.667

CGATTAATCG

14.5

ATTACCGATC

14.312

AATCGATCTC

13.6

50nM [protein]

50mM [salt]

ATCGGTAATC

20.867

GATTACCGA

19.31

ATCGGCAATC

17.5

ATCGGTATTC

16.952

GGCTTACCGA

15.529

50nM
[protein]

100mM [salt]

ATCGGTAATC

14.125

GATTACCGA

13.478

GGATTACCGA

12.8

AGATTACCGA

11.8

GATTGCCGAA

11.667

5nM [protein]

100mM [salt]

GATTACCGA

9.808

AGATTGCCGA

9.6

ATCGGTAATC

9.375

GATTGCCGA

6.333

ATCGATTAA

5.871

350nM [protein]

100mM
[salt]

GATTACCGA

11.162

ATCGATTAC

11.029

GATTGCCGA

11

AATCGATTA

10.514

TAATCGATTA

9.474

15


Table 2.
Zinc finger

Miz
-
1 Motif

Consensus

Sequences

Identified by Bind
-
n
-
Seq
. The five
most highly enriched consensus sequences for each binding condition of

the Bind
-
n
-
Seq
assay for the zinc finger Miz
-
1 construct is shown along with the enrichment score.


Binding Condition

Consensus Sequence

Fold
-
Enrichment

50nM [protein]

1mM [salt]

ATCGATTAAT

25.053

TAATCGATTA

23.71

ATAATCGATC

19.611

ATCGGTAATC

16.909

ATCGATTAA

16.81

50nM [protein]

50mM [salt]

AAAAATCGAT

26.2

ATCGGTAATC

17.267

ATCGGCAATC

16.053

ATCGATTAAA

16

ATCGATTAC

14.2

50nM [protein]

100mM [salt]

AACATCGAT

16.421

GATTGCCGA

16.31

AGTAATCGAT

15.053

CATCGATCG

14.864

ATCGATCGAT

13.333

5nM [protein]

100mM [salt]

ATCGATCGAT

19.6

ATCGATCGA

16

GATTGCCGA

14.227

ATCGGTACTC

12.2

ATCGGTACTC

11

120nM [protein]

100mM [salt]

ATCGGTATC

10.75

ATCGATTG

9.619

AATCATCGAT

8.833

GATTGCCGA

7.781

GATTACCGA

6.892




16


motif occurrences with a p
-
value less than 0.0001 respectively for the full
-
length and zinc
finger constructs.
D
atabase for
A
nnotation,
V
isualization and

I
ntegrated
D
iscovery

(
DAVID) analysis of Miz
-
1 bound genes containing motif occurrences identified by
FIMO shows enriched clusters of functionally related genes. Significant gene ontology
clusters are outlined for both full
-
length and zinc finger constructs in Table 4 and
inclu
des genes involved in cell growth, differentiation, including Hox genes, and other
developmental associated genes, regulation of transcription and DNA binding and
chromatin structure including acetylation.

EMSA Supports Miz
-
1 Binding ATCGAT and GATTACCGA
.
Electrophoretic mobility
shift analysis is a long
-
standing, robust method of confirming protein
-
DNA interaction
and serves as a means of validating the motifs ATCGAT and GATTACCGA affinity for
the human Miz
-
1 protein. Poly
-
dI/dC non
-
specific inhibitor olig
onucleotide was added to
the binding reactions and results were analyzed by polyacrylamide DNA retardation gel
electrophoresis. SYBR green DNA stain revels that ATCGAT and GATTACCGA
containing probes were bound and shifted in the gel in the presence of ful
l
-
length Miz
-
1
protein, while reactions not containing Miz
-
1 protein ran unaltered.

MBP
-
Miz
-
1 Pull
-
down Mass Spectrometry A
nalysis
.
MBP
-
Miz
-
1 pull
-
down experiments
were performed in order to screen for novel Miz
-
1 cofactors in hESCs. To assess the
possibi
lity of performing a pull
-
down experiment with MBP
-
Miz
-
1 constructs, MBP
-
Miz
-
1 proteins were incubated with a known interactor, c
-
Myc, and analyzed by western
blot (Figure 3). c
-
Myc is shown to bind MBP
-
Miz
-
1 full
-
length construct, but does not

17


Table 3.
P
utative Miz
-
1 DNA Binding Motifs
. Consensus seed motifs from Bind
-
n
-
Seq
were analyzed by GLAM2 and the highest scoring motifs for the full
-
length and zinc
-
finger constructs are shown.


