The role of the oncoprotein DEK in DNA replication stress and damage repair

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The role of the oncoprotein DEK in DNA replication stress and damage repair

Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften

(Doctor rerum naturalium)

Vorgelegt von

Anja Deutzmann

an der Universität Konstanz

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie

Konstanz, 2013

Tag der mündlichen Prüfung: 20.12.2013 1. Referentin: Prof. Dr. Elisa May 2. Referent: Prof. Dr. Alexander Bürkle

3. Referent: Prof. Dr. Michael O. Hottiger, Universität Zürich

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-259705

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ABBREVIATIONS

AML acute myeloic anemia

APC anaphase promoting complex

APH aphidicolin

A-T (D) Ataxia-telangiectasia (complementation group D)

cDNA coding DNA

CldU 5-chloro-2′-deoxyuridine

CPT camptothecin

CREST acronym for main features of CREST-syndrome: Calcinosis, Raynaud’s syndrome, Esophageal dysmotility, Sclerodactyly, Telangiectasia

DNA Deoxyribonucleic acid

DSB DNA strand break

EdU 5-ethynyl-2’-deoxyuridine

EGFP enhanced GFP

EJC exon-exon junction complex Er:fiber Erbium-doped fiber

FANC Fanconi anemia

FBS fetal bovine serum

FRAP fluorescence recovery after photobleaching GFP green fluorescent protein

HDAC histone deacetylase

HIV human immunodeficiency virus

HR homologous recombination

HU hydroxyurea

IC50 half maximal inhibitory concentration

IdU 5-ido-2′-deoxyuridine

MSO mitotic shake-off

NCS neocarzinostatin

NHDF normal human dermal fibroblasts NHEJ non-homologous end joining NMR nuclear magnetic resonance

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ABBREVIATIONS IV

OPT Oct-1, PTF, transcription

PAGE poly-acrylamide gel electrophoresis PAGFP photoactivatable GFP

PAR poly(ADP-ribose)

PCR polymerase chain reaction PTM post-translational modification

RNA ribonucleic acid

ROI region of interest

SAC spindle assembly checkpoint

SAP SAF-A/B, Acinus and PIAS

SDS sodium dodecyl sulfate

shRNA small hairpin RNA

ssDNA single stranded DNA

SV40 Simian virus 40

TALEN transcription activator-like effector nuclease TBB 4,5,6,7-tetrabromobenzotriazole

TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling

UV ultra violet

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1 PUBLICATIONS

Parts of this thesis and additional results, not explicitly mentioned in this work but obtained during my doctoral studies, are published or submitted for publication:

Deutzmann A, Ganz M, Schönenberger F, Vervoorts J, Kappes F, Ferrando-MayE (2013) The oncoprotein DEK promotes replication fork progression and facilitates proliferation under DNA replication stress. Submitted

Saha AK, Kappes F, Mundade A, Deutzmann A, Rosmarin DM, Legendre M, Chatain N, Al-Obaidi Z, Adams BS, Ploegh HL, Ferrando-May E, Mor-Vaknin N, Markovitz DM (2013) Intercellular trafficking of the nuclear oncoprotein DEK. Proc Natl Acad Sci U S A 110: 6847-6852

Tomas M, Blumhardt P, Deutzmann A, Schwarz T, Kromm D, Leitenstorfer A, Ferrando- May E (2013) Imaging of the DNA damage-induced dynamics of nuclear proteins via nonlinear photoperturbation. J Biophotonics 6: 645-655 (cover story)

Beneke S, Meyer K, Holtz A, Hüttner K, Bürkle A (2012) Chromatin composition is changed by poly(ADP-ribosyl)ation during chromatin immunoprecipitation. PLoS One 7:

e32914, doi: 10.1371/journal.pone.0032914

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2 SUMMARY

The DEK oncoprotein is a chromatin architectural factor that has essential functions in the maintenance of heterochromatin integrity. It is an abundant and unique chromatin constituent showing no sequence homology to any other known protein, and is highly conserved among multicellular eukaryotes. DEK binds to DNA, RNA, and interacts with various chromatin components including histones. The affinity of DEK to its binding partners is determined by posttranslational modifications, predominantly by phosphorylation as well as poly(ADP-ribosyl)ation. When interacting with DNA, DEK preferentially binds to cruciform DNA structures which arise during perturbed DNA replication and the repair of DNA strand breaks.

Several lines of evidence, among others the frequent overexpression in highly malignant tumors and the positive correlation between DEK expression levels and chemoresistance, have led to the definition of DEK as a bona fide oncogene. On the other hand, investigation of a potential role of this protein in DNA repair revealed that DNA strand breaks are repaired less efficiently when DEK expression is downregulated leading to hypersensitivity towards genotoxic insults. DEK’s function as a positive regulator of DNA repair has been difficult to reconcile with a genuine tumor promoting activity, since cancer development is most often characterized by defects in DNA repair and genomic instability. This thesis aimed at elucidating this apparent conundrum by a detailed investigation of the impact of DEK on DNA damage susceptibility. The study focused on DNA replication stress as a characteristic source of DNA damage in hyperproliferating tumors.

The data obtained show that DEK renders cells less sensitive to DNA replication stress counteracting accumulation of replication stress-induced DNA damage. DEK facilitates replication fork progression, in particular under conditions of mild but prolonged replication stress, as occurring at the early stages of transformation. For this function, DEK was shown to depend on PARP1/2 activity. DEK’s proliferation promoting activity was also confirmed in the human cancer model of c-myc-induced replication stress.

Furthermore, DEK was demonstrated to reduce damage transmission through mitosis to the next cell generation. Evidence for a role of DEK in the regulation of mitosis progression and chromosome congression was also gained. Finally, DEK localization was shown to be drastically and persistently affected by DNA replication stress, an effect that might be mediated by SUMO1-modification.

In sum, the study presented here proposes a model for DEK´s tumorigenic activity in which this protein contributes to circumvent the cell´s intrinsic barrier against cancerogenesis imposed by the DNA damage response thereby enabling proliferation under stress and eventually tumor growth and malignancy. This study discloses a novel mode of action of this protein frequently deregulated in aggressive tumors that bears the potential for new therapeutic approaches.

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3 ZUSAMMENFASSUNG

Das Onkoprotein DEK ist ein abundanter und einzigartiger Bestandteil des Chromatins, der keinerlei Sequenzhomologie zu anderen Proteinen aufweist und dennoch in multizellulären Eukaryoten hoch konserviert ist. DEK beeinflusst die Architektur des Chromatins und hat essentielle Funktionen in der Erhaltung der Heterochromatinintegrität. DEK bindet sowohl DNA als auch RNA und interagiert außerdem mit verschiedenen Chromatinproteinen, unter anderem mit Histonen. Das Bindeverhalten von DEK mit diesen Interaktionspartnern wird durch post-translationale Modifikationen, besonders durch Phosphorylierung und Poly(ADP-ribosyl)ierung, bestimmt. Interessanterweise ist die Bindung von DEK an DNA ist nicht sequenz-, sondern strukturspezifisch: DEK bindet bevorzugt an kreuzförmige DNA Strukturen, die während einer gestörten DNA Replikation oder der Reparatur von DNA Strangbrüchen auftreten.

