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Characterization of neocentromere formation in Drosophila melanogaster [Elektronische Ressource] / Agata Olszak

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96 pages
Ajouté le : 01 janvier 2010
Lecture(s) : 22
Signaler un abus

Max-Planck-Institute of Immunobiology
Freiburg im Breisgau

Albert-Ludwigs-Universitat Freiburg
Faculty of Biology







Characterization of neocentromere formation
in Drosophila melanogaster



Agata Olszak









Dissertation submitted to obtain a
Degree of Doctor rerum naturalium by the
Albert-Ludwigs-Universität
Faculty of Biology
Freiburg im Breisgau, Germany




Supervisor
Dr. Patrick Heun
Max-Planck-Institute of Immunobiology
Freiburg im Breisgau 2010

Erklärungen
Die vorliegende Arbeit wurde im Zeitraum von März 2006 bis April 2010 am Max-
Planck-Institut für Immunbiologie in Freiburg im Breisgau unter Anleitung von Herrn Dr.
Patrick Heun angefertigt.

Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und
ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus
anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter
Angabe der Quelle gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche
Hilfe von Vermittlungs- beziehungsweise Beratungsdiensten (Promotionsberater oder
anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder
mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem
Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In- noch im
Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.

Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität
Freiburg sind mir bekannt; insbesondere weiß ich, dass ich vor Vollzug der Promotion
zur Führung des Doktortitels nicht berechtigt bin.

Dekan der Fakultät: Prof. Dr. Ad Aertsen
Promotionsvorsitzender: Prof. Dr. Eberhard Schäfer

Betreuer der Arbeit: Dr. Patrick Heun

Referent: Prof. Dr. Rudolf Grosschedl
Koreferent: Prof. Dr. Karl-Friedrich Fischbach
3. Prüfer: Dr. Patrick Heun