Construct

Consensus

Motif

Score

Full
-
length

GATTACCGA

G

⡁⽃)

⡔⽁/


⽁/

(䄯䜩

⡃⽔(

C

G

A

ㄹ㠮㌶1

Z楮挠
晩湧e牳

䅔C䝁T

A

T

C

G

⡁⽇/

⡔⽃)

ㄸ㠮㘲1







18


Table 4.

Gene Ontology Clusters Identified by DAVID A
nalysis
.

Gene lists generated by
FIMO for full
-
length and zinc finger constructs respectively were submitted for DAVID
analysis, the most si nificant ene ontolo y clusters are shown (p ≤ 0.00 ) alon with the
percentage of genes that comprise the cluster and the
enrichment score.


Gene Ontology Cluster

Miz
-
1
Construct

% Total
Genes in
GO
category

P
-
Value

Fold
-
Enrich
ment

Positive regulation of cell
proliferation

FL

8

2.50x10
-
05

3.5

Regulation of transcription

FL

23

4.10
x10
-
04

1.6

Regulation of cell proliferation

FL

9.9

7.80
x10
-
04

2.3

Embryonic organ development

FL

4.2

8.90
x10
-
04

4.5

Transcription cofactor activity

FL

7

1.30
x10
-
04

3.4

Transcription regulator activity

FL

16.4

2.30
x10
-
04

1.9

DNA binding

FL

22.1

3.20
x10
-
04

1.7

Transcription corepressor activity

FL

4.2

3.80
x10
-
04

5.1

Transcription factor binding

FL

8

4.80
x10
-
04

2.7

Nucleus

FL

36.2

7.80
x10
-
07

1.7

DNA
-
binding

FL

18.8

2.90
x10
-
05

2

Homeobox

FL

5.6

5.70
x10
-
05

4.7

P
hosphoprotein

FL

48.8

1.30
x10
-
04

1.3

A
cetylation

FL

22.5

2.00
x10
-
04

1.7

T
ranscription regulation

FL

17.8

7.00
x10
-
04

1.8


Chromatin

ZF

0.5

4.20
x10
-
04

5

Transcription factor activity

ZF

1.2

6.00
x10
-
04

2.3

Transcription regulator activity

ZF

1.6

7.70
x10
-
04

1.9

P
hosphoprotein

ZF

4.8

3.60
x10
-
05

1.4

Nucleus

ZF

3.2

1.80
x10
-
04

1.6

Acetylation

ZF

2.1

9.30
x10
-
04

1.7



19




Figure 2:
DAVID Analysis of Motif
-
containing Miz
-
1 Bound G
enes.
DAVID analysis
gene ontology clusters of genes both bound by Miz
-
1 and containing motifs.

0
1
2
3
4
5
6
positive regulation of cell
proliferation
regulation of transcription
regulation of cell proliferation
embryonic organ development
transcription cofactor activity
transcription regulator activity
DNA binding
transcription corepressor activity
transcription factor binding
nucleus
DNA-binding
Homeobox
phosphoprotein
acetylation
transcription regulation
chromatin
Fold
-
Enrichment

DAVID Analysis of Motif
-
Containing Miz
-
1 Bound Genes

Miz-1 ZF
Miz-1 FL
20



Figure
3
.
EMSA Indicates Miz
-
1 Binding Motif
-
containing

Oligonucleotide Probes
.
Oligonucleotide probes and polydI/dC nonspecific competitor nucleotide DNA was
incubated with or without full
-
length Miz
-
1 protein and subsequently separated by non
-
denaturing polyacrylamide get electrophoresis and DNA visualized
by SYBR green,
300nm UV transillumination.

Protein
-
containing lanes show a retardation of the short
oligonucleotides containing the motifs ATCGAT and GATTACCGA.