Viele Beobachtungen, unter anderem die Überexpression von DEK in vielen bösartigen Tumoren und die Korrelation des Expressionsniveaus von DEK mit der Resistenz gegenüber chemotherapeutischer Behandlung, haben dazu geführt, dass DEK als ein bona fide Onkogen angesehen wird. Andererseits hat das Niveau der DEK Expression aber auch Einfluss auf DNA Reparaturprozesse: DNA Strangbrüche werden interessanterweise in Zellen mit verminderter DEK Expression nicht effizient repariert, was dazu führt, dass diese Zellen besonders sensitiv auf genotoxische Substanzen reagieren. Dieser positive Einfluss von DEK auf die Effizienz der DNA Reparatur ist nur schwer mit der weithin publizierten Tumor-fördernden Aktivität dieses Proteins in Einklang zu bringen. Denn die Entwicklung eines Tumors ist sehr häufig durch Defekte in der DNA Reparatur und daraus folgende genomische Instabilität gekennzeichnet. Das Ziel dieser Arbeit war es, diesen offensichtlichen Widerspruch aufzuklären, indem genau untersucht wurde, wie sich DEK auf die Anfälligkeit von Zellen gegenüber DNA Schädigung auswirkt. Hierbei stand vor allem Replikationsstress im Mittelpunkt, weil dieser eine charakteristische Quelle von DNA Schäden in hyperproliferativen Tumoren darstellt.

Im Zuge dieser Arbeit konnte gezeigt werden, dass DEK Zellen vor Replikationsstress schützt und so der Anhäufung von durch Replikationsstress induzierten DNA Schäden entgegen wirkt. Dabei begünstigt DEK das kontinuierliche Fortschreiten der Replikationsgabel besonders unter Bedingungen, die zu mildem aber anhaltendem Replikationsstress führen und daher vergleichbar sind mit den Bedingungen, die in frühen Phasen der Tumorentstehung herrschen. Zusätzlich konnte die Abhängigkeit dieser Funktion von der Aktivität der Proteine PARP1/2 gezeigt werden. DEK’s unterstützende Wirkung auf die Zellproliferation wurde außerdem in einem humanen Krebsmodellsystem,

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3 ZUSAMMENFASSUNG 4

dem c-myc-induzierten Replikationsstressmodell, bestätigt. Außerdem konnte gezeigt werden, dass DEK die Verschleppung von DNA Schaden über die Mitose hinweg und somit die Weitergabe an die Tochterzellen verringert. Auch wurden Hinweise für eine Rolle von DEK in der Kongression von Chromosomen und der Regulation des Mitoseverlaufs erhalten. Schließlich konnte noch gezeigt werden, dass die zelluläre Lokalisation des DEK Proteins drastisch und lang anhaltend durch DNA Replikationsstress beeinflusst wird. Dieser Effekt könnte durch eine post-translationale Modifikation von DEK durch Sumoylierung bedingt sein.

Die vorgelegte Arbeit schlägt ein Modell für die tumorfördernde Eigenschaft von DEK vor: DEK unterstützt das Umgehen der zelleigenen Barriere gegen Krebsentwicklung, welche durch die DNA Schadensantwort errichtet wird. Somit ermöglicht DEK Zellproliferation unter Replikationsstressbedingungen und damit letztendlich auch Tumorwachstum und -entartung. Diese Arbeit beschreibt somit eine neuartige Funktionsweise des DEK Porteins, die neue Ansätze für die Therapie von aggressiven Tumoren, in denen DEK häufig dereguliert ist, aufzeigt.

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4 INTRODUCTION

4.1 The human oncoprotein DEK

4.1.1 Protein structure

The protein DEK was discovered in 1992 in a patient (Derek K.) suffering from acute myeloid leukemia (AML) as part of the DEK-CAN fusion protein also known as DEK- NUP214 (von Lindern et al, 1992b). DEK-CAN results from the chromosome translocation (6;9)(p23;q34) which leads to a single DEK-CAN transcript in which the last 26 amino acids of DEK are replaced by two thirds of the CAN nucleoporin. The leukemogenic properties of the DEK-CAN fusion protein are not well understood, but it was recently demonstrated that the fusion protein is able to initiate leukemogenesis from hematopoietic stem cells (Oancea et al, 2010). A second fusion product originating from a chromosomal translocation leads to the expression of two splice variants of another CAN- fusion protein; SET-CAN which is associated with the development of acute undifferentiated leukemia. Therefore, CAN was supposed to be the oncogenic part in the CAN-fusion proteins and responsible for leukemia development (von Lindern et al, 1992a). If and how DEK contributes to the oncogenic properties of DEK-CAN is not known.

The DEK protein is highly conserved in mammals and has orthologous in most multicellular eukaryotes but not in yeast. In human cells, the DEK protein exists in two splice variants, has no sequence homologues and is therefore considered the only protein of its kind. The NMR-structures of an N-terminal (78-208 aa) (Devany et al, 2008) and a C-terminal (309-375 aa) (Devany et al, 2004) segment of DEK are solved, but the overall protein structure of DEK is not known. This may be due to the presence of low-complexity regions which can hinder crystallization. The full-length human DEK protein consists of 375 amino acid residues and comprises a SAP (SAF-A/B, Acinus and PIAS) which is a putative DNA/RNA binding domain and was shown to be involved in chromatin organization (Aravind & Koonin, 2000). The SAP domain can be found in more than 40 nuclear proteins with diverse functions. In addition to the SAP domain, DEK contains a pseudo-SAP domain which is similar to the SAP domain in structure but not in amino acid sequence. Together, both domains form the major DNA-binding region within the DEK protein and are responsible for DEK’s structure specific binding of double stranded DNA (Devany et al, 2008). DEK preferentially binds to unusual DNA structures which include supercoiled and distorted DNA as well as structures that resemble four-way junctions (Böhm et al, 2005; Waldmann et al, 2003). Furthermore, DEK contains a C-terminal DEK_C domain which folds in a three-helix-bundle (Devany et al, 2004). The DEK_C domain shows similarity to the DNA-binding domain of the transcription factor DP2 (E2F

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4 INTRODUCTION 6

dimerization partner 2) (Figure 4.1) and constitutes another DNA binding as well as a multimerization domain within the DEK protein.

Figure 4.1 | Overlay of DEK_C domain (light green) and the DNA-binding domain of DP2 (light blue) in complex with DNA. PDB references: 1Q1V, DEK_C; 1CF7, DP2.

