FAT10 and JunB co-localize at the nuclear membrane and in the cytosol Our previous experiments revealed clear evidence that FAT10 becomes stably attached to

JunB and inhibition of the proteasome led to a significant accumulation of the conjugate.

To further investigate the localization and fate of either JunB or FAT10 when co-expressed and to analyze the role of FAT10ylation in this process, we performed confocal laser-scanning microscopy. Therefore, we transfected HEK293 cells with either a pcDNA3.1-HA-FAT10 or pCMV6-JunB-MYC-FLAG construct alone, or co-transfected them together, treated them with or without MG132 for 6 hours and carried out a series of co-localization experiments using a directly labelled coupled Alexa Fluor 488 antibody to visualize HA-FAT10 expression and a rabbit polyclonal antibody to JunB (ab31421) followed by a Alexa Fluor 546-coupled secondary goat-anti-mouse antibody, to stain for JunB.

126 (a) (b)

(c) (d)

(e)

(f)

Results

127

Figure 37: FAT10 modification leads JunB to the nuclear membrane

HEK293 cells were transiently transfected with MYC-FLAG-tagged JunB and a HA-tagged FAT10 construct alone or co-transfected together and treated with or without MG132 (10 µM) for 6 h and fixed with 4 % PFA. (a) HA-FAT10 transfected cells were stained with a 6,85 µg ml^-1 dilution of directly labelled HA-coupled Alexa Fluor 488 antibody (green) and (b) treated additionally with MG132. (c) JunB transfected cells were stained with a 5 µg ml^-1 dilution of a rabbit polyclonal antibody to JunB (ab31421), followed by a Alexa Fluor 546-coupled secondary goat-anti-mouse antibody (red) and (d) treated additionally with MG132. (e) Double transfected cells were treated subsequently with Alexa488-HA antibody and a rabbit polyclonal Ambra1 (ab31421) antibody followed by a Alexa Fluor 546-coupled secondary goat-anti-mouse antibody and (f) treated additionally with MG132. Confocal microscopy images are shown. Scale bar: 25 µm. Images are representatives of several cells examined in three independent experiments.

In singly transfected cells, FAT10 was evenly distributed throughout the cytosol and showed varying degrees of localization to the nucleus (see Figure 37 (a)). Kalveram et al. (Kalveram et al., 2008) previously described, that FAT10 localizes in aggresomal structures under proteasomal inhibiton, which unfortunately could not be confirmed here (see Figure 37 (b)).

JunB by contrast, was completely excluded from the cytosol and showed thoroughly nucleolar localization (see Figure 37 (c)). Strikingly, treatment with MG132 resulted in JunB dislocation towards the nuclear membrane and to the cytosol. Moreover, cells seem to be stressed, which is apparent in increased membrane ruffle formation (see Figure 37 (d)).

Interestingly, co-expression of FAT10 together with JunB resulted in a clear co-localization of both proteins in favor to the nuclear membrane (see Figure 37 (e)).

In double transfected and MG132 treated cells, cells showed again increased membrane ruffle formation. Interestingly, JunB dislocalizes from the nucleus to the cytosol (visible in modified nucleus) like previously seen in Figure 37 (d) and JunB co-localized with FAT10 near the nuclear membrane. However, for the majority of cells, JunB and FAT10 co-localization was predominantly detectable in the cytosol (see Figure 37 (f)). Unfortunately, a quantitative analysis could not be performed due to a lack of time.

Our results substantiate the precedent data that JunB and FAT10 interact and we strongly suggest that modification with FAT10 regulate JunB localization and function. Moreover, we assume that interaction of JunB and FAT10 occurs preferentially at the nuclear membrane, which requires the translocation of JunB away from the nucleus towards the nucleolic membrane. Strikingly, treatment with MG132 leads to a cumulative displacement of JunB to the nuclear membrane and into the cytosol which implicate as functional consequence, that the transcription of JunB transactivating genes could be impaired. Further work will determine whether FAT10ylation is a cause or a consequence of this localization. This issue was further investigated in reporter assays to reveal the impact of FAT10ylation on JunB transcriptional activities on minimal AP-1 driven reporter genes (see chapter 6.2.18).

