Table Of ContentAnalysis of the cell cycle dependent
dynamics of Dnmt1 and Np95
in living cells
Andrea Rottach
München 2009
Analysis of the cell cycle dependent
dynamics of Dnmt1 and Np95
in living cells
Andrea Rottach
Dissertation
an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München
vorgelegt von
Andrea Rottach
aus Garmisch-Partenkirchen
München, den 29. Oktober 2009
Erstgutachter: Prof. Dr. Heinrich Leonhardt
Zweitgutachter: PD. Dr. Berit Jungnickel
Tag der mündlichen Prüfung: 18.12.2009
CONTENTS
Contents
Summary 3
1. Introduction 5
1.1. DNA methylation-mediated epigenetic control 5
1.1.1. DNA methylation in vertebrates 6
1.1.2. Connection between DNA replication and DNA methylation 10
1.1.3. Mechanisms of DNA methylation-mediated transcriptional repression and their
interconnection with other epigenetic pathways 15
1.2. Tools and techniques to monitor Dnmt1 and interacting proteins in their natural environment 21
1.2.1. Antibody based protein detection 21
1.2.2. Fluorescent protein tags and photobleaching techniques 27
1.2.3. Mouse embryonic stem cells as model system to analyze protein dynamics, interactions
and complex protein networks in vivo 31
1.3. Aims of the work 32
2. Results 33
2.1. Generation and Characterization of a Rat Monoclonal Antibody against multiple red
fluorescent proteins. 33
2.2. Generation and Characterization of a Rat Monoclonal Antibody specific for PCNA. 43
2.3. Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative
maintenance of DNA methylation. 55
2.4. DNMT1 but not its interaction with the replication machinery is required for maintenance of
DNA methylation in human cells. 77
2.5. Structure and function of the mouse Dnmt1 CXXC zinc finger domain. 87
2.6. The multi-domain protein Np95 connects DNA methylation with histone modification. 99
2.7. DNA methylation-mediated epigenetic control. 137
3. Discussion 149
3.1. Monitoring Dnmt1 and interacting proteins in their natural environment 149
3.2. Dnmt1 dynamics and cell cycle dependent regulation in living cells 155
3.2.1. Functional and structural consequences of the PCNA interaction 157
3.2.2. Role of the CXXC zinc finger in Dnmt1 mobility and activity 161
3.2.3. Coordinated binding kinetics of Dnmt1 regulatory subdomains 167
3.3. Role and regulation of the multi-domain protein Np95 in living cells 169
4. Annex 177
4.1. References 177
4.2. Abbreviations 195
4.3. Contributions 199
4.4. Acknowledgements 203
5. Curriculum Vitae 205
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CONTENTS
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SUMMARY
Summary
Epigenetic marks like DNA methylation, post-translational histone modifications and
associated chromatin states modulate gene expression, chromatin structure and
cellular differentiation. DNA methyltransferase 1 (Dnmt1) plays an essential role in
the maintenance of DNA methylation patterns after DNA replication and is tightly
regulated by a multitude of intra- and intermolecular interactions.
In this study, I analyzed the cell cycle dependent localization, activity and dynamics
of Dnmt1 and its interaction partners PCNA and Np95 in living cells. The goal was to
achieve a better understanding of the complex regulation of Dnmt1 in vivo and how
these interactions are involved in the stable inheritance of DNA methylation patterns.
For this purpose, I developed tools and techniques to monitor Dnmt1, PCNA and
Np95 in their natural, cellular environment. More specifically, I established advanced
live cell microscopy and photobleaching techniques in somatic and embryonic stem
(ES) cells and generated specific monoclonal antibodies to study Dnmt1, PCNA and
red fluorescent fusion proteins in different cell biological and biochemical applications.
The association of Dnmt1 with PCNA throughout S phase was proposed as an
efficient mechanism, coupling the replication of genetic and epigenetic information.
Whether the PCNA binding domain (PBD) mediated interaction with the replication
machinery is strictly required for the faithful propagation of DNA methylation marks
in vivo was still elusive. I have addressed this question by comparing the subcellular
localization, binding kinetics and activity of GFP-tagged wt Dnmt1 and PCNA-binding
deficient mutants via live cell photobleaching assays and in vivo Dnmt activity
measurements (trapping assay). I could show that the interaction of Dnmt1 with
PCNA is highly transient, increases the efficiency of postreplicative methylation by
twofold, but is not strictly required for maintaining global CpG methylation. I propose
that the dynamic binding of Dnmt1 to PCNA may increase local protein concentration
at replication sites and may thereby serve as additional safety mechanism.
As part of the large N-terminal region of Dnmt1, the zinc finger (ZnF) domain was
suggested to play an essential role in Dnmt1 enzyme activation and DNA substrate
recognition. In this study, I observed a nuclear binding of the isolated ZnF domain,
whereas it seemed to be dispensable for the overall Dnmt1 localization, dynamics
and activity in context of the full-length protein.
Recently, Np95 emerged as key regulator of DNA methyltransferases. The
recognition of hemimethylated DNA substrates via Np95 and subsequent targeting of
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SUMMARY
Dnmt1 to these DNA sites is considered a crucial step in the DNA methylation
maintenance process. Hence, I investigated the influence of different methylation
levels on Np95 localization, activity and dynamics in living wt and knockout ES cells.
Surprisingly, Np95 localization and binding kinetics were not affected by either
-/- -/- -/- -/-
reduced (dnmt1 ) or completely absent (TKO; dnmt1 3a 3b ) DNA methylation
compared to wt (J1) ES cells. I found a strong binding of Np95 to pericentric
heterochromatin that was dominated by the SRA domain and suggest that the stable
interaction of Np95 with chromatin may serve as anchor point for the transient
binding of Dnmt1. Furthermore, in vitro data showed that Np95 preferentially binds
trimethylated but not acetylated H3 N-terminal histone tails (H3K9) via a tandem
Tudor domain that accommodates the histone tail in an aromatic cage similar to that
of HP1β. Finally, the interplay of Np95 with Dnmts and histone modifying enzymes,
together with its binding to DNA and repressive histone marks, point to a central role
in the integration of various epigenetic silencing mechanisms and the mediation of
epigenetic crosstalk.
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