DLX3 is the only member of the DLX family containing putative SUMOylation sites, one of which is conserved among several vertebrates
The DLX3 amino acid sequence was analyzed and two potential SUMOylation sites ψKXE, where ψ represents a hydrophobic amino acid, X represents any amino acid, were discovered at lysine 83 (pkse) and lysine 112 (vkee) (). Because proline is less hydrophobic than valine, we hypothesized that lysine 83 is less susceptible to SUMOylation than lysine 112. The DLX3 amino acid sequence in several vertebrates was aligned, and K112 was found to be conserved among human, mouse, chicken, zebrafish, and xenopus (). An amino acid sequence comparison was made between all human DLX proteins. DLX3 is the only member of the DLX family containing these two potential SUMOylation sites (). This specificity and the conservation among vertebrates led us to further investigate the effect of SUMOylation on DLX3.
Potential SUMOylation sites in the DLX3 protein sequence
DLX3 is SUMOylated by SUMO1
To determine the functionality of the two potential SUMOylation sites in DLX3, we co-expressed V5DLX3 (DLX3 tagged with a V5 epitope) with wildtype SUMO1 in Saos2-TetOFF cells. As a control, we also co-expressed V5DLX3 with a mutated form of SUMO1 lacking the C-terminal double glycine which forms an isopeptide bond with the target protein (SUMO1-ΔGG). SUMO1 and SUMO1-ΔGG are tagged with both a His tag and a c-Myc tag. Western blot was performed to analyze the size pattern of c-Myc tagged and V5-tagged proteins in these cells (). Results monitored with anti-cMyc showed a low molecular weight band, around 15 kDa, corresponding to the monomer of SUMO1 or SUMO1-ΔGG, as well as a smear corresponding to all endogenous proteins SUMOylated by SUMO1, but not SUMO1-ΔGG (, left panel). The anti-V5 blot revealed two major bands for V5DLX3 when co-expressed with SUMO1: the lower band corresponding to the normal size of V5DLX3, and the upper corresponding to a V5DLX3 derivative with additional 15-20 kDa (, right panel). This upper band was not detected with SUMO1-ΔGG. These observations suggest that V5DLX3 is SUMOylated by SUMO1. To confirm that the upper complex detected is indeed SUMOylated V5DLX3, we performed the same western blot analysis after purifying the protein extracts with a Ni-column (Ni-NTA, binding His-tagged SUMO1) to pull-down all SUMOylated proteins. Using this approach, we detected SUMOylated V5DLX3 among the proteins purified on the Ni-column (). These results demonstrate that V5DLX3 is SUMOylated by SUMO1. Moreover, the fact that we detected only one band for SUMOylated V5DLX3 suggests that only one lysine is involved in DLX3 SUMOylation.
Detection of DLX3 SUMOylation by SUMO1
DLX3 is SUMOylated by SUMO1 on K112, but not K83
To test which of the two potential SUMOylation sites in DLX3 is actually involved in SUMOylation, we mutated the lysine residues at positions 83 and 112 into arginines (). Thus, we generated two single mutants (DLX3K83R and DLX3K112R) as well as a double mutant in which the two lysine residues were mutated (DLX32K). In Saos2-TetOff cells, we co-expressed SUMO1 or SUMO1-ΔGG with DLX3WT, DLX3K83R, DLX3K112R or DLX32K, respectively. Western blot analysis was performed using anti-cMyc and anti-V5 antibodies, both on whole extracts and on Ni-column purified protein fraction (). This assay revealed that mutation in position 83 does not preclude SUMOylation of V5DLX3, since a band corresponding to SUMOylated DLX3 appears for both DLX3WT and DLX3K83R. This band, however, was absent for DLX3K112R. As predicted, the double mutant DLX32K is not SUMOylated. These observations demonstrate that lysine K112 is the only SUMOylation site in DLX3.
Identification of the lysine involved in DLX3 SUMOylation
SUMOylation does not affect DLX3 subcellular localization
In order to assess the effect of SUMOylation on DLX3 subcellular localization, we performed immunohistochemical analysis on Saos2-TetOFF cells co-transfected with V5DLX3 and SUMO1 or SUMO1-ΔGG (). Immunostaining using anti-cMyc antibody showed that SUMO1 was present in both the cytoplasm and the nucleus: the nuclear expression was rather diffuse, however, it tended to be stronger at the nuclear periphery, while in the cytoplasm it formed dense aggregates (). SUMO1-ΔGG exhibited a completely different distribution pattern: it was distributed throughout the cytoplasm in a filament-like pattern and was also present in the nucleus (). The distribution pattern of V5DLX3 in the nucleus appeared the same in the presence of wild-type and mutant SUMO1 (). This observation suggests that SUMO1 does not dramatically affect DLX3 subcellular localization. However, we observed a partial co-localization between DLX3 and SUMO1 at the periphery of the nucleus (). We used cell fractionation and western blot analysis to corroborate our immunohistochemical observations (). The anti-cMyc blot revealed that the cytoplasm contained both monomeric SUMO1 and SUMO1 conjugated with target proteins, while in the nucleus SUMO1 was essentially present in its conjugated form. SUMO1-ΔGG, that can only be monomeric, exhibited a much stronger expression in the cytoplasm than in the nucleus. The anti-V5 blot showed that both V5DLX3 and SUMOylated V5DLX3 were accumulated almost exclusively in the nuclear fraction. These data confirm that SUMO1 does not dramatically affect DLX3 subcellular localization. We then investigated whether DLX3 subcellular localization was affected by its inability to be SUMOylated. To address this question, immunohistochemical analysis was carried out with Saos2-TetOff cells transfected with V5DLX3WT or V5DLX3K112R. As shown in , both wild type and K112R mutant were exclusively located in the nucleus. Thus, preventing DLX3 from being SUMOylated does not affect its subcellular localization. Taken together, these data suggest that SUMOylation does not play a significant role in determining the distribution pattern of DLX3 in cells.