21


bind the MBP
-
Miz
-
1 zinc
-
finger construct. Although the zinc
-
finger construct lacks the
BTB/P
OZ domain, it does contain the reported c
-
Myc interaction domain
(
Sakamuro &
Prendergast, 1999
)
. Stringent
washing conditions with high ionic strength buffer (2 M
NaCl) did not disrupt the inte
raction between MBP
-
Miz
-
1 and c
-
Myc, however,
treatment with detergent abolished any interaction. Less stringent washing with ZBA
yielded background bands in the bead
-
only control. The MPB
-
Miz
-
1 pull down mass
spectrometry analysis was performed with an MB
P only control and the results are
summarized in Table 5. Significant matches were identified in the full
-
length Miz
-
1
construct, while the MBP only and Miz
-
1 zinc finger constructs yielded far fewer
peptides, all present in the MBP only control. Miz
-
1 rec
ombinant protein was readily
detected and comprised the most abundant protein in the samples, as expected. Other
ribosomal and collagen associated proteins identified as top hits in the scaffold 3 analysis
can be disregarded as background from the hESC nuc
lear extracts and contamination
respectively. However, nucleophosmin (NPM) and developmental pluripotency
-
associated protein 4 (DPPA4) are of interest as putative Miz
-
1 cofactors, but further
validation by western blot is needed.



22















Figure 4.
MBP
-
Miz
-
1 P
ull
-
down

of c
-
Myc
. Western blot of MBP
-
Miz
-
1 Pull
-
down with
c
-
Myc (72 kDa) input under increasingly stringent washing conditions

reveals specific
binding under high salt
. Under 2M NaCl salt washing condition c
-
Myc appears to
maintain i
nteraction with Miz
-
1. NP
-
40 detergent abolishes any interaction and ZBA
wash buffer yields higher background.



23


Table 5.
MBP
-
Miz
-
1 Mass Spectrometry A
nalysis by Scaffold 3.

Percent probability of
proteins identified by MS is given for targets of interest.

Two putative cofactors were
identified by MS
. DPPA4 is of interest for its role in mediating pluripotency in mouse
embryonic stem cells and NPM is

another target of interest with important known
transcriptional regulatory functions.

Further validation by
direct pull
-
down and western
blot is needed to confirm these interactions.


Protein

Coverage

Protein
Identification
Probability

Miz
-
1

9%

100%

NPM

8%

100%

DPPA4

8%

50%




24








Figure 5.
Current Model P
roposed for Miz
-
1

Target Gene R
egulation
.

Miz
-
1 gene
repression involves the recruitment of protein cofactors including Myc, HDAC and
Dnmt3A, while Miz
-
1 gene activation involves co
-
activators p300 and NPM. Adapted
from
Varlakhanova et al
, 2011.





INR

ATCGAT

GATTACCGA


Miz
-
1


Miz
-
1






Myc

HDAC

Max



NPM

p300

Dnmt3a

Dppa4

Growth

Cell cycle

Chromatin structure

Acetylation

Transcriptional control


Development

Hox

25


DISCUSSION


Essential to the success of regenera
tive medicine therapies is our basic scientific
understanding of hESCs. The complex regulatory network that governs pluripotency and
self
-
renewal, distinguishing characteristics of hESCs, is an important topic of basic stem
cell research. c
-
Myc, the well
-
s
tudied oncogene, is also a key player in the maintenance
of pluripotency and self
-
renewal in stem cells
(
N. V. Varlakhanova, Cotterman, R.F.,
deVries, W.N., Morgan, J., Donahue, L.R., Murray, S., Knowles, B.B., Knoepfler, P.S.,
2010
)
. Myc regulation of pluripotency and self
-
renewal is evidenced by its function in the
generation of iPS cells
(
Takahashi, et al., 2007
;
Takahashi & Yamanaka, 2006
)
. Myc
regulates target gene expression both loc
ally by classical mechanisms
,

and globally
through euchromatin remodeling
(
Knoepfler, et al., 2006
)
. In this way, Myc can affect
gene expression on a large enough scale to reprogram differentiated cells into iPS cells.
Miz
-
1
, named for its interaction with Myc,

is known to bind

initiator sequences
in the
promoters of target gene core promoters

thereby modulating their expression
(
Kime &
Wright, 2003
;
Peukert, 1997
;
Seoan
e, Le, & Massague, 2002
)
.

