Development of new methods to study DNA methyltransferase function and protein interactions in living cells [Elektronische Ressource] / vorgelegt von Kourosh Zolghadr

Kourosh Zolghadr

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 22 Mo

Extrait

Development of new methods to study
DNA methyltransferase function and
protein interactions
in living cells

Kourosh Zolghadr












Dissertation
an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München



vorgelegt von
Kourosh Zolghadr
aus Teheran





München, den 18.12.2007































Erstgutachter: Prof. Dr. Heinrich Leonhardt
Zweitgutachter: Prof. Dr. Harry MacWilliams

Tag der mündlichen Prüfung: 19.03.2008
INDEX
SUMMARY .............................................................................................................................................3
CHAPTER I. INTRODUCTION................................................................................................................5
I.1. How to detect protein interactions?....................................................................................................6
I.1.1. Antibodies and derivates thereof to identify proteins ...................................................................6
I.1.2. Biochemical methods to study protein interactions .....................................................................8
I.1.3. Genetic yeast two-hybrid screens to detect protein-protein interactions ....................................9
I.1.4. Fluorescently labeled proteins and applications in living cells..................................................10
I.2. DNA methylation in mammalian cells ..............................................................................................16
I.2.1. Function and localization of Dnmt1...........................................................................................18
I.2.2. Molecular mechanism of DNA methylation...............................................................................20
CHAPTER II. RESULTS.............................................................................................................................25
II.1. A fluorescent two-hybrid (F2H) assay for direct visualization of protein-protein
interactions in living cells .................................................................................................................25
II.2. Targeting and tracing antigens in live cells with fluorescent nanobodies......................................61
II.3. A versatile nanotrap for biochemical and functional studies with fluorescent
fusion proteins...................................................................................................................................77
II.4. Trapped in action: direct visualization of DNA methyltransferase activity
in living cells........................97
II.5. Regulation of DNA methyltransferase 1 ........................................................................................109
CHAPTER III. DISCUSSION...................................................................................................................123
III.1. A new assay to visualize protein-protein interactions in vivo .....................................................123
III.2. A new antibody-format to follow endogenous antigens in living cells .......................................125
III.3. A GFP-binding protein for biochemical and functional studies ..................................................127
III.4. Direct visualization of enzyme-activity in living cells .................................................................128
III.5. Combinatorial approaches and outlook........................................................................................130
CHAPTER IV. ANNEX.............................................................................................................................135
IV.1. Abbreviations .................................................................................................................................135
IV.2. Contributions137
IV.2.1. Declarations of Contributions to II.1 ......................................................................................137
IV.2.2. Declarations of Contributions to II.2137
IV.2.3. Declarations of Contributions to II.3137
IV.2.4. Declarations of Contributions to II.4138
IV.2.5. Declarations of Contributions to II.5138
IV.2.6. Declaration according to the
"Promotionsordnung der LMU München für die Fakultät Biologie” .....................................138
IV.3. Acknowledgements ........................................................................................................................139
IV.4. References ......................................................................................................................................140
CHAPTER V. CURRICULUM VITÆ ......................................................................................................155
V.1. Publications .....................................................................................................................................156
V.2. Awards and conference contributions ...........................................................................................157


-2- Summary
Summary
Interactions between proteins are central to essentially all cellular processes. Many methods for
the investigation of proteins have been developed in the last years, requiring either removal of
the proteins from their normal cellular environment, perturbation of the cells or costly
instrumentation and high technical expertise. Therefore we aimed at designing assays for in
vivo studies of protein interaction, distribution, dynamics, characterization, manipulation and
enzyme activity focusing on proteins essential for DNA methylation.
During this thesis we developed a novel fluorescent two-hybrid (F2H) assay for a direct analysis
of protein-protein interactions in real-time in mammalian cells using a simple co-localization
read-out of fluorescent bait and prey proteins at a stably integrated chromosomal lac operator
array. The F2H assay can in principle be automated and therefore be used for high-throughput
protein-protein interaction analysis in living cells.
To study localization and mobility of endogenous proteins inside cells we took advantage of
antibodies derived from alpacas (Lama pacos). Alpacas and other Camelidae posses so-called
heavy-chain antibodies that lack light chains. We isolated the heavy chain variable domain
(V H) of these single chain antibodies, fused this antigen-binding fragment to a fluorescent H
protein and expressed the resulting chimeric protein (referred to as chromobody) in cells.
Chromobodies allow for the first time targeting and tracing of endogenous antigens, i.e.
proteins as well as non-protein components in living cells.
An additional, highly versatile application of V Hs is to couple them on solid support for H
biochemical methods or to fuse them to a structural protein. We generated a V H against the H
green fluorescent protein GFP (referred to as GFP-binding protein; GBP) and tethered it to the
nuclear lamina (referred to as GFP-nanotrap). With this intracellular GFP-nanotrap we could
ectopically capture GFP-fusion proteins and their interacting endogenous factors. Such directed
manipulation of cellular components opens up new possibilities to study protein dynamics,
function and regulation including the role and organization of nuclear architecture.
To gain insight into the regulation and maintenance of cytosine-5 methylation by DNA
methyltransferases (Dnmts), an epigenetic hallmark leading to transcriptional silencing, we
developed an in vivo trapping assay that directly visualizes the activity and subnuclear targeting
of Dnmts in the context of dynamic chromatin structures and simultaneously ongoing DNA
replication. The trapping assay offers an approach to test new types of Dnmt inhibitors
including small molecules.
In summary, we developed new, simple, versatile and efficient methods and tools to study the
regulation and function of proteins involved in complex cellular processes such as DNA
methylation in living cells in real-time.
-3- Summary
-4- CHAPTER I Introduction

CHAPTER I. Introduction
The understanding of cellular processes and regulatory networks requires quantitative data on
all cellular components including their concentration, modifications, interactions, distributions,
dynamics and activity in living cells. Biological processes such as DNA replication or DNA
methylation are strictly coordinated and mostly take place in complex, dynamic networks and
clusters where many proteins are involved. In the case of DNA replication, being an essential
part of the cell cycle, this implies assembly of several thousand replication clusters throughout
the cell which comprise specialized proteins to unwind the DNA, stabilize the single strand
state and precisely duplicate each and every base pair of the genome exactly one time on the
leading strand as well as on the lagging strand where in additi