Acquisition and loss of chromatin modifications during an Epstein-Barr Virus infection [Elektronische Ressource] / Anne Schmeinck. Betreuer: Dirk Eick





DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES
DER FAKULTÄT FÜR BIOLOGIE
DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN






ACQUISITION AND LOSS OF CHROMATIN MODIFICATIONS
DURING AN EPSTEIN-BARR VIRUS INFECTION

















ANNE SCHMEINCK



































Dissertation eingereicht am 28. April 2011
Erstgutachter: Prof. Dr. Dirk Eick
Zweitgutachter: Prof. Dr. Heinrich Leonhardt

Tag der mündlichen Prüfung: 25.10.2011 

TRICKERKLÄRUNG

Hiermit erkläre ich, dass die vorliegende Arbeit mit dem Titel

„ACQUISITION AND LOSS OF CHROMATIN MODIFICATIONS DURING AN EPSTEIN-BARR VIRUS
INFECTION“

von mir selbstständig und ohne unerlaubte Hilfsmittel angefertigt wurde, und ich mich dabei
nur der ausdrücklich bezeichneten Quellen und Hilfsmittel bedient habe. Die Arbeit wurde
weder in der jetzigen noch in einer abgewandelten Form einer anderen Prüfungskommission
vorgelegt.

München, 28. April 2011






Anne
Schmeinck 


CK

































BRIGHT LIGHTS IN BLACK HOLES

TRICKCONTENT
1.
 INTRODUCTION 
 1

1.1 
 Epstein ­Barr
virus
 –
discovery
and
basic
principles 
 1

1.1.1
 EBV
and
cellular
mimics
 2 

1.2
 Epigenetics 
 5

1.2.1
 DNA
methylation 
 7

1.2.2
 Nucleosomes:
More
than
just
beads
on
a
string 
 11

1.2.3
 Histone
modifications
 16

1.2.4
 The
epigenetic
memory
 20 

1.3 
 Epigenetics
in
EBV
 22

1.3.1
 EBV’s
life
cycle
 22 

1.3.2
 Important
checkpoints
of
EBV’s
life
cycle
rely
on
epigenetic
mechanism
s 24 

1.4
 Scope
of
my
thesis
work
 25

2.
 MATERIAL
 26

2.1
 Plasmids
 26

2.2
 Antibodies 
 26

2.3
 Oligonucleotides
 27

2.4
 Bacterial
strains
 27

2.5
 Eukaryotic
cell
lines 
 27

2.6
 Cell
culture
media
and
additives
 27

2.6.1
 Media
for
the
cultivation
of
bacteria 
 27

2.6.2
 Media
for
the
cultivation
of
eukaryotic
cells 
 28

2.7
 Chemicals
and
enzymes
 28

2.8
 Buffers
and
solutions
 29

2.9
 Commercial
kits
 30 

2.10
 Software
 30 

2.11 
 Devices
and
consumables
 31 

3.
 METHODS
 32

3.1 
 Bacterial
culture
 32

3.2
 Eukaryotic
cell
culture 
 32

3.2.1
 Cell
culture
conditions
 32 

3.2.2
 Storage
of
eukaryotic
cells 
 33

3.2.3
 Electroporation
of
eukaryotic
cells 
 33

 CONTENT
 II

3.2.4
 Establishment
of
stablce
ll
lines
 33

3.2.5
 Isolation,
separation
and
infection
of
human
B
cells
 33

3.2.6
 Collection
of
B95.8
virus
stocks
 34

3.2.7 
 Flow
cytometry
 34

3.2.8
 Sorting
of
GFP
expressing
cells
 34

3.3 
 Nucleic
acid
techniques 
 35 

3.3.1
 DNA
purification
from
E.coli 
 35

3.3.2
 DNA
purification
from
eukaryotic
cells 
 35

3.3.3
 Purification
of
DNA
from
PCR
products
and
agarose
gels 
 35

3.3.4
 Polymerase
chain
reaction
(PCR) 
 36

3.3.5
 Quantitative
real
time
PCR 
 36

3.3.6
 Isolation
of
RNA
from
cells 
 37

3.3.7
 Reverse
transcription