In the current model
(Figure 5
),
Miz
-
1 is thought to form a co
-
repressor complex with Myc, silencing Miz
-
1
target genes
(
Peukert, 1997
)
, and alternately, Miz
-
1 forms a co
-
activating complex with
p300 and NPM, activating target genes
(
M. Wanzel, Herold, S., Eilers, M., 2003
;
M.
Wanzel, Russ, A.C., Kleine
-
Kohlbrecher, D., Colomb
o, E., Pelicci, P.G., Eilers, M.,
2008
)
.
Still, relatively little is known about the function of Miz
-
1 as a transcriptional
regulator and recent gene expression and epigenetic analysis in human ESCs suggest
26


Miz
-
1 binds alternative sequences, not associa
ted with the initiator sequences of target
gene promoters
(
N. V. Varlakhanova, et al, 2011
)
.

CEAS analysis of Miz
-
1 ChIP
-
chip data failed to identify putative m
otifs for Miz
-
1 DNA binding.
While not surprising given the size and complexity of the data, finding
motifs
is

still vital to the study of Miz
-
1 in hESCs.
An
in vitro
approach to finding DNA
binding motifs is an efficient and comprehensive
alternative
way to examine
Miz
-
1
-
DNA
binding.
The production of MBP
-
Miz
-
1 fusion protein allows

for flexible analysis using
both
protein
-
DNA and protein
-
protein
biochemical
assays
.

Bind
-
n
-
Seq overcomes
problems associated with other motif
-
finding approaches including
limitations on
in vivo

detection and sensitivity, and time and labor
-
intensive
in vitro

approaches.

Instead, Bind
-
n
-
Seq employs massively parallel sequencing and a MBP purification scheme to
renovate

de novo
motif finding

into a high
-
throughput
in vitro

as
say
.

Identification of novel human
Miz
-
1 DNA binding motifs by Bind
-
n
-
Seq assay
(
Zykovich, 2009
)

has revealed highly
enriched DNA binding motifs for Miz
-
1 relating to both full
-
length and zinc
-
finger
containing protein constructs.
Currently, the Bind
-
n
-
Seq assay is optimized for the study
of zinc
-
finger containing proteins and even then, results high
ly depend on the specific
properties of each protein fusion construct. Additionally, Bind
-
n
-
Seq analysis is likely to
only identify the most highly enriched DNA motifs, while there may be several motifs for
a given protein based on its structural conformat
ion. Because of these limitations, Bind
-
n
-
Seq serves best as a stepping off point for further analysis of important protein
-
DNA
interactions
,

and it is important to incorporate other analyses to corroborate Bind
-
n
-
Seq
findings.
The
consensus
motifs
identif
ied for Miz
-
1,
ATCGAT and G
A
TTACCGA
,

were
27


both highly enriched over background and were further analyzed for their ability to bind
Miz
-
1 protein
in vitro

and their presence near Miz
-
1 bound genes, determined from
Varlakhanova

(
N. V. Varlakhanova, et al, 2011
)

hESC ChIP
-
chip data. EMSA analysis
confirmed Miz
-
1 binding ATCGAT and G
A
TTACCGA containing DNA probes in the
presence of poly
-
dI/dC non
-
specific competitor
oligonucleotide. Probing Miz
-
1 ChIP
-
chip data

by FIMO

reveled an abundance of motif
-
containing sequences related to the
Miz
-
1 ChIP
-
chip peaks. In total
,

over 2000 and 3000 sequences contained significant
matches to
the

motifs
identified by GLAM2

respective
ly. Of th
ese thousands of hits,
many are
sequences corresponding
to
genes

that were present in replicate in the ChIP
-
chip data

or belong to genes that have yet to be fully annotated.
Respectively,
166 and
2
12 well
-
annotated genes
identified by FIMO
were submitted for DAVID analysis
for the
zinc finger and full length constructs.

Significant, functionally related, gene ontology
clusters had overlap between both constructs including:
acetylation, phosphoprotein, and
transcriptional regulators.
The full
-
length construct revealed more significant matches in
both FIMO and DAVID analysis
, yet
it is important to note that there is significant
overlap between both constructs.
Across both constructs, t
he most highly enriched gene
ontology

clusters
from DAVID a
nalysis
include: chromatin, homeobox, transcription
corepressor, embryonic organ development, and cell proliferation associated genes.
These
results are in

agreement

with
known functional role
s

of Miz
-
1 in hESC and support the
hypothesis that Myc maintains

hESC

pluripotency and self
-
renewal in part through a co
-
repression

program with Miz
-
1.