In human cells, the DEK_C domain is only present in DEK itself and in protein phosphatase slingshot homologues with approximately 30% sequence identity (Hunter et al, 2012). The DEK_C-domain is of special interest because this part of DEK was demonstrated to complement mutagen sensitivity, hyper-recombination, and radio-resistant DNA synthesis of Ataxia-telangiectasia complementation group D fibroblasts (Meyn et al, 1993). How DEK accomplishes the reversion of these phenotypic abnormalities of A-T D fibroblasts is not known. Interestingly, a dominant negative mutant of human topoisomerase 3 alpha (TOP3α) also complemented for abnormal phenotypes of A-T D fibroblasts (Fritz et al, 1997). TOP3α in concert with BLM (Bloom syndrome protein which is a RecQ-like DNA helicase), dissolves double Holliday junctions avoiding cross- over products and thereby limits sister chromatid exchange and loss of heterozygosity (Raynard et al, 2006; Wu & Hickson, 2003). Both DEK and the dominant negative TOP3α mutant complemented for the same defects in A-T D fibroblasts. Additionally, DEK preferentially binds to branched DNA structures, which allows speculating about a role of DEK in the processing of DNA replication intermediates during the process of their dis- or resolution.

4.1.2 Posttranslational modifications

The DEK protein is a target for heavy posttranslational modification. To date, 50 of DEK’s 375 amino acids were shown to be targeted by posttranslational modification using site specific or high-throughput mass-spectrometry approaches (Hornbeck et al, 2012).

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4 INTRODUCTION 7

Figure 4.2 | Schematic of the DEK oncogene. Shown is the linear DEK sequence and functional domains within the protein (SAP: SAF-A/B, Acinus and PIAS; psSAP: pseudo SAP; DEK_C: DEK C-terminal domain). Covalent posttranslational modifications (PTMs): Black line, phosphorylation; blue dotted line, acetylation; red dashed line, ubiquitination. Non-covalent PAR interaction: yellow boxes, PAR binding motifs; green boxes, PAR interacting peptides.

The most abundant modification within the DEK protein is the phosphorylation of serine and threonine residues and it is presently verified in vitro and in vivo for 18 amino acids (Kappes et al, 2004; Soares et al, 2006). CK2 was shown to be one, if not the most important kinase responsible for DEK phosphorylation (Kappes et al, 2004). Other covalent modifications comprise acetylation, methylation and ubiquitination. In addition, there is evidence for PARP1-mediated covalent modification of DEK with poly(ADP- ribose) (PAR) in the course of apoptotic cell death (Kappes et al, 2008). The covalent modification with PAR drastically decreases DEK’s affinity to DNA leading to its displacement from chromatin (Gamble & Fisher, 2007; Kappes et al, 2008). The amino acids in DEK targeted for covalent PARylation are so far unknown. DEK is not only covalently modified with PAR but also interacts with PAR in a non-covalent fashion. Non- covalent PAR binding was shown for three regions, one within and one near the SAP domain and one region within the DEK_C domain (Fahrer et al, 2010). The PAR-DEK interaction is dependent on the chain length of PAR and promotes the formation of DEK- DEK complexes but does not affect the overall binding capability of DEK. Also P/CAF (p300/CBP-associated factor)-dependent acetylation of DEK was shown to reduce DEK’s affinity to DNA and to lead to redistribution of DEK into interchromosomal granule clusters or nuclear speckles (Cleary et al, 2005). Posttranslational modifications of DEK detected so far are summarized in Figure 4.2. Generally, posttranslational modification of DEK can change DEK’s affinity to itself, to other interacting proteins, and to DNA. The fact that so many residues (at least 13% of the whole protein) are targets for different types of modifications provides a multitude of combinatorial possibilities to modulate and regulate DEK’s localization, interaction properties and functions.

4.1.3 Expression, localization, and molecular functions

4.1.3.1 Functions of DEK in chromatin remodeling

The DEK protein is a non-histone chromatin constituent. The majority of the protein is bound to DNA: DEK binds to active DNA which is accessible for micrococcal nuclease (MN) as well as to inactive chromatin which is refractory for MN treatment (Kappes et al, 2001). DEK is bound to chromatin during all stages of the cell cycle including all mitotic stages and the predominant amount of DEK can be eluted from chromatin with 250 mM

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4 INTRODUCTION 8

NaCl (Kappes et al, 2001). Approximately 10% of the DEK protein pool is bound to RNA and is found in complexes with RNA splicing factors together forming the exon-exon junction complex (Le Hir et al, 2000; McGarvey et al, 2000). Posttranslational modifications are believed to determine DEK’s binding and interaction behavior towards DNA or rather different chromatin compartments, RNA and interacting proteins (Fahrer et al, 2010; Gamble & Fisher, 2007; Mor-Vaknin et al, 2011; Privette Vinnedge et al, 2013).

Chromatin-associated functions of DEK are based on its ability to change chromatin topology. DEK introduces constrained positive supercoils in naked DNA as well as in nucleosome containing SV40 minichromosomes in vitro (Waldmann et al, 2002) and thereby leads to a compaction of DNA and chromatin, respectively. In human cells, DEK directly interacts with HP1α and thereby markedly enhances the binding of HP1α to trimethylated histone H3 (H3K9me3). HP1α is a component of constitutive heterochromatin and facilitates heterochromatin formation and transcriptional repression, by serving as a binding platform for chromatin modifiers and other HP1α-interacting proteins that are involved in heterochromatin maintenance. One interaction partner recruited by HP1α is the H3K9-specific histone methyltransferase SUV39H1/2 which is responsible for the spreading of H3K9 tri-methylation. Thus, downregulation of DEK expression leads to the displacement of HP1α from chromatin and its accumulation in the nucleosolic fraction. The consequences are a massive loss of H3K9 trimethylation and a drastic decrease in global heterochromatin stability. This disruption of the establishment of a proper histone code impacts on epigenetic gene silencing and leads to reexpression of otherwise silenced genes (Kappes et al, 2011). In addition to its function in heterochromatin maintenance, DEK binds to core histones in vitro with a preference for histones H2A and H2B (Alexiadis et al, 2000) and to the histone variant macroH2A1.1 (Timinszky et al, 2009). DEK also possesses histone chaperone activity: in the Drosophila melanogaster model of ecdysone-induced gene expression, which is accompanied by massive chromatin decondensation, DEK facilitated H3.3 assembly in a CK2-dependent manner (Sawatsubashi et al, 2010). The histone chaperone activity of DEK was also demonstrated in mammalian cells and again shown to be dependent on DEK phosphorylation by CK2 (Kappes et al, 2011).

DEK is also a component of a high molecular weight chromatin remodeling complex named B-WICH which only assembles when transcription is active suggesting a specific role of this multi-protein machinery in transcription (Cavellán et al, 2006). In this case, DEK interacts with WSTF which is an atypical tyrosine-kinase and a core component of the WICH complex (WSTF-ISWI chromatin remodeling complex). WSTF forms the WICH complex together with SNF2H, a helicase with intrinsic ATP-dependent nucleosome-remodeling activity, (Poot et al, 2004). The B-WICH complex, which consists of WSTF-ISWI and six additional protein subunits as well as ribosomal RNAs, is involved in RNA polymerase I and III-dependent transcription and consequently is necessary for the transcription of short, untranslated RNAs. Since WSTF is part of the WICH complex which functions in transcription, DNA repair and replication of heterochromatin, DEK

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4 INTRODUCTION 9

might also play a role in these WICH-dependent chromatin remodeling processes (Barnett

& Krebs, 2011; Bozhenok et al, 2002; Poot et al, 2005).