Effect of SUMOylation on DLX3 subcellular localization
SUMOylated DLX3 is able to bind DNA
To determine the role of SUMOylation on DLX3 function, we next explored the effect of SUMOylation on DLX3 binding activity. We first asked if SUMOylated DLX3 was able to bind DNA. To address this question, we first generated a recombinant SUMO-DLX3 fusion protein and tested its ability to bind to the DLX3 consensus binding site in an Electrophoresis Mobility Shift Assay (EMSA), using a probe containing a DLX3 binding site. A protein-DNA complex was formed, that could be competed using an excess of non-radioactive probe (self competitor), but not using an excess of a mutated non-radioactive probe (mutant competitor) (, lanes 2-4). To confirm that this protein-DNA complex contained SUMO-DLX3, we performed a supershift assay using anti-DLX3 antibody and were able to detect a protein-DNA-antibody complex (, compare lanes 5 and 6). Even though the SUMO-DLX3 fusion protein does not perfectly mimic the tertiary structure of SUMOylated DLX3, this strategy is commonly used in the field [Ouyang et al., 2009
] and gives a preliminary suggestion that having SUMO bound to DLX3 does not seem to affect its ability to bind DNA. To test this hypothesis in a more physiologically meaningful context, we performed EMSA using nuclear extracts from cells expressing V5DLX3 with SUMO1 or SUMO1-ΔGG (, inset). In both cases (wild-type and mutant SUMO1), we could detect a protein-DNA complex formed between DLX3 and the probe (, arrow). In order to detect if SUMOylated DLX3 is involved in a complex with the probe, we performed a supershift assays using anti-cMyc antibody (recognizing SUMO1 and SUMO1-ΔGG). As shown in , a partial supershift could be detected in the presence of anti-cMyc when DLX3 was expressed with SUMO1, but not with SUMO1-ΔGG (, arrowhead, compare lanes 3 and 5). We performed a similar experiment in which we prepared nuclear extracts from cells expressing SUMO1 with DLX3WT
(, inset), and performed an EMSA as described above. As expected, using anti-cMyc antibody, we could detect a partial supershift for DLX3WT
but not for DLX3K112R
(, compare lanes 3 and 5). These observations demonstrate that when SUMO1 is bound to DLX3, DLX3 is still able to bind to its consensus binding site. We also showed that DLX3K112R
is able to bind DNA (, arrowhead, lanes 4 and 5), demonstrating that preventing DLX3 from being SUMOylated does not affect its ability to bind DNA.
Effect of SUMOylation on DLX3 DNA binding activity
SUMOylation has a positive effect on DLX3 transcriptional activity
In order to test the effect of SUMOylation on DLX3 transcriptional activity, we performed luciferase reporter assays. The first question we asked was what is the effect of overexpressing SUMO1 on DLX3 transcriptional activity. To address this question, we transfected Saos2-TetOFF cells with pCMV-V5DLX3 (constitutive expression of V5DLX3), pTRE2-SUMO1 (inducible expression of SUMO1), pGL3-3xDRE (3 copies of the DLX3 responsive element upstream of the Firefly luciferase reporter gene), and pRL-TK (constitutive expression of Renilla luciferase gene used for normalization). After transfection, the cells were grown for 24 hours with or without doxycycline, and assayed for Firefly and Renilla luciferase activity. Using this strategy, we found that DLX3 transcriptional activity was slightly higher in the presence of SUMO1 (−Dox) than in its absence (+Dox), but this difference was not statistically significant (data not shown). Considering that this moderate effect of SUMO1 overexpression on DLX3 transcriptional activity could be due to the effect of SUMO1 on other endogenous targets that may interact with DLX3, we decided to focus on the comparison between DLX3WT and DLX3K112R. In this next assay, Saos2-TetOFF cells were transfected with pBi-GFP, pBi-V5DLX3WT/GFP or pBi-V5DLX3K112R/GFP, together with pTRE2-SUMO1, pGL3-3xDRE, and pRL-TK. After transfection, the cells were grown for 24 hours with or without doxycycline, and relative luciferase activity (Firefly/Renilla) was measured (). As expected, in the absence of doxycycline, we detected a significant increase in relative luciferase activity in the presence of DLX3WT compared to the GFP control. The transcriptional activity measured for DLX3K112R was significantly lower than that of DLX3WT, demonstrating that preventing DLX3 SUMOylation in a context where SUMO1 is active significantly reduces its transcriptional activity. In the presence of doxycycline that shuts down the expression of the transgenes in Saos2-TetOff cells, the relative luciferase activity was reduced to a basal level in all conditions, confirming that the transcriptional activities measured are related to the transgenes expression. Taken together, these data suggest that SUMOylation potentially promotes DLX3 transcriptional activity.
Effect of SUMOylation on DLX3 transcriptional activity