Analysis of protein interactions controlling DNA methyltransferases [Elektronische Ressource] / vorgelegt von Karin Fellinger

Karin Fellinger

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Analysis of Protein Interactions
Controlling DNA Methyltransferases


Karin Fellinger


















München 2009  








Analysis of Protein Interactions
Controlling DNA Methyltransferases


Karin Fellinger







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



vorgelegt von
Karin Fellinger
aus München





München, den 28. Januar 2009




























Erstgutachter: Prof. Dr. Heinrich Leonhardt
Zweitgutachter: Prof. Dr. Peter Becker

Tag der mündlichen Prüfung: 20.03.2009
Contents
SUMMARY 1 
1. INTRODUCTION 3 
1.1  Protein-Protein Interactions 4 
1.2  Establishment and Maintenance of DNA Methylation 13 
1.3  The Epigenetic Protein Network 18 
2.  RESULTS 25 
2.1  A Mutagenesis Strategy Combining Systematic Alanine Scanning with Larger
Mutations to Study Protein Interactions 27 
2.2  Biochemical Analysis of Intramolecular N-C Terminal Dnmt1 Interactions 36 
2.3  Dimerization of DNA Methyltransferase 1 is Mediated by its Regulatory Domain 41 
2.4  Np95 Controls Maintenance of DNA Methylation by Interaction with DNA
Methyltransferase 1 68 
2.5  Np95 Interacts with de novo DNA Methyltransferases Dnmt3a and 3b and Mediates
Epigenetic Silencing 77 
3. DISCUSSION 101 
3.1  Mutagenesis Strategies to Study Protein-Protein Interactions 101 
3.2  Biochemical Analysis of N-C Terminal Dnmt1 Interactions 104 
3.3  The N-Terminal TS Domain Mediates Dnmt1 Dimerization 109 
3.4  Np95 is a Key Regulator of DNA Methyltransferases 111 
4.  ANNEX 117 
4.1 References 117 
4.2 Contributions 129 
4.3  Declaration According to the “Promotionsordnung der LMU München für die Fakultät
Biologie” 131 
4.4 Abbreviations 133 
4.5  List of Expression Constructs 135 
4.6 Acknowledgements 139 
5.  CURRICULUM VITAE 143 



Summary

Summary
Epigenetic mechanisms control a multitude of processes in mammalian development such as
X-chromosome inactivation, genomic imprinting and cellular differentiation, but if
misregulated they also cause diseases as cancer. Complex molecular networks regulate
patterns of DNA methylation and histone modifications that give rise to distinct gene
expression profiles.
In this study we analyzed the family of DNA methyltransferases (Dnmts) that are responsible
for the establishment and maintenance of DNA methylation patterns. To elucidate their role
and regulation in the epigenetic protein network we identified and characterized intra- and
intermolecular interactions of Dnmts. For this purpose, we first developed a fast and robust
alanine scanning mutagenesis method that allowed the streamlined generation of point,
deletion and insertion mutants and the establishment of a Dnmt protein variant library as
basis for further analysis.
As the methyltransferase activity of Dnmt1 requires allosteric activation of the C-terminal
catalytic domain through its regulatory N-terminal domain, we determined the relevant
regions for this interaction within the N-terminal domain. While the CXXC zinc finger (amino
acids (aa) 648-694) and phosphorylation of serine 515 were dispensable, a major part of the
N-terminal regulatory domain was necessary for interaction with the catalytic domain. These
results point to the importance of structural integrity of the regulatory domain for allosteric
activation of Dnmt1. Moreover, we observed that Dnmt1 forms stable dimers through its N-
terminal regulatory domain. Mutational analyses mapped the dimerization domain to a
bipartite interaction surface in the targeting sequence domain (TS, aa 310-629) that is also
known to be responsible for recruiting Dnmt1 to heterochromatin.
Similar to the targeted disruption of the dnmt1 gene, knockout of Np95 was reported to
result in global hypomethylation in ES cells (ESCs). Therefore, we examined the role of Np95
in the regulation of DNA methylation. We mapped and characterized the interaction between
Dnmt1 and Np95 and found that the TS domain of Dnmt1 mediates this interaction and a
small deletion within the highly conserved core region of the TS domain abolished the
interaction with Np95. This Np95 interaction mutant showed catalytic activity on
oligonucleotide DNA in a radioactive methyltransferase assay. However, it failed to restore
-/-methylation patterns in dnmt1 ESCs. These results indicate that Np95 facilitates access of
Dnmt1 to DNA target sites in chromatin.

  1 Summary

In addition, we found an interaction of Np95 with the N-terminal domains of the de novo
DNA methyltransferases Dnmt3a and Dnmt3b which is even stronger than the interaction
-/- -/- -/- -/-with Dnmt1. Interestingly, we observed no transgene silencing in np95 or dnmt1 3a 3b
-/-(TKO) ESCs in contrast to wildtype or dnmt1 ESCs indicating that both, Np95 and the de
novo DNA methyltransferases are required for promoter silencing in ESCs. These results
assign a crucial role to Np95 in epigenetic silencing and make it an interesting target for
epigenetic reprogramming.


In summary, we developed a versatile mutagenesis strategy that allows
efficient generation of protein variant libraries. We mapped and
characterized intra- and intermolecular interactions of DNA
methyltransferases. The TS domain of Dnmt1 that is located in the center of
the N-terminus harbors several regulatory functions: It is necessary for
allosteric activation of the catalytic domain, mediates dimerization of
Dnmt1 and its interaction with Np95 recruits Dnmt1 to pericentric
heterochromatin. Beside a complex regulation of Dnmts through
interactions within the Dnmt family, other chromatin factors such as Np95
function as key regulators for DNA methylation.




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