4.1.3.2 DEK’s role in transcription and RNA splicing

First evidence of DEK being involved in the regulation of transcription was found while investigating replication of viral HIV-2 DNA in the host cell. In this study, DEK was found to bind to peri-ets sites in the HIV-2 enhancer (Fu et al, 1997). In contrast to transcription of proviral HIV-1 DNA, where the transcription factor NFk-B is sufficient, transcription of the HIV-2 genome requires additional factors to stimulate transcription.

Since DEK specifically binds to a sequence within the HIV-2 regulatory enhancer elements, which are target binding sites for transcriptional co-regulators, a regulatory function of DEK in HIV-2 transcription was assumed. In the following years, the impact of DEK on transcription was confirmed by various groups. It was demonstrated that DEK accumulates at promoter regions of actively transcribed genes (Hollenbach et al, 2002) and is able to activate (Campillos et al, 2003) as well as repress (Kim et al, 2010) the transcription of certain genes. The mechanisms by which DEK interferes with transcription are diverse: modulation of transcription factor transactivation activities (Campillos et al, 2003; Sammons et al, 2006), coactivation of a nuclear receptor via its histone chaperone properties (Sawatsubashi et al, 2010), and induction of an inactive chromatin structure have been reported. The latter is achieved by influencing the acetylation status of histone tails by either recruiting histone deacetylases (Hollenbach et al, 2002) or decreasing histone acetyltransferase activity of p300 and PCAF (Ko et al, 2006). Some of these studies also showed that phosphorylation of DEK is needed for its regulatory function in gene transcription. Furthermore, DEK not only alters p300 and PCAF-mediated histone acetyltransferase activity, but it is itself a target for p300 and PCAF-mediated acetylation in vitro (Cleary et al, 2005). Acetylation within the first 70 amino acid residues of the DEK protein was shown to lead to a decrease in DNA binding affinity and to DEK’s relocalization to interchromatin granule clusters which are small sub-nuclear bodies believed to be storage centers for factors involved in RNA processing.

As mentioned above, DEK predominantly binds to DNA but approximately one-tenth of nuclear DEK is bound to RNA. Initially, DEK was shown to associate with splicing complexes, most likely via interaction with serine/arginine-repeat proteins (McGarvey et al, 2000). Furthermore, it was shown that DEK is one component of the exon-exon junction complex (EJC) which acts as a binding platform for proteins that are responsible for post-splicing events, such as RNA decay or RNA export (Kim & Dreyfuss, 2001; Le Hir et al, 2001; Le Hir et al, 2000). However, these reports were called into question since the presence of DEK in the EJC could not be confirmed by mass spectrometry analysis (Reichert et al, 2002). It was also pointed out that the DEK antibody (established in Grossfeld laboratory) used for previous immunoprecipitation studies, which formed the experimental basis for demonstrating the DEK-EJC interaction, actually cross-reacted with other components of the EJC (Reichert et al, 2002). The authors concluded that DEK is not a stably bound component of the EJC. Nevertheless, DEK was shown to be required for

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4 INTRODUCTION 10

intron removal via enforcement of 3’ splice site discrimination by the splicing factor U2AF. To allow for specific mRNA cleavage, DEK provides a proofreading function that allows association of U2AF at AG-rich consensus 3’ splicing sites instead of non- consensus regions (Soares et al, 2006). The interaction of DEK and U2AF as well as efficient intron-removal was dependent on the phosphorylation status of DEK, once again emphasizing the tremendous impact of posttranslational modifications on DEK function.

4.1.3.3 DEK is an auto-antigen and a chemotactic factor

In its physiological state, DEK is a nuclear protein that mostly localizes to chromatin and to a minor extent to RNA. However, in some autoimmune diseases, such as systemic lupus erythematodes and juvenile idiopathic arthritis, DEK is also found extracellularly evoking the production of anti-DEK auto-antibodies (Sierakowska et al, 1993; Wichmann et al, 1999; Wichmann et al, 2000). DEK can be secreted either actively in its free form, in exosomes (Mor-Vaknin et al, 2006) or it is released during apoptosis in a highly PARylated state (Kappes et al, 2008). CXCL8, which is an important cytokine secreted predominantly by macrophages but also by synovial stromal cells, stimulates DEK secretion by activated human monocyte derived macrophages. While CXCL8 itself acts as chemoattractant for neutrophils and peripheral blood monocytes into the inflamed synovium, secreted DEK protein also acts as chemotactic factor and recruits CD8+ T-cells and CD56+ natural killer cells (Mor-Vaknin et al, 2006). Mor-Vaknin and colleagues also demonstrated that the anti-inflammatory glucocorticoid dexamethasone as well as the immunosuppressant cyclosporine A inhibits the secretion of DEK by monocyte derived macrophages.

4.1.3.4 DEK impacts on DNA damage repair

The facts that a C-terminal fragment of DEK rescues multiple phenotypes of A-T fibroblasts (Meyn et al, 1993), and that DEK downregulation sensitizes HeLa cells towards genotoxic agents (Kappes et al, 2008; Saha et al, 2013), are hints towards a function of DEK in DNA damage repair pathways. Furthermore, DEK is a target for PARylation (Kappes et al, 2008), which also points to a function of DEK in DNA damage repair. Many proteins involved in DNA repair are PARylated or interact with PAR which acts as a recruitment platform for other proteins containing PAR-binding modules (Barkauskaite et al, 2013). PARP activation can be triggered by different cellular stress situations, such as DNA damage, metabolic stress, and growth hormone-mediated ERK signaling (Cohen- Armon et al, 2007; D'Amours et al, 1999; Herceg & Wang, 2001; Luo & Kraus, 2012; and references therein). Especially, DNA base damage and single strand breaks trigger PARP1 binding to DNA and its activation to produce PAR. Mechanistically, the negatively charged PAR polymer can change biochemical properties of target or binding proteins, often affecting protein structure as well as protein localization (Campalans et al, 2013;

Couto et al, 2011; Gamble & Fisher, 2007; Timinszky et al, 2009). DEK also interacts with PAR in a non-covalent fashion and prefers long PAR chains (54mer) over shorter ones (18mer). Three of the five PAR interacting consensus sequences present in DEK were

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4 INTRODUCTION 11

biochemically confirmed as PAR binding sites; all three residing within functional domains of the DEK protein: in the DNA-binding SAP domain, within the nuclear leading sequence, and in the C-terminal DNA-binding/multimerization domain. Whereas covalent modification with PAR - the target residues are still matter of ongoing investigation - decreases DEK’s affinity to DNA, non-covalent interaction of DEK and PAR has no impact on DEK’s DNA binding but it leads to a reduced multimerization activity of DEK (Fahrer et al, 2010). Recently, it was shown that DEK modulates the response to DNA damage: downregulation of DEK expression leads to increased DNA damage signaling via activated ATM but to decreased DNA-PKcs signaling when cells were challenged with etoposide, a topoisomerase II inhibitor (Kavanaugh et al, 2011). An impaired recruitment of Ku70/80 to DNA strand break sites which is a prerequisite for DNA-PKcs activation was determined as the cause for decreased DNA-PKcs signaling. Kavanaugh and colleagues also demonstrated a substantial defect in non-homologous end joining (NHEJ) in DEK knockout mouse embryonic fibroblasts which is a consequence of impaired DNA- PKcs signaling.

