Quantitative and functional analysis of chromosome dynamics [Elektronische Ressource] : influence of the nucleolus on the regulation of gene expression / Axel B. Berger

Axel Berger

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• Biologie

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 87
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Doctorate in molecular cell biology

UNIVERSITE PARIS XI UNIVERSITÄT REGENSBURG
Ecole Doctorale "Gènes, Génomes, Cellules" Naturwissenschaftliche Fakultät III




Presented by :

Axel B. Berger
To obtain the grade of DOCTEUR de l’Université Paris XI and
DOKTOR RER. NAT. der Universität Regensburg


Thesis title :
Quantitative and functional analysis of chromosome
dynamics: Influence of the nucleolus on the
regulation of gene expression.


Defended on December 12, 2008



Jury :
Prof. Giuseppe BALDACCI President
Prof. Danièle HERNANDEZ-VERDUN Rapporteur
Dr. Xavier DARZACQapporteur
Prof. Herbert TSCHOCHNER Thesis Director / Rapporteur
Dr. Olivier GADAL ThesisDirector









This PhD has been executed at the Institut Pasteur, Department of Cell Biology and
Infection, Unité de Biologie Cellulaire du Noyau, Paris, France and
the University of Regensburg, Institute for Biochemistry, Genetics and Microbiology,
Lehrstuhl für Biochemie III, Regensburg, Germany.


The work was supervised by Dr. Olivier Gadal, University of Toulouse, LBME of
CNRS, Equipe Organisation et Dynamique Nucléaire, Toulouse, France and
Prof. Dr. Herbert Tschochner, University of Regensburg, Institute for Biochemistry,
Genetics and Microbiology, Lehrstuhl für Biochemie III, Regensburg, Germany.







Hereby I assure that this thesis has been written autonomously and that no sources
other than the ones indicated have been used. This work has not been part of another
examination procedure; other attempts to obtain a PhD have not been undertaken.


Paris, November 3, 2008


ACKNOWLEDGEMENTS

First of all I want to thank Xavier Darzacq, Danièle Hernandez-Verdun and
Giuseppe Baldacci for having kindly accepted to be part of my thesis committee.

I want to thank Ulf Nehrbass for giving me the opportunity to work in his laboratory
for all the last years. I thank Olivier Gadal for his supervision from near and far
during all this time, his encouragement and enlightening discussions. I also want to
thank Herbert Tschochner for having accepted me as a student in his laboratory, his
personal supervision and interest in my work.

I next want to thank Frank Feuerbach-Fournier for his great support in all regards
inside and outside the lab. I also want to thank the other lab members Ghislain
Cabal, Vincent Galy, Sophie Bachellier-Bassi, Musa Mhlanga, Alper Romano for
having substantially influenced my work, as well as the other “non-yeast” lab
members for their support; Brigitte David-Watine, Françoise Donnadieu, Harry
Doualot, and Isabelle Dulieu for providing a really pleasant working atmosphere.

I especially want to thank Christophe Zimmer for the great and very close
collaboration with him. It was enormously interesting and enlightening to live this
true interdisciplinarity over the last years. I also want to thank his lab members
Mickaël Lelek and Tarn Duong for having joined the project so dedicatedly.

I want to thank Alain Jacquier, Laurence Decourty, Cosmin Saveveanu and last
but not least Micheline Fromont-Racine for a very productive collaboration, very
interesting discussions, and especially for my ‘tutrice’, for always following my
welfare on campus and abroad.

Thank you very much to Emmanuelle Fabre and Pierre Thérizols for having shared
the passion for yeast nuclear cell biology on campus and for the good collaboration
over the last years.



I want to thank Spencer Shorte and Christophe Machu for providing access to great
microscopes and for excellent technical assistance; as well as to Jean-Yves Coppée
for providing access to microarray scanners in his facility.

Now, a big thank you to all the people in Regensburg for a very warm welcome in
Bavaria; for the generous scientific advice teaching me real biochemistry Philipp
Milkereit, Achim Griesenbeck, Max Felle; interesting discussions and productive
collaborations Gernot Längst, Attila Nemeth and Anna Schrader; and every day
assistance everybody, but especially Katharina Gmelch, Andreas Neueder, as well
as my girls from room 50.17 Juliane Merl, Kristin Hergert, Jochen Gerber,
Hannah Götze and Robert Steinbauer. Thank you all for having made my stay so
pleasant!

