A complex interplay of regulatory domains controls cell cycle dependent subnuclear localization of DNMT1 and is required for the maintenance of epigenetic information [Elektronische Ressource] / von Hariharan P. Easwaran

Aus dem Max-Delbrück-Centrum für molekulare Medizin
der Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin


A complex interplay of regulatory domains controls cell cycle dependent
subnuclear localization of DNMT1 and is required for the maintenance
of epigenetic information

DISSERTATION
Zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach BIOLOGIE
eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Hariharan P. Easwaran
geb. am 16.09.1974 in Mumbai (Indien)











Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jürgen Mlynek

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Michael Linscheid

Gutachter:
Prof. Dr. Harald Saumweber
Prof. Dr. Heinrich Leonhardt
Prof. Dr. Peter B. Becker

Eingereicht: 23. April 2003
Tag der mündlichen Prüfung: 1. October 2003
Table of Contents

Table of contents

ABBREVIATIONS......................................................................................................1
SUMMARY ..................................................................................................................5
ZUSAMMENFASSUNG .............................................................................................6
1. INTRODUCTION....................................................................................................7
1.1. GENETIC AND EPIGENETIC INFORMATION ............................................................7
1.2. REPLICATION OF GENETIC INFORMATION .............................................................7
1.2.1. DNA replication origins...............................................................................7
1.2.2. DNA replicates in discrete sites in the nucleus called replication foci .......8
1.2.3. Temporal and spatial order of DNA replication..........................................9
1.2.4. Specific protein sequences mediate assembly of replication factors at RF
..............................................................................................................................10
1.3. EPIGENETIC INFORMATION.................................................................................12
1.3.1. Types of epigenetic information.................................................................12
1.3.2. Role of DNA methylation ...........................................................................14
1.3.3. Regulation of DNA Methylation ................................................................15
1.3.4. Enzymes involved in methylating DNA......................................................17
1.3.5. Organisms that lack DNA methylation22
1.4. QUESTIONS ADDRESSED IN THIS WORK...............................................................22
2. MATERIALS AND METHODS ..........................................................................25
2.1. CONSTRUCTION OF PLASMIDS ENCODING VARIOUS FUSION PROTEINS ................25
2.2. CELL CULTURE AND TRANSFECTION...................................................................26
2.2.1. Drosophila cells.........................................................................................26
2.2.2. Mammalian cells........................................................................................26
2.3. CELL EXTRACTS AND WESTERN BLOT ANALYSIS ................................................27
2.4. CELL CYCLE AND IMMUNOFLUORESCENCE ANALYSIS ........................................27
2.4.1. BrdU labeling of replication foci and immunostaining .............................27
2.4.2. BrdU pulse-chase for identification of cells in G2 phase..........................28
2.4.3. Localization of DNMT1 at mitotic chromatin............................................29
2.4.4. Obtaining cells in G1.................................................................................29
2.5. MICROSCOPY .....................................................................................................30
2.6. LIVE CELL MICROSCOPY.....................................................................................31
2.7. SEQUENCE ANALYSIS .........................................................................................31
2.7.1. Search for DNA Ligase I homologue in Drosophila..................................31
2.7.2. Multiple sequence alignments, profiles and profile search .......................31
2.7.3. PSI-BLAST searches ..................................................................................32
3. RESULTS ...............................................................................................................33
3.1. ASSOCIATION OF PROTEINS WITH RF IN DROSOPHILA.........................................33
3.1.1. PCNA is highly conserved in S. cerevisiae, D. melanogaster and mammals
..............................................................................................................................33
3.1.2. Human PCNA and Drosophila PCNA can associate with RF across the
two organisms ......................................................................................................35
3.1.3. Subnuclear localization of DNMT1 in Drosophila cells ...........................38
Table of Contents

3.1.4. Search for an RFTS in Drosophila ............................................................44
3.2. REGULATION OF SUBCELLULAR LOCALIZATION OF DNMT1 IN MAMMALIAN
CELLS THROUGHOUT THE CELL CYCLE ......................................................................51
3.2.1. Targeting preference of the three targeting domains of DNMT1..............51
3.2.2. TS associates with late replicating pericentric heterochromatin57
3.2.3. Subnuclear localization of DNMT1 throughout the cell cycle...................61
3.2.4. Comparison of dynamics of TS with DNMT1: what determines release of
DNMT1 from pericentric heterochromatin during G1? ......................................65
3.3. BIOLOGICAL EFFECTS OF TS OVEREXPRESSION..................................................70
3.3.1. Effect on cell viability ................................................................................70
3.3.2. Effect on nuclear organization and morphology .......................................71
3.4. EVOLUTIONARY CONSERVATION OF THE REGULATORY DOMAIN OF DNMT1.....74
3.4.1. PBD is present only in metazoan DNMT1 family......................................74
3.4.2. TS is a unique domain present only in DNMT1 family from animals, plant
and fungi ..............................................................................................................76
4. DISCUSSION .........................................................................................................81
4.1. THE MAMMALIAN DNMT1 DOES NOT ASSOCIATE WITH RF IN DROSOPHILA
CELLS........................................................................................................................81
4.2. TARGETING OF DNMT1 TO RF IN MAMMALIAN CELLS IS DRIVEN SOLELY BY THE
PBD..........................................................................................................................82
4.3. THE TS MEDIATES CELL CYCLE DEPENDENT BINDING OF DNMT1 TO
PERICENTRIC HETEROCHROMATIN.............................................................................83
4.4. ALTERNATIVE MECHANISM OF INHERITANCE OF DNA METHYLATION IN PLANTS
AND FUNGI ................................................................................................................84
4.5. MECHANISM OF MAINTENANCE OF EPIGENETIC INFORMATION BY DNMT1 IN
MAMMALS.................................................................................................................86
5. OUTLOOK.............................................................................................................89
6. POSTFACE ............................................................................................................90
7. VIDEOS ..................................................................................................................92
8. BIBLIOGRAPHY..................................................................................................93
APPENDIX...............................................................................................................104
A) ACKNOWLEDGEMENTS.......................................................................................104
B) CURRICULUM VITÆ .....................................................................................105
C) STATEMENT .......................................................................................................106

Abbreviations
Abbreviations

A

Ab antibody
AdoHCy S-adenosyl-L-homocysteine
AdoMet S-adenosyl-L-methionine
ADP adenosine diphosphate
Ar argon
5AzaC 5-azacytidine
B

BAH bromo adjacent homology domain (also called PBHD)
BDGP Berkeley Drosophila Genome Project
BLAST Basic Local Alignment Search Tool
bp base pairs
BrdU 5-bromodeoxyuridine
C

C2C12 mouse myobl