4.1.3.5 DEK is involved in cancer development and progression

The DEK protein is expressed at low level in nearly all healthy tissues and with (slightly) elevated levels in immune cells, stem cells and epithelial progenitor cells (McCall et al, 2011; Wu et al, 2009). However, DEK is heavily overexpressed in various tumor types such as melanoma, breast cancer, ovarian cancer, retinoblastoma, colon cancer, acute myeloid leukemia and non-small lung cancer (Carro et al, 2006; Grasemann et al, 2005;

Han et al, 2009; Larramendy et al, 2002; Liu et al, 2012; Paderova et al, 2007; Vinnedge et al, 2012; Vinnedge et al, 2011; Wang et al, 2013). In some of these malignant lesions, DEK correlates with tumor progression and invasiveness. Because of this, DEK is suggested to serve as a biomarker for malignant melanoma and bladder cancer (Datta et al, 2011; Khodadoust et al, 2009). A chemical carcinogenesis mouse model provided the first in vivo evidence that DEK displays tumor-promoting properties (Wise-Draper et al, 2009a) and DEK was therefore suggested to be a bona fide oncogene. Additionally, using different cellular model systems it could be demonstrated that DEK interferes with cellular mechanisms that protect against cancer formation and development: differentiation, senescence as well as apoptosis. Cell differentiation in keratinocytes was demonstrated to be accompanied by DEK downregulation whereas overexpression of DEK resulted in hyperplasia and a delay of the differentiation process (Wise-Draper et al, 2009b). Micro- RNA-mediated DEK downregulation is essential for maintaining quiescence of muscle stem cells (Cheung et al, 2012) and DEK acts as a senescence inhibitor in cervical cancer cells and life-time expansion of primary keratinocytes (Wise-Draper et al, 2005). In addition to DEK counteracting cell differentiation and senescence, there are contradicting reports about a role of DEK in apoptotic cell death. Downregulation of DEK expression leads to the induction of apoptosis in HeLa cells but has only little effect on the survival of SAOS-2 cells (Wise-Draper et al, 2006). Since HeLa cells express low levels of proapoptotic p53 and SAOS-2 cells are completely p53 negative, the authors suspected a connection between DEK and p53. They could show that downregulation of DEK

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4 INTRODUCTION 12

expression leads to an increased transcription of various p53-reporter constructs containing endogenous p53-responsive promoters whereas over expression of DEK leads to their transcriptional repression. The underlying reason is the stabilization of p53 protein upon DEK downregulation. In other words, DEK inhibits apoptotic cell death by destabilizing p53 resulting in accelerated p53 degradation. In line with an antiapoptotic function of DEK was the observation that downregulation of DEK expression was accompanied by transcriptional repression of the antiapoptotic protein MCL-1 whereas the protein levels of the related antiapoptotic factors BCL-2 and BCL-XL were not affected (Khodadoust et al, 2009). On the other hand, a Drosophila transgenic model showed a proapoptotic function of DEK when overexpressed in the developing eyes of the flies (Lee et al, 2008). It was demonstrated that DEK overexpression leads to hypo-acetylation of histones H3 and H4, most likely by DEK’s inhibitory effect on the histone acetyltransferases p300/CBP and PCAF (Ko et al, 2006). In turn, this H3/H4 hypo-acetylation negatively affected the transcription of the anti-apoptotic protein BCL-2 which suggested a pro-apoptotic function of DEK.

Taken together, there are two aspects of DEK that seem contradictory in the context of tumor development: on the one hand DEK promotes the repair of DNA damage which is generally important to assure genomic stability and protects from unregulated cell proliferation and cancer development. On the other hand, DEK promotes cancer development in vivo and is overexpressed in many malignancies originating from diverse tissues. DEK expression correlates with cancer aggressiveness and mediates chemoresistance. In turn, downregulation of DEK expression in cancer or primary cells is accompanied by induction of senescence and apoptosis or differentiation, respectively.

Therefore, it is unclear whether DEK’s function in DNA damage repair is at all beneficial for cancer development. Genomic instability as a result of impaired DNA repair is believed to be a hallmark of cancerogenesis. Because of this, it remains an open question if and how DEK’s role in promoting DNA repair fits in the concept of DEK being a tumor promoting factor (or even a bona fide oncogene).

4.2 Chromatin architecture in mammalian cells

In eukaryotic cells, the genetic information in form of DNA is condensed in an orderly manner with the aid of multiple accessory proteins. This condensed DNA-protein conglomerate is called chromatin. Chromatin density depends on many factors such as DNA base modifications and abundance or activity of chromatin-interacting proteins. On the other hand, nearly every fundamental cellular process is either directly or indirectly affected by chromatin structure. The configuration of chromatin impacts on gene transcription, on DNA replication, as well as DNA damage repair. During all of these processes, chromatin needs to undergo drastic structural changes: decondensation and re-

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4 INTRODUCTION 13

establishment of the chromatin structure are events that need to be very efficient and well- orchestrated to allow for normal and healthy proliferation.

Figure 4.3 | Schematic of DNA compaction in eukaryotes. The DNA double helix is compacted via interactions with nucleosomes. Histone tails protrude from the nucleosome core particles and are targets for post-translational modifications that affect DNA-histone and histone-protein interactions. The linker histone H1 further stabilizes the nucleosome-DNA interaction leading to the formation of the “10 nm fiber”. Further compaction leads to the appearance of higher order chromatin structures with differing densities. The classical, but nowadays challenged model is the folding into a 30 nm fiber which further condenses by coiling into larger fibers. Maximum density is reached in the formation of the chromosome during mitosis.

4.2.1 Chromatin composition and structure

The first level of DNA condensation is achieved by the interaction of negatively charged DNA with positively charged nucleosomes. Nucleosomes are octameric protein complexes consisting of two copies of each of the four core histones H2A, H2B, H3, and H4. 147 base pairs of DNA are wrapped around one nucleosome in positive helical turns forming a nucleosome core particle. Short DNA stretches link these particles. The resulting 10 nm fiber is stabilized further by binding of the linker histone H1 which restricts nucleosome mobility and inhibits the activity of chromatin remodelers (Bustin et al, 2005; Catez et al, 2006). There are differing models about higher-order chromatin compaction: the classical textbook knowledge is that the 10 nm fiber is further coiled resulting in a 30 nm fiber. To achieve an even higher degree of condensation, the 30 nm fiber is assumed to wind up in even larger fibers involving many accessory proteins. These kind of higher-order fibers can be visualized by cryo-electron microscopy of chromatin spreads (Olins & Olins, 2003) and also initial data obtained by small-angle X-ray scattering pointed to their existence in

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4 INTRODUCTION 14

interphase nuclei (Langmore & Paulson, 1983). However, the existence of these fibers has recently become a matter of debate (Bancaud et al, 2012; Fussner et al, 2011; Grigoryev &

Woodcock, 2012; Hansen, 2012; Joti et al, 2012; Lieberman-Aiden et al, 2009; McNally &

Mazza, 2010; Mirny, 2011; Nishino et al, 2012). New techniques as well as structure modeling approaches led to the proposal of new models of chromatin compaction. One of these models suggests a rather irregular folding of the 10 nm fiber into higher-order chromatin in which fractal globules are believed to be the fundamental unit. Self- organization by spatial repeat of self-similar structures is an increasingly emerging concept in microscopic as well as macroscopic nature. However, it might be possible that the mode of chromatin compaction differs between cell types.