I also want to thank my friends from Pasteur for having made life on- and off-campus
really nice: Gernot Sellge, Neetu Gupta, Stefano Cairo, Patricia Rueda, Gilles
Gheusi, Neha Issar, Annie Olry & Laurent and, of course Ghislain Cabal & ‘son’
Léa for all these unforgettable moments we spent and will spend together!

I thank my Parisian friends Rafael & Nadège, Shabnam and Sophie for having
shared the good and the bad moments that spread over from work to private life. I also
want to thank my friend Thomas Weitz for frequently sharing thoughts about our
scientific future albeit not being in Paris.

In the end I want to thank my fortune for having arranged the collaboration with
Regensburg where I met Anna. And I want to thank Anna for her deep love and
constant support for the last part of my PhD (and hopefully far beyond).

Last but least, I want to thank from all my heart my parents and my brother Moritz;
for having made this whole thing possible by their presence and true encouragement.
Thank you so much!



SUMMARY

Chromatin is distributed non-randomly within the cell nucleus. Its spatial organization
has been demonstrated to be important for nuclear metabolism such as, DNA
replication, reparation or transcription. However, little is known about the functional
implications resulting from its organization or the molecular driving forces
responsible for chromatin organization.
In the course of this thesis, I studied the budding yeast HMG-box protein Hmo1. An
initial screen demonstrated that this chromatin-associated protein is genetically linked
to the RNA polymerase (Pol) I, to genes coding for ribosomal proteins (RPGs) as well
as to genes implicated in stress response. I could show that Hmo1 physically interacts
with the rRNA coding gene transcribed by Pol I and with a subset of RPG promoters.
Global expression analyses showed a clear dependence on Hmo1 for the expression of
a sub-set of RPGs. An hmo1 deletion strain is also largely alleviated in repressing
RPG transcription after TOR complex 1 inhibition. These results suggested that Hmo1
is implicated in Pol I transcription as well as RPG regulation. Preliminary in vitro
transcription assays suggest a role of Hmo1 in Pol I initiation and elongation events.
Since Hmo1 is a bona fide nucleolar factor, I wanted to test the hypothesis if Pol II
transcribed RPGs associated with Hmo1 are localized in the proximity of the
nucleolus, as previously reported for Pol III encoded tRNA genes. Due to the small
size of the yeast nucleus and the stochastic, sub-diffusive movement of DNA, we first
needed to develop a new method allowing determination of gene localization
probabilities with very high accuracy and with respect to the nucleolus. As a result of
this collaborative approach, we could demonstrate by analyzing thousands of cells,
that genes are confined into sub-nuclear volumes. These ‘gene territories’ show a
locus specific size and can be remodeled upon transcriptional activation. Applying
this new method to Pol II transcribed genes required for ribosome biogenesis, such as
the RPGs, indicates that the localization of the gene on the chromatin fiber has
important implications for its three dimensional positioning. Genes in proximity to the
centromere localize in front of the spindle pole body while RPGs further away from
the extremities of the chromosome arm can also occupy a nucleolar-close territory.
Furthermore, it also seems that neighboring genes are important positioning
determinants.
All together, these results show that Hmo1 participates in mediating Pol I and RPG
transcription regulation in response to growth conditions and that this important cross-
talk between different RNA polymerases could be mediated by spatial co-positioning
of the genes. Further analyses will be required to reveal the potential function of
Hmo1 in this interplay.
1
RESUME

Au sein du noyau des cellules eucaryotes, la chromatine, support de l’information
génétique, n’est pas distribuée de façon aléatoire. Son organisation spatiale est
étroitement liée aux métabolismes nucléaires, tels que la réplication de l’ADN, la
réparation ou la transcription. Cependant, les mécanismes gouvernant l’organisation
tridimensionnelle de la chromatine restent mal connus. De même, les rôles
fonctionnels de cette architecture restent à explorer.
Au cours de mon travail de thèse, j’ai étudié la protéine Hmo1, une protéine à boîte
HMG de la levure Saccharomyces cerevisiae.