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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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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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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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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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 pro-apoptotic 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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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 chemo-resistance. 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).