It is important not to think of chromatin as a static macromolecular complex. Not the core histones, but all the accessory proteins, including the linker histone H1, are highly mobile and continuously compete for chromatin binding sites. Therefore, shaping of chromatin architecture is a dynamic process that happens constantly within a living cell.

Figure 4.4 | Recent alternative model of chromatin compaction: fractal self-organization. (A) Folding of a linear polymer (top; colors code distance from one endpoint) into an equilibrium globule (bottom left) and a fractal globule (bottom right). In the equilibrium globule, proximal regions of the polymer do not need to be close in the three-dimensional globule. In contrast, fractal organization is achieved by favoring three- dimensional proximity of regions that are nearby in the unfolded polymer. (B) Fractal model of genome architecture at three increasing scales. Chromosomes (blue, cyan, green) occupy chromosome territories and the same territory displays open (light colors) and condensed (dark colors) chromatin regions in an interphase nucleus. A fractal globule is suggested to form the smallest unit within chromatin. (modified from Lieberman-Aiden et al)

4.2.2 Chromatin structure during DNA replication and DNA damage repair Chromatin architecture is a key regulator for all DNA-dependent processes. The efficiency of chromatin rearrangement has significant impact on the accessibility of DNA, especially for proteins that directly bind to DNA. Binding of transcription factors and DNA repair proteins to their DNA substrates, as well as replication fork progression are two examples of crucial processes that are significantly affected by chromatin structure. On the scale of the 10 nm fiber, nucleosomes have to be slid along the DNA or even completely removed from the fiber to allow other proteins access to the DNA. Furthermore, chromatin architecture needs to be restored after the completion of DNA-dependent processes to

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4 INTRODUCTION 15

avoid unfavorable or unscheduled actions on DNA. Nucleosome dynamics can be affected in at least three different ways: (1) DNA methylation which affects DNA topology and stability and thereby also DNA interaction with histones and other proteins (Bartke et al, 2010; Collings et al, 2013), (2) post-translational modifications of N-terminal histone tails and of histone core domains which affect the histone’s affinity towards DNA and DNA- remodeling factors (Mersfelder & Parthun, 2006), and (3) ATP-dependent chromatin remodeling complexes which slide complete nucleosomes or disassemble histone octamers from the chromatin fiber (Bowman, 2010). In addition, there is crosstalk between these different remodeling pathways leading to a complex network of chromatin remodeling activities. In the following, two important DNA-dependent processes – DNA replication and DNA damage repair – will be outlined with respect to chromatin remodeling necessary for their effective operation.

4.2.2.1 Chromatin structure and DNA damage repair

One vital DNA-dependent process that relies on chromatin reorganization is the repair of DNA lesions. The scale of chromatin remodeling required for effective damage repair depends on the nature and the extent of the damage. The eukaryotic cell has evolved highly specialized DNA repair pathways for all kinds of DNA lesions and each of these pathways comprises varying chromatin remodeling processes. How chromatin structure is affected by DNA repair and vice versa will be described here exemplarily for the repair of DNA strand breaks (DSBs). Many of the processes described in this context also apply of other repair pathways (reviewed in (Cann & Dellaire, 2011; Lafon-Hughes et al, 2008; Sulli et al, 2012)).

When DNA is lesioned in a way that breaks covalent bonds within the DNA backbone, the cell needs to react instantaneously to ensure genome integrity. To this end, chromatin has to relax to allow repair factors to gain access to the damage (Cann & Dellaire, 2011; Ziv et al, 2006). How exactly DSBs are recognized and which structure or topology change leads to the initiation of the following cascade of repair events is still not completely understood.

So far, the earliest detectable reaction after induction of DSBs is the PARylation of N-terminal histone tails of core histones and of multiple other acceptor proteins at or near the damage site. The consequences of PAR formation at the site of DNA damage are multiple and diverse. They range from chromatin remodeling (interestingly, both, chromatin condensation and decondensation can be triggered by PARylation), to protein recruitment and protein retention. This mixture of even counteractive effects points to a highly dynamic process which is dependent on timing and location of PAR formation and degradation. One of the first initial steps of DSB repair is also the activation of the key kinase ATM (ataxia telangiectasia mutated). Activated ATM phosphorylates the histone variant H2AX at S139 (then designated γH2AX) directly at the damage site but also in

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4 INTRODUCTION 16

vicinity surrounding region of up to several megabases up- and downstream of the damage (Rogakou et al, 1999). Additionally, H2AX can be phosphorylated by other kinases which also can be activated in the course of DSB repair: the ATM-related kinases ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase). γH2AX then serves as a binding platform for many sensor proteins which – once recruited – are often targets for phosphorylation themselves and are required for subsequent DNA repair steps. One of these sensors was shown to recruit ATP-dependent chromatin remodelers to the site of DNA damage (van Attikum et al, 2007). Other histone modifications that directly affect chromatin structure and composition in response to DSB induction are methylation and acetylation of histone tails. Especially acetylation is associated with local chromatin relaxation. Additionally, histone tail acetylation enhances ATM activity establishing a positive feedback loop, (Bhoumik et al, 2008; Sun et al, 2005). DSBs also lead to local recruitment of histone deacetylases (HDACs), a finding that further supports the concept of a highly dynamic rearrangement of the chromatin fiber in response to DSBs. Underscoring these dynamics, the recruitment of HDACs was recently demonstrated to favor DSB repair via non-homologous end joining with respect to homologous recombination. This observation clearly shows that DNA damage not only causes massive changes in chromatin architecture, but chromatin structure in turn affects the manner a DSB is repaired (Lukas et al, 2011b; Miller et al, 2010).

4.2.2.2 Chromatin structure and DNA replication

A eukaryotic cell cycle comprises different phases. During G1 phase, a cell prepares for genome replication which takes place during S phase. In the following G2 phase, the entire genome is completely duplicated and distributed equally between two daughter cells during mitosis (or M phase) which then reside again in G1 phase. In the course of this cycle, all chromosomes need to be decondensed in a timely fashion so that the replication machinery can produce a copy of the entire genome. After completed replication the chromosomes recondense again. To enable replisome progression, the chromatin fiber has to be disassembled and the DNA helix unwound in front of the replication fork. Given an approximate replication speed of 2 to 3 kb per minute (Méchali, 2010), 10 to 15 nucleosomes have to be removed from the chromatin fiber every minute to guarantee effective replication fork progression (Alabert & Groth, 2012). First, the linker histone H1 which stabilizes the 10 nm fiber has to be removed before nucleosome dismantling.