Tag der mündlichen Prüfung: 29.06.2010

 II

Abbreviations

A
 Ampere

nd2L
 left arm of 2 Drosophila chromosome

nd2R
 right arm of 2 

3D
 three-dimensional

rd3L
 left arm of 3 Drosophila chromosome

rd3R
 right arm of 3 

Ab
 antibody

APS
 aminopropyltriethoxysilane

ATP
 adenosine triphosphate

BAC
 bacterial artificial chromosome

bp
 base pair

C. albicans
 Candida albicans

C. elegans Caenorhabditis elegans
CAD
 CENP-A-nucleosome distal

CATD
 Centromere protein A targeting domain

CCAN
 constitutive centromere-associated network

CEN
 centromere

CenH3
 centromere specific histone H3 variant

CENP-A-W
 centromere protein A-W

chr
 chromosome

CID 
 centromere identifier

Da
 Dalton

DAPI
 ',6-diamidino-2-phenylindole

DMSO
 dimethyl sulfoxide

DNA
 deoxyribonucleic acid

dNTP
 deoxyribonucleotide triphosphate

DSB
 double strand break

dsRNA
 double stranded RNA

DTT
 dithiothreitol

dUTP
 deoxyuridine triphosphate

E. coli
 Escherichia coli

EDTA
 thylenediaminetetraacetic acid

EM
 electron microscopy

eu
 euchromatin

F
 forward

FACS
 fluorescence-activated cell sorting

FCS
 Fetal Calf Serum

FISH
 fluorescence in situ hybridization

FP
 fluorescence protein

GFP
 green fluorescence protein

H3K9me2
 dimethylation on lysine 9 of histone H3


 III
H4Ac
 acetylation on histone H4

HA
 haemagglutinin

HAC
 human artificial chromosome

het
 heterochromatin

HFD
 histone fold domain

HJURP
 holliday junction recognition protein

hygro
 hygromycin

IF
 immunofluorescence

kMT
 kinetochore microtubule

LacI
 Lac repressor

LacOp
 Lac Operator

Mbp
 mega base pair

MT
 microtubule

mut
 mutated

NAC
 nucleosome associated complex

NPM1
 Nucleophosmin1

PBS
 phosphate buffered saline

PCR
 polymerase chain reaction

PEV
 position effect variegation

PFGE
 pulse field gel electrophoresis

Pol
 polymerase

prox
 proximity

puro
 puromycin

rDNA
 ribosomal DNA

rev
 reverese

RFP
 red fluorescence protein

RNAi
 RNA interference

rpm
 revolutions per minute

RT
 room temperature

S. cerevisiae
 Saccharomyces cerevisiae

S. pombe
 Schizosaccharomyces pombe

SDS
 sodium dodecyl sulfate

TdT terminal deoxynucleotidyl transferase
tel
 telomere

TEMED
 tetramethylethylenediamine

trx
 transcript

UV
 ultra violet

WT
 wild type










 IV



 V
Erklärungen.................................................................................................................................................II

Abbreviations............III

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

1.1
 Chromatin.....................................................................................................................................1

1.1.1
 Nucleosome..........................1

1.1.2
 Higher
order
structure....2

1.2
 The
centromere................................................................................................4

1.2.1
 Specification
of
centromere..........................7

1.3
 Organization
of
centromeric
chromatin..........................................................................9

1.3.1
 The
centromere
specific
histone
variant
(CenH3)..............9

1.3.2
 Composition
of
CenH3
nucleosomes.....11

1.3.3
 Histone
modifications
at
centromeres..................................................................13

1.3.4
 CenH3
deposition...........................................13

1.4
 The
kinetochore.......................................................16

1.4.1
 Constitutive
and
cycle‐dependent
components
of
kinetochore................17

1.5
 Neocentromeres......................................................................................20

1.6
 Aim
of
the
thesis................................22

2
 MATERIALS
&
METHODS............24

2.1
 Cloning.........................................................................................................................................24

2.2
 Cell
culture.26

2.2.1
 Cultivation
of
cells..........26

2.2.2
 Stable
transfection.........................................................................................................26

2.2.3
 Protein
expression.........28

2.3
 Live
imaging..............................................................28

2.4
 Image
acquisition
and
analysis.........................................................................................30

2.5
 Fluorescent
Activated
Cell
Sorting
(FACS)...................................31

2.6
 Cytological
preparations......................................................................31

2.7
 Microtubules
fixing................................................32

2.8
 Immunofluorescence.............32

2.9
 Metamorph®
Analysis..........................................................................35

2.10
 Laser‐Microsurgery.............................................35

2.11
 HP1
depletion
by
RNA
interference
(RNAi).............................................................35

2.12
 Western
analysis..................................................................................36

2.13
 Genomiphi
V2
DNA
amplification.................................................37

2.14
 Fluorescence
in
situ
Hybridization
(FISH)................................................................38

2.14.1
 FISH
probes
preparation..........................................................38

2.14.2
 Probes’
purification
and
precipitation...............................40

2.14.3
 Hybridization
reaction..............................................................40

2.15
 Minichromosome
rescue
with
telomeric
sequences............................................42

3
 Results..................................................................................................................43

3.1
 Pulse
induction
of
CID
overexpression
leads
to
the
formation
of
ectopic
“CID

islands”
with
kinetochore
function............................................................43

3.2
 CID
islands
self‐propagate
for
multiple
cell
generations......................................48

3.3
 Overexpression
of
CID
combined
with
the
histone
H3.3
variant
or
bearing

mutations
in
the
CATD
domain
of
CID
interfere
with
CID
islands
formation..........50
3.4
 Ectopic
kinetochores
preferentially
form
near
telomeres
and
pericentric

heterochromatin
regions................................................................................................................53

3.5
 Formation
of
functional
ectopic
kinetochores
protects
CID
islands
from

clearing...................................................54

3.6
 High
levels
of
HP1
expression
interfere
with
CID
islands
formation...............59

3.7
 Local
targeting
of
HP1‐LacI
to
Lac
Operator
DNA
sequences
creates
a
new

hotspot
for
CID
deposition.............................................................................................................61