128 6.2.18 FAT10ylation controls JunB transcriptional activities on minimal AP-1

driven reporter genes

In order to understand, if JunB FAT10ylation has an impact on biological functions of JunB we next aimed to determine, if transcription is affected through this post-translational modification. JunB belongs to the AP-1 family, which collectively describes a class of structurally and functionally related proteins, characterized by a basic DNA-binding domain and a basic leucine zipper (bZIP) dimerization motif which require dimerization before binding to a common DNA target sites, called TPA response elements (TRE) (Wagner, 2001). Previously Garaude et al. (Garaude et al., 2008) described, that post-translational modification of JunB with SUMO regulates the transcriptional activity of JunB in T lymphocytes and plays a critical role in T cell activation. In comparison to c-Jun, JunB has a ten-fold decreased activity to activate AP-1-responsive genes containing single AP-1 binding sites, due to a small number of amino acid changes between its DNA-binding and dimerization motifs (Deng and Karin, 1993). But strikingly, JunB appears to be as effective as c-Jun in trans-activating reporter genes containing multiple AP-1 binding sites which suggests, that trans-activation by JunB may require synergistic interactions between multiple homodimers bound to adjacent sites (Angel and Karin, 1991; Chiu et al., 1989).

To address either potential similarities or differences in biological functions, when JunB becomes attached to FAT10, we conducted a luciferase reporter assay in Jurkat cells on reporter genes containing 4x TRE binding sites, namely AP-1 (4xTRE). Our previous results indicated that K237 was the primary site for FAT10ylation; when this site was mutated to arginine, we detected a complete abrogation of the higher conjugate band of FAT10ylated JunB. The triple mutant JunB-K3R was barely FAT10ylated, as not only the upper conjugate completely disappeared, but also the lower conjugate band was barely detectable (see chapter 6.2.14). Therefore, we decided to use JunB wt as positive, or the triple mutant JunB-K3R as negative control, to investigate the role of FAT10ylation on the transcriptional activity of JunB. Furthermore, co-transfection with FAT10∆GG served as a further negative control, where JunB FAT10ylation is prevented.

Twenty million Jurkat cells were transfected via electroporation with 5 µg expression vectors encoding for HA-tagged JunB wt, HA-tagged JunB-K3R mutant, FLAG-tagged FAT10 or FLAG-tagged FAT10∆GG. In each experiment, cells were transfected with the same total amount of 12 µg DNA by adding the required quantities of pcDNA3 empty vector, along with 2 µg of a reporter gene controlled by the AP-1 (4xTRE-luc) promoter or a pGl3 vector which served as a normalization vector, as indicated. 1/600 TK-Renilla-Luc was used as a control for transfection efficiency and luciferase activity is expressed relative to Renilla luciferase.

Results

129 Two days after transfection, cell extracts were prepared in 100 μl Passive Lysis Buffer (PLB) Dual-Luciferase^® Reporter (DLR™) Assay System (see 5.1.2) and luciferase activity was determined using Berthold Lumat LB9501.

Figure 38: FAT10ylation affects JunB transcriptional activity on minimal AP-1-driven reporter genes in JURKAT cells

Twenty million Jurkat cells were co-transfected with 5 µg of expression vectors for HA-tagged JunB wt, JunB-K3R, FLAG-tagged FAT10 or FAT10∆GG, as indicated. In each experiment, cells were transfected with the same total amount of 12 µg DNA by adding the required quantities of pcDNA3 empty vector, together with 2 µg of a reporter gene controlled by the AP-1 (4xTRE-luc) promoter, as indicated. Transfection of 2 µg pGl3 vector together with 10 µg pcDNA3 served for normalization. 1/600 TK-Renilla-Luc was used as a control for transfection efficiency and luciferase activity is expressed relative to Renilla luciferase. Two days after transfection, luciferase activities were measured. The data are presented as the mean ± SD of five independent experiments. n.s indicates not significant. The asterisks represent the level of significance as calculated with a paired two-tail P value test.