28


Additionally
, t
he motif, ATCGAT, is of particular interest because of its
similarity to known transcription factor binding motifs,
including

c
-
Myc. ATCGAT is
a
palindro
mic

sequence like
that of
the Myc E
-
box,
CACGTG
.
However, ATCGAT has
not

yet

been previously
associated with
other
human
proteins

in the literature
.

Palindromic motifs in DNA are common and not unique to just tran
scription factor
binding motifs. T
hey
can
be

high
ly conserved across many species

and important for
mobile, repetitive DNA elements like transposons
.

These shared features of palindromic
DNA motifs may imply a
n important

function and significance for the
DNA
-
binding
motifs of master transcriptiona
l regulators like Myc and Miz
-
1.


The MBP
-
Miz
-
1 fusion protein
construct
also allowed for detection of n
ovel Miz
-
1 protein interactors
by
MBP pull
-
down mass spectrometry analysis.
Pull
-
down of hESC
nuclear extracts followed by mass spectrometry identificat
ion of proteins revealed a
couple candidate cofactors for Miz
-
1 including
DPPA4 and NPM
.

DPPA4 is not well
studied, however
,

it has been shown to be important to mESC
pluripotency

and self
-
renewal, whereby overexpression resulted in
cell proliferation and inhibition of
differentiation
(
Masaki, 2007
)
.

Alter
nately, NPM is a
better
-
characterized

protein

that
functions in diverse cellular processes from histone assembly and cell proliferation to

regulation of important tumor suppressors like p53 and ARF

(
Okuwaki, 2008
;
Sw
aminathan V., 2005
;
Wang H.F., 2011
)
.
The
coordinate
rol
e
of Miz
-
1 and NPM
is

only characterized by their association
in a

co
-
activating complex

(Figure 4
)
,
and

further
investigation

is of particular interest
.
Additional

biochemical
analysis
, like direct
immunoprecipitation or pull
-
down and weste
rn blot detection,

will need to be conducted
29


in order to validate putative

protein

cofactors.

Like the Bind
-
n
-
Seq assay, MBP pull
-
down mass spectrometry analysis serves as a great starting point for
more in
-
depth

biochemical and
in vivo

studies
.


Bind
-
n
-
S
eq has revealed important INR
-
independent DNA binding functions of
Miz
-
1
and MBP pull
-
down mass spectrometry
h
as identified
interesting

putative
cofactors of Miz
-
1

in hESCs
, while supporting the antagonistic functions of Myc and
Miz
-
1 in hESCs
.
Though

these important analyses require further validation and study
in
vivo
, the work represents an
essential

advance in the understanding of an important
master transcriptional regulator

and Myc antagonist

in hESCs.

Continued study of
the
basic regulation of p
luripotency and self
-
renewal in hESCs is vital to our understanding
of their purpose and potential in regenerative medicine so that therapies may be safe and
effective.




30


LITERATURE CITED


Brenner, C., Deplus, R., Didelot, C., Loriot, A.
, Vire, E., De Smet, C., et al. (2005). Myc
represses transcription through recruitment of DNA methyltransferase
corepressor. [print].
EMBO (European Molecular Biology Organization) Journal,
24
(2), 336
-
346.

Chaudhary, J., Michael K. Skinner. (1999). Basic Helix
-
Loop
-
Helix Proteins Can Act at
the E
-
Box within the Serum Response Element of the c
-
fos Promoter to Influence
Hormone
-
Induced Promoter Activation in Sertoli Cells.
Molecular Endocrinology
12
(5), 774
-
78
6.

Cotterman, R., Jin, V. X., Krig, S. R., Lemen, J. M., Wey, A., Farnham, P. J., et al.
(2008). N
-
Myc Regulates a Widespread Euchromatic Program in the Human
Genome Partially Independent of Its Role as a Classical Transcription Factor.
Cancer Research, 68
(23).

