Identification of novel regulators of TNF-α [TNF-alpha] signaling using genome-wide RNAi screens [Elektronische Ressource] / by Dorothee Nickles

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Identification of Novel Regulators of TNF-Signaling using Genome-wide RNAi ScreensDissertationbyDorothee Nickles2009Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural Sciencespresented byDipl.-Biol. Dorothee Nicklesborn in Landau/Pfalz, Germanydate of oral examination: 22.01.2010Identification of Novel Regulators of TNF-Signaling using Genome-wide RNAi ScreensDissertationbyDorothee NicklesReferees:Prof. Dr. Lutz GissmannProf. Dr. Herbert SteinbeisserAcknowledgmentsFirst of all I want to thank my supervisor Michael Boutros for giving me this interestingand challenging project. I am especially thankful that he gave me the opportunity to visitseveral international conferences and courses from which I benefited a lot.I acknowledge my referees, Lutz Gissmann and Herbert Steinbeisser, for taking over thecorrectorship of this thesis and their advice during PhD committee meetings. I am alsograteful to Anne Regnier-Vigouroux for her constant support ever since I worked with heras a student.My thank goes to all - former and current - members of the "Heidelberg branch" ofthe Boutros group for providing a nice working atmosphere.

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Published 01 January 2009
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Identification of Novel Regulators of TNF-
Signaling using Genome-wide RNAi Screens
Dissertation
by
Dorothee Nickles
2009Dissertation
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
Dipl.-Biol. Dorothee Nickles
born in Landau/Pfalz, Germany
date of oral examination: 22.01.2010Identification of Novel Regulators of TNF-
Signaling using Genome-wide RNAi Screens
Dissertation
by
Dorothee Nickles
Referees:
Prof. Dr. Lutz Gissmann
Prof. Dr. Herbert SteinbeisserAcknowledgments
First of all I want to thank my supervisor Michael Boutros for giving me this interesting
and challenging project. I am especially thankful that he gave me the opportunity to visit
several international conferences and courses from which I benefited a lot.
I acknowledge my referees, Lutz Gissmann and Herbert Steinbeisser, for taking over the
correctorship of this thesis and their advice during PhD committee meetings. I am also
grateful to Anne Regnier-Vigouroux for her constant support ever since I worked with her
as a student.
My thank goes to all - former and current - members of the "Heidelberg branch" of
the Boutros group for providing a nice working atmosphere. Even though I think almost
everyone in the lab helped me at some point with either reagents, protocols or advice, I am
especially indebted to Nadege Pelte, who was a great teacher, Anan Ragab, with whom
I had critical as well as reassuring discussions, Dierk Ingelfinger and Sandra Steinbrink,
who introduced me to RNAi screening in human cells, David Kuttenkeuler, who had lots
of good advice concerning assay design and data analysis, as well as Thomas Sandmann
who supported me greatly in finalizing my thesis.
I also want to thank Tobias Haas and Markus Brechmann for fruitful discussions.
I owe many thanks to Wolfgang Huber as he agreed on taking a me as a guest for a
week in his group and contributed a lot to raising my interest into R. I also want to thank
the whole Huber group who was always very friendly and helpful whenever I turned with
questions towards them. Special thanks go to Florian Hahne for his patience with my li-
mited programming skills and for sharing insights into the Bioconductor world.
I would like to thank Sheena Pinto for unforgettable lunch breaks. I am extraordinarily
grateful to my family and friends for their support, cheering-up words and interest in my
work. I especially thank Patrick König for always being there.Abstract
Signaling pathways are crucial for multicellular organisms: they are necessary for cell
communication and development, they enable cells to specialize and act together. TNF-
signaling is one such pathway crucial for orchestrating the body’s response to cellular
stress or invasion by pathogens. TNF- signaling mediates inflammation, a key event for
an efficient innate immune response. As inflammation has to be tightly controlled in order
to avoid damage to the host, deregulation of TNF- signaling has been implicated in the
pathogenesis of many inflammatory diseases and cancer.
TNF- exerts effects by binding to its receptor, TNFR1. Upon binding,
several proteins including DD (death domain) proteins are recruited to TNFR1 to form the
TNFR complex, resulting in the activation of the IB kinase complex (IKK). Subsequently,
inhibitor of B (IB) proteins are phosphorylated and degraded, releasing transcription
factors of the NF-B family. NF-B then controls the expression of hundreds of different
genes required for inflammation and innate immunity.
The aim of my PhD project was to identify new factors required for TNF- signaling.
In order to monitor NF-B transcriptional activity upon stimulation with TNF-, I estab-
lished a cell-based dual luciferase assay. This assay was suited to measure TNF- signaling
activity in miniaturized format necessary for large-scale experiments. For finding novel reg-
ulators of TNF- signaling, I used the cell-based dual luciferase assay in two genome-wide
RNAi screens. These screens identified several candidates potentially implicated in NF-B
activation by TNF-. I next established secondary assays for confirming the requirement
of these candidates in TNF- signaling. On the basis of the results of these secondary
assays, I selected three candidates, SPP1, GAB3 and CASP4, for further characterization.
Epistasis experiments revealed that SPP1, GAB3 and CASP4 are required for the acti-
vation of the IB kinase complex. Further experiments demonstrated that the candidates
are not essential for proper TNFR1 cell surface expression. SPP1, a multifunctional pro-
tein, has been described to interact with the DD protein MyD88 during Toll-like receptor
signaling. It could thus interact with DD proteins present in the TNFR complex. GAB3
belongs to a family of scaffold proteins of which one member, GAB2, has been shown to
interact with RANK, a TNFR family member. Analogously, GAB3 could act as a scaffold
at TNFR1 supporting the recruitment of signaling molecules. CASP4 is an inflammatory
caspase. My results indicate that CASP4 catalytical activity is dispensable for its role
in TNF- signaling. CASP4 could thus serve as another scaffold protein in the TNFR
complex as described for other caspases. Future experiments will identify the interaction
partners of SPP1, GAB3 and CASP4, clarifying the molecular details of their mode of
action in TNF--induced activation of NF-B.