JunB wt and JunB-K3R showed indistinguishable activity on the reference AP-1 reporter gene, driven by four canonical binding sites upstream of the TATA box, namely AP-1 (4xTRE-luc), indicating that the mutation did not affect basal functions. In comparison, neither FAT10 nor FAT10ΔGG trans-activated the AP-1 reporter gene, showing that binding of either JunB wt or JunB-K3R to the 4xTRE sites is specific. Strikingly, co-expression of JunB wt and FAT10 resulted in a significantly decreased reporter activity in comparison to JunB alone. Co-expression of either mutant JunB-K3R with FAT10 or FAT10ΔGG, or JunB wt and FAT10ΔGG (negative controls) resulted only in a slightly decreased activity in comparison JunB alone, suggesting that JunB FAT10ylation specifically provoke the reduced

130 activity. Taken together, these results suggest that FAT10ylation repress JunB transcriptional activity on AP-1 driven promoter with minimal 4xTRE binding sites.

To evaluate the effect of FAT10 conjugation for JunB transactivating capabilities in a different cell line, we performed co-transfection experiments with the AP-1 (4xTRE) reporter in HEK293 cells. 3 x 10⁵ HEK293 cells were transiently transfected with 1,5 µg vectors expressing for HA-tagged JunB wt, HA-tagged JunB-K3R mutant, FLAG-tagged FAT10 or FLAG-tagged FAT10∆GG. In each experiment, cells were transfected with the same total amount of 4,5 µg DNA by adding the required quantities of pcDNA3 empty vector, along with 1 µg of a reporter gene controlled by the AP-1 (4xTRE-luc) promoter or a pGl3 vector which served as a normalization vector, as indicated. 1/600 TK-Renilla-Luc was used as a control for transfection efficiency and luciferase activity is presented relative to Renilla luciferase.

One day after transfection, cell extracts were prepared in 500 μl Passive Lysis Buffer (PLB) Dual-Luciferase® Reporter (DLR™) Assay System (see 5.1.2) and luciferase activity was determined using Berthold Lumat LB9501.

Figure 39: FAT10ylation affects JunB transcriptional activity on minimal AP-1-driven reporter genes in HEK293 cells

3x10⁵ HEK293 cells were co- transfected with 1,5 µg of expression vectors for HA-tagged JunB wt, JunB-K3R, FLAG-tagged FAT10 or FAT10∆GG, as indicated. In each experiment, cells were transfected with the same total amount of 4,5 µg DNA by adding the required quantities of pcDNA3 empty vector, together with 1 µg of a reporter gene controlled by the AP-1 (4xTRE-luc) promoter or a pGl3 vector which served as a normalization vector.

Transfection of 1 µg pGl3 vector together with 3 µg pcDNA3 served for normalization. 1/600 TK-Renilla-Luc was used as a control for transfection efficiency and luciferase activity is expressed relative to Renilla luciferase. 24 h post-transfection, luciferase activities were measured. The data are presented as the mean ± SD of five independent experiments. n.s indicates not significant. The asterisks represent the level of significance as calculated with a paired two-tail P value test.

Results

131 In HEK293 cells, JunB-K3R transactivated reporter genes driven by these composite sites at the same level as JunB wt. Like previously seen in JURKAT cells, co-expression of JunB together with FAT10 resulted in significantly diminished AP-1 promoter activity and co-expression of either mutant JunB-K3R with FAT10 or FAT10ΔGG, or JunB wt and FAT10ΔGG (negative controls) resulted in a similar transactivation of AP-1 (4xTRE), as JunB wt. Taken all the experiments together it seems, that JunB-FAT10 conjugation (as seen in previous experiments) has a direct consequence on transactivating activities of JunB under these conditions.