of
RNA 
 37

3.3.8
 Transfer
of
DNA
to
membranes
(Southern
blot)
 38

3.3.9
 Radioactive
labeling
of
DNA
 38

3.3.1
0 DNA ‐DNA
hybridization 
 38

3.4
 Methylated
DNA
immunoprecipitation
(MeDIP)
 39 

3.4.1
 Immunoprecipitation
of
methylated
DNA
(MeDIP) 
 39

3.4.2
 Quantification
of
MeDIP
DNA
by
real
time
PCR
(qPCR) 
 39

3.4.3
 Genome‐wide
analysis
of
MeDIP
DNA
by
microarray
hybridization
(MeDIP ‐
on ‐ChIP) 
 39

3.5
 Bisulfite
sequencing
 41

3.5.1
 Bisulfite
modification
of
DNA 
 41

3.5.2
 PCR
of
bisulfite
modified
DNA 
 41

3.5.3
 Deep
bisulfite
sequencing 
 41

3.5.4
 Data
analysis
 41

3.6
 Analysis
of
nucleosome
occupancy
 42

3.6.1
 MNase
digestion
of
chromatin 
 42 

3.6.2
 Labeling
of
DNA
for
microarray
hybridization 
 43

3.6.3
 Microarray
hy bridization 
 43

3.6.4
 Data
analysis
 43

3.7
 Chromatin
Immunoprecipitation
(ChIP)
 44

3.7.1
 Chromatin
preparation 
 44

3.7.2
 Chromatin
immunoprecipi tation
and
purification
of
ChIP
DNA 
 45

3.7.3
 Quantification
of
 ChIP
 DNA
by
real
time
PCR
(qPCR)
 45

3.8
 Indirect
endlabeling 
 46

3.9
 Western
blot
immunodetection
 of
RNA
Pol
II
 46

4.
 RESULTS
I
 47

4.1
 Early
epigenetic
events
can
be
monitored
in
infection
experiments
with
primary
B

cells
 in
vitro
 48

4.2
 EBV’s
DNA
methylation
is
a
slow,
but
precise
process
and
can
be
followed
in
vitro
 49

4.2.1
 MeDIP
experiments
in
the
cell
line
Raji
 49 

4.2.2
 Kinetics
of
DNA
methylation
in
primary
infected
B
cells 
 52 

4.3
 EBV’s
DNA
is
governed
by
nucleosomes
very
early
after
infection 
 55

4.3.1
 Micrococcal
nuclease
is
a
tool
to
study
nucleosomal
DNA 
 56

4.3.2
 A
Southern
blot
analysis
detects
EBV
DNA
in
nucleosomes
nine
days
p
i 56

4.3.3
 OriP
nucleosomes
are
detected
as
early
as
three
days
pi
in
a
qPCR
approac
h 57

5.
 RESULTS
II
 60

5.1
 EBV’s
DNA
methylation
at
the
nucleotide
level 
 61 

5.1.1
 Deep
bisulfite
sequencing
assesses
EBV
DNA
methylation
at
the
nucleotide
level 
 62 

5.1.2
 Lytic
induction
does
not
change
the
DNA
methylation
of
EBV
promoters 
 67

5.2
 Nucleosome
occupancies
in
EBV
 70

 CONTENT
 III 

5.2.1
 Mononucleosomal
DNA
on
Chip
(MND ‐on ‐Chip)
experiments
assess
the
nucleosomal

occupancy
of
viral
DNA 
 70

5.2.2
 Nucleosome
occupancy
in
EBV’s
BZLF1‐responsive
promoters
 71

5.2.3
 Indirect
endlabeling
and
Chromatin
Immunoprecipitation
experiments
confirm
the
loss

of
nucleosomes
at
ZREs
 78

5.3
 Histone
modifications
and
chromatin
modifying
enzymes
in
EBV
 81

5.3.1
 Histone
H3
and
its
post‐translational
modifications
at
EBV
promoters 
 82

5.3.2
 Polycomb
proteins
set
up
the
repressed 
state
in
EBV’s
lytic
promoters
 86

5.4
 Occupation
of
EBV
promoters
with
RNA
polymerase
II
 87

5.4.1
 ChIP
experiments
of
RNA
Pol
II
and
its
CT‐mD odified
versions
 90

5.4.2
 The
Pol
IIB
form
cannot
be
detected
in
western
blomt
imunodetection
after
induction
of

the
lytic
phase 
 91

5.4.3
 The
binding
of
RNA
Pol
II
at
promoters
of
early