Eisenman, R. N. (2001). Deconstructing Myc.
Genes and Development, 15
, 2023
-
2030.

Evans, M. J., Kaufman, M.H. (1981). Establishment in culture of pluripotent cells from
mouse embryos.
Nature, 292
, 154
-
156.

Frith, M. C., Saunders, N.F., Kobe, B., Bailey, T.L. (2008). Discovering sequence motifs
with arbitrary insertions and deletions.
PLoS Computational Biology, 4
(5).

Grandori, C., Cowley, S.M., James, L.P., Eisenman, R.N. (2000). The MYC/MAX/MAD
network and
the transcriptional control of cell behavior. [Review].
Annual Review
of Cell Developmental Biology, 16
, 653
-
699.

Grant, C. E. B., T.L., Noble, W.S. (2011). FIMO: Scanning for occurrences of a given
motif.
Bioinformatics, 27
(7), 1017
-
1018.

Huang, D. W., Sh
erman, B.T., Lempicki, R.A. (2009a). Bioinformatics enrichment tools:
paths toward the comprehensive functional analysis of large gene lists.
Nucleic
Acids Research, 37
(1), 1
-
13.

Huang, D. W., Sherman, B.T., Lempicki, R.A. (2009b). Systematic and integrati
ve
analysis of large gene lists using DAVID Bioinformatics Resources.
Nature
Protocols, 4
(1), 44
-
57.

Ji X, L. W., Song J, Wei L, Liu XS. . (2006). CEAS: cis
-
regulatory element annotation
system.
Nucleic Acids Research, 1
(34), 551
-
554.

31


Kim, J. B., Sebastian
o, V., Wu, G., Araúzo
-
Bravo, M.J., Sasse, P., Gentile, L., Ko, K.,
Ruau, D., Ehrich, M., van den Boom, D., Meyer, J., Hübner, K., Bernemann, C.,
Ortmeier, C., Zenke, M., Fleischmann, B.K., Zaehres, H., Schöler, H.R. (2009).
Oct4
-
induced pluripotency in adu
lt neural stem cells.
Cell, 136
(3), 411
-
419.

Kime, L., & Wright, S. C. (2003). Mad4 is regulated by a transcriptional repressor
complex that contains Miz
-
1 and c
-
Myc. [print].
Biochemical Journal, 370
(1),
291
-
298.

Knoepfler, P. S. (2007). Myc goes global:
New tricks for an old oncogene.
Cancer
Research, 67
(11), 5061
-
5063.

Knoepfler, P. S., Zhang, X.
-
y., Cheng, P. F., Gafken, P. R., McMahon, S. B., &
Eisenman, R. N. (2006). Myc influences global chromatin structure.
EMBO
(European Molecular Biology Organizat
ion) Journal, 25
(12).

Masaki, H., Nishida, T., Kitajima, S., Asahina, K. and Teraoka, H. (2007).
Developmental Pluripotency
-
associated 4 (DPPA4) Localized in Active
Chromatin Inhibits Mouse Embryonic Stem Cell Differentiation into a Primitive
Ectoderm Line
age.
Journal of Biological Chemistry, 282
, 33034
-
33042.

McMahon, S. B., Wood, M.A., Cole, M.D. (2000). The essential cofactor TRRAP
recruits the histine acetyltransferase hGCN5 to c
-
Myc.
Molecular and Cellular
Biology, 20
, 556
-
562.

Meyer, N., Penn, L.Z. (2
008). Reflecting on 25 years with MYC. [Review].
Nature
Review Cancer, 8
(12), 976
-
990.

Nakagawa M., K. M., Tanabe K., Takahashi K., Ichisaka T., Aoi T. Okita K., Mochiduki
Y., Takizawa N., Yamanaka S. (2008). Generation of induced pluripotent stem
cells wi
thout Myc from mouse and human fibroblasts.
Nature Biotechnology,
26
(1), 101
-
106.

Okuwaki, M. (2008). The structure and functions of NPM1/Nucleophsmin/B23, a
multifunctional nucleolar acidic protein.
Biochemistry, 143
(4), 441
-
448.