Dissociation of H1 from the nucleosome is induced by its phosphorylation by the S phase- CDK1 (cyclin-dependent kinase 1) which is activated by CYCA (cyclin A) (Thomson et al, 2010). The following disassembly of nucleosomes ahead to the replication fork takes place only in very close proximity to the fork and is therefore thought to be a direct consequence of a collision of the replicative helicase with a nucleosome. Whether other proteins are involved in nucleosome disruption is so far unknown. Histones that are evicted in front of

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4 INTRODUCTION 17

the replication fork are not simply lost: they are precious raw material for the condensation of the newly synthesized DNA daughter strands. By passing on old histones – including their post-translation modification pattern which was termed the “histone code” – the cell does not only economize but passes on valuable information in addition to the DNA sequence. This information is prerequisite for re-establishing the proper chromatin structure in the daughter cells. The current model of redistribution of parental histones to daughter DNA strands suggests that the parental H3/H4-tetramers are randomly integrated into the new strands where they are combined with either new or old H2A/H2B-dimers (Ransom et al, 2010; Xu et al, 2010). The shuttling of old histones from ahead of the fork to the newly synthesized DNA strands is aided by specialized histone binding proteins. So- called histone chaperones bind to histones or histone multimers and form complexes with components of the replisome, e.g. the replicative helicase (Jasencakova et al, 2010). The so far best studied histone chaperones which directly impact on replication efficiency are ASF1 (anti-silencing function protein 1), FACT (facilitates chromatin transcription) (Bao

& Shen, 2006; Formosa, 2012) and CAF1 (CCR4-associated factor 1). The different histone chaperones either display preferences for binding a specific histone or have multiple histone binding partners. The proportion of new and old histones bound to these chaperones strongly depends on the processivity of the replisome. When replication is disturbed more old histones are “stored” in complex with histone chaperones. A molecular mechanism that explains how histone chaperones escort their histone or histone multimer from ahead of the fork to the other side of the replisome remains to be elucidated.

4.2.3 Oncogene-induced replication stress model of oncogenesis

All situations that force DNA replication forks to prolonged pauses or even initiate their disintegration are referred to collectively as DNA replication stress conditions. DNA replication stress can be induced by an insufficient supply of deoxynucleotides or by the collision of the replisome with any impediment, such as transcription machineries, unrepaired DNA damage sites, or stable DNA secondary structures that are difficult to replicate. The consequence of these stress conditions, however, is the accumulation of DNA damage due to replication fork collapse. The response to replication-associated DNA damage involves specialized DNA damage repair pathways that include fork stabilization mechanisms and the activation of cell cycle checkpoints which slow down cell cycle progression and allow for timely repair. Stabilization of replication forks as well as the repair of replication stress-induced DNA damage again requires rearrangements of chromatin structure as well as changes in DNA topology. Therefore, proteins involved in these processes directly impact on the welfare of the proliferating cell.

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A recent concept, which aims at explaining the elevated presence of DNA lesions in very early precancerous lesions – but their absence in highly proliferating normal tissue – suggests that oncogene-induced hyperproliferation is accompanied by DNA replication stress (Bartkova et al, 2010; Tsantoulis et al, 2008). As long as a cell possesses functional repair pathways and checkpoints, apoptosis or senescence will be initiated if the damage is beyond repair. These mechanisms are considered to pose an intrinsic physiological barrier against cancerogenesis. However, cells that have the means to circumvent this barrier are positively selected for and build the basis for cancer progression. Upon inactivation of cell cycle checkpoints or modulation of DNA repair pathways, cells proliferate although accumulating replication stress-induced DNA damage and consequently, become more and more genomically unstable.

4.3 Aim of this thesis

The DEK protein displays two properties which at first sight appear contradictory: it supports DNA repair but also promotes cancerogenesis. This thesis aims at clarifying DEK’s role in the DNA damage response in order to better understand how it is linked to tumor promotion. As a working hypothesis, DEK was assumed to affect DNA damage and repair in the context of DNA replication stress. To investigate this hypothesis and expand the current knowledge on DEK´s involvement in the DNA damage response this thesis will address the following aspects and pursue the following tasks:

• Establish a cellular system for stable downregulation of DEK expression

• Establish robust readouts for DNA damage and repair at the single cell level that enable the detection of small modulatory effects of DEK on the DNA damage response

• Characterize in more detail the role of DEK on the handling of DNA damage, in particular DNA strand breaks, arising from different types of insults

• Investigate the impact of DEK on DNA damage generated by impaired replication in different models of DNA replication stress.

Overarching goal of the thesis will be to shed light on how DEK, an architectural chromatin factor, acts in cancer development through its role in DNA replication and repair.

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5 RESULTS

5.1 DEK depletion sensitizes cells towards DNA strand break induction by neocarzinostatin but not by multi-photon laser irradiation

In a eukaryotic cell, chromosomal DNA does not exist as a naked molecule within the nucleus but is bound to histones and other DNA binding proteins resulting in highly organized chromatin structures. Depending on epigenetic marks on either DNA or histones chromatin may be preset in varying densities: heterochromatic regions contain up to four times more DNA per volume than decondensed euchromatin (Bohrmann et al, 1993).

These diverse chromatin environments significantly affect DNA damage and repair: DNA repair processes partly involve different repair proteins and proceed with different kinetics in euchromatin as compared to the more densely organized heterochromatin, most likely due to steric effects.

Since DEK is important for heterochromatin integrity and downregulation of DEK expression leads to the loss of heterochromatic regions, it was investigated how DEK expression impacts on DNA damage susceptibility and handling using two different techniques to induce DNA strand breaks (DSBs): (1) treatment with neocarzinostatin (NCS), a single strand break inducing antibiotic that again leads to the formation of double strand breaks when a replication fork reaches the damage site, and (2), non-linear excitation via irradiation with femtosecond laser pulses which induce DSBs through the formation of solvated electrons and reactive oxygen species (Tomas, 2013; Vogel et al, 2005). The application of these two techniques, combined with time-resolved detection of the recruitment of DNA repair factors, such as XRCC1, at DSBs enables to discriminate between the effects of DEK on DNA damage induction on the one hand, and DNA damage signaling and repair on the other hand.