lytic
genes
strongly
increased
gene

transcription 
 92 

6.
 DISCUSSION
 95

6.1
 State
of
the
art 
 98

6.2
 Scope
and
aim
of
my
thesis
work
 100 

6.3
 How
to
establish
and
maintain
latency?
 101 

6.3.1
 The
temporal
establishment
of
an
epigeentic
pattern
on
EBV
DNA 
 101

6.3.2
 Epigenetic
modifications
help
to
maintain
the
latent
state
 103

6.4
 How
to
escape
the
latent
state?
 105 

6.4.1
 Primary
infected
B
ces
llare
ready
for
lytic
reactivation
two
weeks
 pi 
 105

6.4.2
 Chromatin
changes
at
defau‐lotff
promoters
after
lytic
induction
 105

6.4.3
 Two
default­off 
promoters
in
a
close‐up:
Epigenetic
regulation
 at
 BBLF4 
and
 BMRF1 
 108

6.4.4
 Chromatin
changes
atd
efault­on 
and
 poised­on 
promoters
upon
lytic
induction
 111

6.5
 Open
questions
and
outlook
 112 

6.5.1
 Why
are
certain 
parts
of
the
EBV
genome
kept
in
an
open
configuration? 
 112

6.5.2
 What
is
the
mechanism
of
chromatin
remodeling
upon
lytic
induction? 
 112

6.5.3
 What
is
the
mechanism
of
poised­on 
and
 default­on 
pr omoter
activation
upon
lytic

induction?
 113

6.5.4
 What
is
the
nature
of
RNA
Polymerase
II
during
lytic
replication
of
EBV? 
 114

6.5.5
 Which
concepts
of
EBV
can
be
transferred
to
cellular
epigenetic 
mechanisms? 
 114

7.
 SUMMARY 
 115 

8.
 ABBREVIATIONS
 117 

9.
 LITERATUR
 120 

10.
 APPENDIX 
 127 

10.1 
 Oligonucleotides 
 127 

10.1.1
 RT‐PCR
Primer
 127

10.1.2
 qPCR
Primer
 128

10.1.3
 Deep
bisulfite
sequencing
primer 
 129 

10.2
 Deep
bisulfite
sequencing
analys is 
 131 

10.2.1
 Matlab
script
 131

10.2.2
 Deep
bisulfite
sequencing
results
of
all
CpG
sites
in
the
analysis
 132 

10.3 
 Nucleosome
occupancies
at
BZLF1
responsive
promoters
 139 

11. 
 PUBLICATIONS
 140

12.
 CURRICULUM
VITAE
 141

1. INTRODUCTION
1.1 Epstein-Barr virus – discovery and basic principles
Epstein-Barr virus (EBV), also called human herpesvirus 4 (HHV 4), belongs to the family of
γ-herpesviruses. EBV infects very efficiently human B cells due to the strong interaction of
the viral glycoprotein gp350/220 with the complement receptor CD21, which is presented on
the surface of B cells and certain epithelial cells (Nemerow et al., 1987).
EBV’s discovery is closely related to an important characteristic of the virus: the association
with different human tumors and lymphomas. In 1958, Denis Burkitt investigated a malignant
tumor of children in equatorial Africa (Burkitt, 1958), which became to be known as Burkitt’s
lymphoma (BL). Because of the conspicuous coincidence of this tumor disease with the
distribution area of malaria, he assumed the connection of an infectious agent spread by
arthropods to this disease (Burkitt, 1962). In 1964, the virologist Anthony Epstein and his
student Yvonne Barr as well as R. J. Pulvertaft were able to cultivate a B cell line out of those
tumor samples (Epstein et al., 1964; Pulvertaft, 1964). Epstein was capable to prove the
existence of herpesviru