Peukert, K., Staller, P.,

Schneider, A., Carmichael, G., Hanel, F., Eilers, M. (1997). An
alternative pathway for gene regulation by Myc.
EMBO (European Molecular
Biology Organization) Journal, 16
, 5672
-
5686.

Pierce, G. B. J., Dixon, F.J. Jr., Verney, E.L. (1960). Tetracarcinogeni
c and tissue
-
forming potentials of the cell types comprising neoplastic embryoid bodies.
Laboratory Investigation, 9
, 583
-
602.

32


Sakamuro, D., & Prendergast, G. C. (1999). New Myc
-
interacting proteins: A second
Myc network emerges. [print].
Oncogene, 18
(19),

2942
-
2954.

Seoane, J., Le, H.
-
V., & Massague, J. (2002). Myc suppression of the p21Cip1 Cdk
inhibitor influences the outcome of the p53 response to DNA damage. [print].
Nature (London), 419
(6908), 729
-
734.

Seoane, J., Pouponnot, C., Staller, P., Schader,
M., Eilers, M., & Massague, J. (2001).
TGFbeta influences Myc, Miz
-
1 and Smad to control the CDK inhibitor
p15INK4b. [print].
Nature Cell Biology, 3
(4), 400
-
408.

Stadtfeld, M., Hochedlinger, K. (2010). Induced pluripotency: history, mechanisms, and
applica
tion. [Review].
Genes & Development, 24
(20), 2239
-
2263.

Staller, P., Peukert, K., Kiermaier, A., Seoanet, J., Lukas, J., Karsunky, H., Moroy, T.,
Bartek, J., Massague, J., Hanel, F., Eilers, M. (2001). Repression of p15INK4b
expression by Myc through assoc
iation with Miz
-
1.
Nature Cell Biology, 3
, 392
-
399.

Swaminathan V., K. A. H., Febitha K.K., Kundu T.K. (2005). Human histone chaperone
nucleophosmin enhances acetylation
-
dependent chromatin transcription.
Molecular and Cellular Biology, 25
(17), 7534
-
7545.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., et al.
(2007). Induction of pluripotent stem cells from adult human fibroblasts by
defined factors.
Cell, 131
(5), 861
-
872.

Takahashi, K., & Yamanaka, S. (2006). Induction of pluri
potent stem cells from mouse
embryonic and adult fibroblast cultures by defined factors.
Cell, 126
(4), 663
-
676.

Varlakhanova, N. V., Cotterman, R.F., deVries, W.N., Morgan, J., Donahue, L.R.,
Murray, S., Knowles, B.B., Knoepfler, P.S. (2010). myc maintains

embryonic
stem cell pluripotency and self
-
renewal.
Differentiation, 80
, 9
-
19.

Varlakhanova, N. V., et al. (2011). Myc and Miz
-
1 have coordinate genomic functions
including targeting Hox genes in human embryonic stem cells.
Epigenetics and
Chromatin, 4
(20).

Wang H.F., T. K., Nakanishi A., Miki Y. (2011). BRCA2 and nucleophosmin coregulate
centrosome amplification and form a complex with the Rho effector kinase
ROCK2.
Cancer Research, 71
(1), 68
-
77.

Wanzel, M., Herold, S., Eilers, M. (2003). Transcription
al Repression by Myc. [Review].
TRENDS in Cell Biology, 13
(3), 146
-
150.

33


Wanzel, M., Russ, A.C., Kleine
-
Kohlbrecher, D., Colombo, E., Pelicci, P.G., Eilers, M.
(2008). A ribosomal protein L23
-
nucleophosmin circuit coordinates Mizl function
with cell growth.

Nature Cell Biology, 10
(9), 1051
-
1061.

Wu, S., Cetinkaya, C., Munoz
-
Alonso, M., von der Lehr, N., Bahram, F., Beuger, V.,
Eilers, M., Leon, J., Larsson, L.G. (2003). Myc represses differentiation
-
induced
p21CIP1 expression via Miz
-
1
-
dependent interaction
with the p21 core promoter.
Oncogene, 22
, 351
-
360.

Zykovich, A., Korf, I., Segal, D.J. (2009). Bind
-
n
-
Seq: high
-
throughput analysis of in
vitro protein
-
DNA interactions using massively parallel sequencing.
Nucleic
Acids Research, 37
(22), e151.