5.1.1 DEK modulates cellular sensitivity towards DSB induction by NCS

To investigate the impact of DEK expression on the formation of DSBs, HeLa S3 cells (kindly provided by Ferdinand Kappes, see Figure 5.4 A) that stably expressed a DEK specific shRNA (shDEK cells) and in cells carrying the correspondent empty vector (control) were treated with NCS. DNA damage was visualized by immunocytochemistry using antibodies specific for phosphorylated histone H2AX (pS139) (γH2AX) which serves as marker for DNA lesions and confocal fluorescence microscopy. The amount of

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DNA damage was determined at single cell level by integrating the γH2AX-specific signal on mean intensity projections of confocal z-stacks spanning the whole nucleus. Low doses (50 and 100 ng/ml) of NCS were applied for a short time (15 and 30 min) to induce DNA damage at a low level and to avoid signal spreading over the whole cell nucleus. This pan- nuclear γH2AX staining is considered a pre-apoptotic stage (de Feraudy et al, 2010). In control cells the γH2AX-specific signal was only marginally increased (1.7-fold) when cells were treated with 100 ng/ml NCS for 30 min. Under the same treatment conditions, cells with downregulated DEK expression displayed a 3.2-fold increase in γH2AX signal.

This increased sensitivity towards NCS caused by DEK downregulation was dose and time dependent (Figure 5.1 A).

DEK belongs to a class of supercharged proteins that can be taken up by cells and then localizes to the cell nucleus (Cronican et al, 2011; Saha et al, 2013). Therefore, DEK levels can be restored in shDEK cells by supplementation of the cell culture medium with recombinant DEK protein ((Saha et al, 2013), see also Chapter 5.2.5). This tool was used to investigate whether exogenous DEK protein may compensate for the increased NCS- sensitivity of shDEK cells (Figure 5.1 B+C). Cells were cultivated in the presence of exogenous His-tagged DEK for two days before further treatment. Pre-incubation with His-DEK protein completely abolished the increased NCS-sensitivity caused by DEK downregulation. Addition of His-DEK to control cells which already express high levels of DEK had no effect (Figure 5.1 B+C). These data suggest that DEK either physically protects DNA from NCS-induced DNA damage or that it limits DNA damage signaling via upstream sensor kinases responsible for the phosphorylation of histone H2AX.

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Figure 5.1 | DEK downregulation sensitizes cells towards DSB induction by NCS. (A) HeLa S3 control and shDEK cells were either left untreated or treated with NCS as indicated. γH2AX was immunolabeled using specific antibodies. DNA was counterstained using Hoechst33342. Mean intensity projections of confocal z- stacks were used for determination of mean γH2AX-specific fluorescence intensity per nucleus. Data is the mean of 2 independent experiments and at least 250 nuclei were evaluated per condition and experiment.

(B) Confocal immunofluorescence images of HeLa S3 control and shDEK cells. Cells were either treated as described in (A) or pre-treated with 1.6 µg/ml recombinant His-DEK protein before NCS-mediated DSB induction. Scale bar: 5 µm. (C) Quantitation of γH2AX-specific fluorescence signal normalized to untreated control. At least 140 cells were evaluated per condition. T-test: *** p≤ 0.001.

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5.1.2 HP1α mobility is not influenced by DEK expression level

One main mediator for DEK´s function in heterochromatin stabilization is the heterochromatin protein HP1α (Kappes et al, 2011). HP1α is responsible for establishing higher order chromatin compaction (Cheutin et al, 2003) and is essential for heterochromatin integrity. Hypoacetylation and tri-methylation of H3K9 are typical marks of heterochromatin. HP1α binds to H3K9me3 and recruits the histone methyltransferase SU(VAR)3-9 (Schotta et al, 2002) which is the main enzyme responsible for H3K9 methylation, thereby leading to heterochromatin spreading (Grewal & Moazed, 2003).

Recently, DEK has been shown to interact with HP1α and act as an auxiliary factor for its binding to H3K9me3 (Kappes et al, 2011). Consequently, when DEK is downregulated heterochromatin marks are significantly reduced and heterochromatin integrity is severely compromised (Kappes et al, 2011).

An altered affinity of HP1α to chromatin would affect chromatin structure and dynamics, and - as a consequence - also impact on DNA repair processes. Data showing that DSBs that occur in heterochromatin are moved out of HP1α-positive domains and relocated to less condensed chromatin regions to allow for complete DNA repair (Chiolo et al, 2011) underscore this link between chromatin structure and DNA repair. DEK´s observed influence on DNA damage and repair might possibly be linked to HP1α. Therefore, the question was investigated whether DEK affects HP1α´s chromatin binding behavior.

Protein mobility was chosen as readout in living cells and was determined by fluorescence recovery after photobleaching (FRAP). FRAP experiments were performed in HeLa S3 control and shDEK cells expressing GFP-tagged HP1α. Bleaching of GFP-HP1α was performed in a rectangular region of interest (ROI) within the nucleus and signal recovery was measured over time (Figure 5.2 A). FRAP curves showed no effect of DEK expression on HP1α mobility: half-time of fluorescence recovery (t_1/2) of both HP1α populations (slow, fast) did not differ significantly in shDEK cells as compared to control cells (Figure 5.2 C). Thus, no significant change in HP1α’s affinity to chromatin with respect to DEK expression was detected. This result points to an HP1α-independent mechanism of DEK that leads to protection from DNA damage induction.

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Figure 5.2 | DEK expression does not affect HP1α mobility in living cells. (A) Fluorescence images of an exemplary FRAP experiment. GFP-HP1α was expressed in HeLaS3 control cells, a rectangular region within the nucleus was bleached and fluorescence recovery was monitored over time as indicated. Scale bar: 5 µm.

(B) Exemplary corrected, normalized FRAP data. Red line shows non-linear regression. (C) Determination of half-time of fluorescence recovery (t1/2) after photobleaching. Graphs show the mean t1/2 values for the slow- (left panel) and fast-diffusing HP1α fractions (right panel) of four independent experiments. At least 10 cells were evaluated per experiment (T-test).

5.1.3 Formation and initial processing of DSBs upon infrared multi-photon absorption is not affected by DEK expression

To investigate whether the observed DEK-mediated protection towards NCS induced DSBs is caused by a reduction of DNA damage levels (less actual breaks) or by limited access of repair proteins to the damaged sites (less signaling leading to decreased H2AX phosphorylation), specific labeling of DNA ends was performed and accumulation of DNA repair proteins at damage sites was followed over time. To this end, DSBs were induced via nonlinear excitation using femtosecond laser pulses. This method offers the following advantages: (1) DNA damage can be induced in a very small spatial volume and the amount of DSBs can be varied with the irradiation power (2) damage induction occurs independently of chromatin structure, and (3) the recruitment of DNA repair proteins at irradiated sites can be directly visualized. Ultrashort infrared laser pulses lead to the formation of solvated electrons which again induce reactive oxygen species. Thus, this mode of DSB induction is thought to be independent from chromatin structure and to be mechanistically comparable to the effect of ionizing irradiation (Vogel et al, 2005).

Therefore, the specific induction of DSBs using non-linear excitation at 1050 nm (as described in (Träutlein et al, 2008; Träutlein et al, 2010)) should occur with comparable efficiencies both in dense and open chromatin. This point was of special interest because DEK downregulation leads to a drastic loss of heterochromatic regions (Kappes et al,

The role of the oncoprotein DEK in DNA replication stress and damage repair