3.8
 Telomere
protection
might
stabilize
chromosome
fragments
with
CID

islands.....................................................63

4
 Discussion...........................................................................................................73

5
 Summary.............79

6
 Acknowledgements........................................................................................................................80

7
 References..........................................82


 VII

1 INTRODUCTION
1.1 Chromatin
All of the genetic information crucial for cellular viability and development of an
organism is encoded by deoxyribonucleic acid (DNA). DNA forms long strands
(up to 3 billion base pairs in humans) that need to be tightly wound in order to fit
into the nucleus of about 10um in diameter. This high compaction is achieved by
forming a nucleoprotein complex called chromatin. Two types of chromatin with
distinctive compression level can be recognized: euchromatin and
heterochromatin [1]. The less condensed euchromatin has a more open
conformation and is often under active transcription. It comprises the most active
portion of the genome within the nucleus of the cell. The highly compacted
heterochromatin is generally silenced for transcription and less accessible for
transcription factors and chromatin remodelers. Two general forms can be
distinguished. Constitutive heterochromatin is usually repetitive and forms
structural elements such as centromeres or telomeres. Facultative
heterochromatin is not repetitive, shares the compact structure of constitutive and its state depends on specific developmental or
environmental signaling.

1.1.1 Nucleosome
The basic unit of chromatin is the nucleosome. It consists of about 146 bp of
DNA that are wrapped in 1.67 left-handed superhelical turns around the histone
octamer. It is composed of two copies of each of the four core histones H2A,
H2B, H3, and H4 [2] (Fig.1.1). All core histones are positively charged and have
therefore a strong affinity to negatively charged DNA. Histones are small proteins
of only 100-140 amino acids and consist of a globular domain containing the
histone fold domain (HFD) and a flexible, unstructured N-terminal tail. The fifth

 1
histone, the linker histone H1, binds outside the core nucleosome at the entry
and exit sites of the DNA, thus locking the DNA into place. Adjacent


complete nucleosome
core with DNA










Figure 1.1 Schematic representation of the assembly of the core histones
into the nucleosome. Histone H3 forms dimers only with histone H4, and
histone H2A only with histone H2B. The histone octamer is made of a tetramer of
histones H3–H4, flanked by two dimers of histones H2A–H2B. The nucleosome
core is completed when DNA wraps around the octamer with 1 3/4 turns.

nucleosomes are joined by a stretch of free DNA termed "linker DNA" which
varies from 10 - 80 bp in length depending on species and tissue type.


1.1.2 Higher order structure
The most basic level of chromatin compaction is accomplished by formation of
the 10 nm fibers or so called beads on a string conformation (Fig. 1.2). This array
of nucleosomes results in an approximately 5-7 fold compaction of DNA and is
further arranged in a 30 nm fiber. This compacted structure has a packing ratio of
~50-fold and its formation is dependent on the presence of the H1 histone. It is
still unclear how the structure of 30 nm fiber is formed, but two models have been

 2
proposed. The solenoid model consists of a one-start helix in which the
nucleosomes are organized around a central axis and histone H1 is in the center.
In the second, zigzag model the nucleosomes are organized in a two-start helix


DNA

add core

histones The nucleosome



Beads-on-a-string
add histone
H1

The 30nm fibre


add further scaffold
proteins

Active chromosome
(Interphase)

add further scaffold
proteins

The metaphase chromosome
(cell division)



Figure 1.2 The major structures in DNA compaction. Histones H2A, H2B, H3,
and H4 form a histone octamer, around which DNA wraps, to form a nucleosome
core. The nucleosome core plus 10-80 bp of linker DNA and one molecule of
histone H1 forms a nucleosome, which is the basic unit of 10 nm fiber (“bead on
a string”) structure. This chromatin can be more highly organized and condensed

 3


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