The Jumonji-C domain containing proteins PHF2 and PHF8 act in concert to stimulate transcription of rRNA genes [Elektronische Ressource] / presented by Weijun Feng

Weijun Feng

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 38
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences












Presented by
M.Sc. Biology Weijun Feng
Born in: Zhejiang, China
Oral-examination:








The Jumonji-C domain containing proteins PHF2 and PHF8
act in concert to stimulate transcription of rRNA genes























Referees: Prof. Dr. Ingrid Grummt
PD Dr. Karsten Rippe








Acknowledgements

I would like to express my sincere gratitude to my Ph.D. supervisor Prof. Dr.
Ingrid Grummt and to thank her for giving me the opportunity to pursue my
Ph.D. in her group. I am indebted to her for her incomparable guidance,
stimulating discussion and unconditional support. I am also very grateful for her
great patience while tutoring my scientific writing during the preparation of
manuscripts and this thesis. Her insistence on the pursue of accuracy and
perfection is a precious gift not only for my scientific career but also for my
whole life.
I owe my gratitude to Dr. Yonggang Zhou for precious advice and
technical assistance from the very beginning of my study in Grummt’s group.
Many thanks go to Dr. Jing Ye, from whom I have received a lot of help both in
the lab and privately. I am indebted to all my colleagues in the group for their
indispensable help and numerous discussions. In particular, I would like to
express my gratitude to Dr. Holger Bierhoff for his helpful suggestions and
critical reading of my thesis. I also would like to thank Mr. Urs Hoffmann-
Rohrer and Mrs. Anne Wohlfahrt for their administrative support and help
during my stay in Heidelberg.
I wish to thank Dr. Masato Yonezawa and Prof. Dr. Thomas Jenuwein,
Reserch Institute of Molecular Pathology (IMP), The Vienna Biocenter, Austria,
not only for providing constructs of PHF2 and PHF8 and other experimental
materials, but also for making this very productive cooperation possible. I am
especially thankful to Dr. Masato Yonezawa for sharing his valuable scientific
experiences, his helpful advice and his active participation in this project.
I would like to extend my gratitude to my Ph.D. thesis committee
members PD Dr. Renate Voit and PD Dr. Karsten Rippe for their scientific
input, insightful discussion, and generous support of my thesis.
I am indebted to all my friends for their kind help and encouragement
during my whole study. My final heartfelt acknowledgement goes to my parents
for their endless support, understanding and constant inspiration through years.


Table of contents
ABBREVIATIONS ................................................................................................. 4
ZUSAMMENFASSUNG......................... 7
SUMMARY ............................................................................................................. 9
1. INTRODUCTION ...........................................................................................11
1.1. Epigenetic regulation of gene expression......................11
1.2. Establishment and interpretation of histone lysine methylation.................... 13
1.2.1. Histone lysine methylation and transcription ......................................... 13
1.2.2. Histone demethylases ............................................ 15
1.2.3. The PHD fingers of chromatin-associated protein recognize different state
of histone lysine methylation............................................. 18
1.3. The structure of rRNA genes and the Pol I transcription apparatus .............. 19
1.4. rRNA genes exist in two distinct epigenetic states....... 21
1.5. The PHF2/PHF8/KIAA1718 subfamily of JmjC domain proteins................ 23
1.6. Objectives ................................................................................................... 25
2. MATERIALS AND METHODS.... 27
2.1. Materials ..................................................................................................... 27
2.1.1. Antibodies............................. 27
2.1.2. Primers.. 29
2.1.3. siRNA oligos.......................................................................................... 31
2.1.4. Standard buffers and solutions............................... 32
2.2. Methods ...................................................................................................... 33
2.2.1. Cloning and constructs.......... 33
2.2.1.1. Plasmid DNA.................................................................................... 33
2.2.1.2. Transformation of bacteria............................... 33
2.2.1.3. Gateway BP reaction........................................................................ 33
2.2.1.4. Gateway LR reaction........ 34
2.2.2. Cell culture and transfection.. 34
2.2.2.1. Cell culture....................................................................................... 34
2.2.2.2. Transient plasmid DNA transfection in HEK293T cells..................... 34
2.2.2.3. siRNA transfection in HEK293T cells............... 35
2.2.3. RNA analysis ......................................................................................... 35
2.2.3.1. RNA extraction................. 35
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Table of contents
2.2.3.2. Northern blot .................................................................................... 35
322.2.3.3. Preparation of P- labeled riboprobes............. 36
2.2.3.4. RT-PCR............................ 36
2.2.4. Chromatin fractionation ........................................................................ 37
2.2.5. Cellular extract preparation.. 37
2.2.6. Glycerol gradient centrifugation............................................................ 38
2.2.7. Immunoblotting ..................................................... 38
2.2.8. Coomassie staining................................................ 38
2.2.9. Immunoprecipitation............. 38
2.2.10. Chromatin immunoprecipitation (ChIP)............... 39
2.2.11. Methylation-sensitive ChIP-chop ......................................................... 40
2.2.12. Immunofluorescence ............................................ 40
2.2.13. Expression of GST fusion proteins........................ 41
3. RESULTS........................................................................ 42
3.1. Generation of antibodies against PHF2 and PHF8....... 42
3.2. PHF2 and PHF8 localize to nucleoli............................................................ 43
3.3. PHF2 and PHF8 are associated with rDNA................. 45
3.3.1. PHF2 and PHF8 bind to the entire rDNA repeat ................................... 45
3.3.2. PHF2 and PHF8 bind to active rRNA genes.......... 47
3.4. PHF2 and PHF8 interact with Pol I and UBF............................................... 48
3.5. PHF2 and PHF8 are required for Pol I transcription..... 49
3.5.1. Overexpression of PHF2 and PHF8 stimulates Pol I transcription ........ 49
3.5.2. Depletion of PHF2 and PHF8 impairs pre-rRNA synthesis.................... 50
3.6. PHF2- and PHF8-dependent activation of rDNA transcription requires the
PHD finger and JmjC domain .............................................................................. 52
3.6.1. Generation of PHF2 and PHF8 mutants................ 52
3.6.2. Mutant PHF2 and PHF8 localize to nucleoli and interact with Pol I and
UBF 54
3.6.3. The PHD finger and JmjC domain are required for PHF2- and PHF8-
dependent activation of rDNA transcription...................................................... 54
3.7. PHF2 and PHF8 are associated with chromatin........... 57
3.8. PHF2 binds to H3K4me3 via the PHD finger.............. 58
3.9. PHF8 demethylates H3K9me2 and H3K9me1............................................. 61
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