SUMO is a multifaceted modifier of chromatin structure. SUMO modification of chromatin proteins regulates a range of cellular processes including transcription, replication, DNA repair and chromosome segregation. SUMOylation has long been believe to be associated with gene silencing or repression. However, global mapping of chromatin binding by SUMO in yeast  and Drosophila , show that SUMOylated proteins are present at transcriptionally active and induced genes. This discovery led to the hypothesis that SUMO functions to prevent super-induction of actively transcribed genes by external factors (in this case, viral infection) to maintain a steady-state level of transcription. However, lower eukaryotes possess only one SUMO isoform, whereas there are two groups of SUMO variants in humans; SUMO-1 and SUMO-2/3. Recently, the global chromatin localization of SUMO-1 through the cell cycle of human HeLa cells has been identified. Similar to that reported in yeast, SUMO-1 tends to cluster around transcriptionally active genes . Although increasing evidence from studies targeting specific cellular factors suggests that there is differential conjugation and functionality among SUMO paralogues, the global functional heterogeneity of human SUMO paralogues seems to be limited in their conjugation dynamics [11, 12] and subcellular localizations . The global functional differences between SUMO paralogues in terms of epigenetic regulation remains a puzzle. In this study, we compared the chromosome-wide labeling of SUMO-1 and SUMO-2/3 proteins before and after herpesvirus reactivation using the ChIP-Seq assay. We found that firstly, on a genome-wide scale, the binding profile of the SUMO paralogues was highly similar in the control cells, but that differences were evident after KSHV reactivation with there being a significant increase in SUMO-2/3 binding while there was only limited changes in the SUMO-1 binding profile. Secondly, the distribution of both SUMO paralogues on the chromatin showed a greater tendency toward being associated with transcription regulatory regions (promoters) and that, furthermore, the binding of SUMO-2/3 onto the promoter regions was significantly increased during viral reactivation. Thirdly, there was a dramatic increase in SUMO-2/3 binding and a slight decrease in SUMO-1 binding onto TFBSs during viral reactivation. Fourthly, the potential SUMO-1 and SUMO-2/3 target TFs highly overlapped in the control cells, while the SUMO-2/3 specific TFs are significantly increased during viral reactivation. Fifthly, three IRFs, “the master regulators of immune responses” show up in the top-10 most important gene-regulating TFs targeted by SUMO-2/3 after KSHV reactivation. Sixth, both the SUMO paralogues are preferentially localized on the promoters of highly expressed genes, and that SUMO-2/3 is predominantly found associated with highly expressed genes that show no change in expression during herpesvirus reactivation. Finally, after viral reactivation, SUMO-2/3 is significantly associated with the promoters of genes in pathways related to cellular immune responses, cytokine signaling, cell growth and apoptosis. To our knowledge, our findings are the first to compare dynamically the global chromatin-binding profiles of SUMO-1 and SUMO-2/3 across the human genome and suggest that, while the binding profile of SUMO paralogues is similarly under un-induced condition, they do change differently during KSHV infection.
Herpesviruses have evolved multiple mechanisms to target SUMOylation pathways, including modulating SUMO conjugation enzymes (SUMO E1 ligase, SUMO E2 ligase and SUMO E3 ligase) and deconjugation enzymes (SUMO-specific proteases; SENP) as well as by directly targeting SUMOylated proteins . Interestingly, KSHV encodes a SUMO E3 ligase in the lytic phase and this enzyme is likely to be the reason behind the increase in SUMO-2/3 paralogues present on chromatin during viral reactivation . However, this hypothesis needs to be rigorously tested via a knock-in recombinant KSHV containing a SIM mutant of K-bZIP that results in a loss of its SUMO E3 ligase activity. This will be an interesting direction to investigate in the future. Moreover, we cannot exclude the possibility that the induction of K-Rta activates host SUMO E3 ligase to deposit SUMO-2/3 at the promoter regions. For example, we have previously identified a host factor, KAP1, is phosphorylated by KSHV vPK during KSHV reactivation  and KAP1 has recently been reported to be a SUMO E3 ligase for IRF-7 .
The complete sequence of the human genome was obtained more than a decade ago; nevertheless, our understanding of this genome is far from complete. The emerging concept from Encyclopedia of DNA Elements (ENCODE) is that biochemical functions of a genome can be assigned by systematically identifying the functional elements within the genome . Patterns in chromatin modification or transcription factor binding onto the functional elements assists with the prediction of their role, particularly when RNA expression is examined. The global but uneven distribution of SUMO modification near TSSs prompted us to study the distribution of SUMO modification on different functional elements of the genome, such as promoters, coding sequences (transcripts), upstream gene regions, downstream gene regions, and intergenic regions. The significant enrichment of SUMO paralogues in promoter regions (Figure 2D and 2E) strongly suggests that SUMOylation may be involved in regulating gene transcription. Consistent with previous reports from lower eukaryotics and another describing SUMO-1 in HeLa cells [7, 46], the correlation between SUMO paralogues binding to promoter region and higher levels of gene transcription, which is also found in the present study (Figure 10), further supports the potential role of SUMOylation in maintaining the expression of constitutively active genes. Moreover, SUMO-1 and SUMO-2/3 may function in a similar manner maintaining the expression of transcriptional active genes in non-reactivated control cells.
SUMO binding onto chromatin must occur via either the modification of chromatin remodeling proteins or the modification of transcription factors, both of which bind to the genome. SUMO shows focal peaks or areas of high occupancy within the promoter region near TSSs. The focal and gene-selective nature of SUMO occupancy resembles the peaks associated with transcription factors, which suggests that there is SUMO modification of TFs. Motif scanning is a powerful method to facilitate the identification of DNA binding motifs (or transcription factor binding motifs) from peaks defined by ChIP-seq. This method has been widely used to distinguish the transcription regulation of one or a few TFs. SUMO modifications are able to occur in many dozens of known TFs as well as being likely to occur in many currently unknown TFs. Using the current findings, it is probably too complex and too time consuming to carrying full scale motif scanning to identify potential SUMO target TFs. Therefore, as an alternative, we used an annotation method that directly annotates SUMO peaks in the promoter region in relation to transcription factor binding sites (TFBS). The details of this method have been submitted in another article . Briefly, the Transfac Matrix Database (v7.0) created by Biobase contains 258 TFBS weight matrices representing the potential DNA binding sites of 176 TFs and this database was chosen to annotate the SUMO peaks. Using this method, we were able to simultaneously identify potential SUMOylated TFs. Potential SUMO target TFs are those TFBSs that show a significant correlation with SUMO peaks and these were identified by the Hampel Identifier. Half of all SUMO-1 and half of the top-20 SUMO-2/3 potential target TFs identified before and after viral reactivation were known SUMO targets. The other half may be potential SUMO targets that have not been identified as yet or proteins containing a SIM domain that provides an additional interaction platform allowing the recruiting of other SUMOylated proteins; both of these situations may be responsible for the TFs identified here. The SUMOylation fraction in a steady state is typically very little in related to the entire pool of transcription factors. Efforts are still needed to confirm the results outlined here and to elucidate the underlying functions of SUMOylation during the regulation of these TFs. Interestingly, when we ranked the potential SUMO-2/3 target TFs by the total number of their regulating genes (Figures 4 and 5), we found three IRFs that were not SUMO-2/3 targets in the control cells that were listed as top 4th, top 5th and top 6th of the SUMO-2/3 target TFs after viral reactivation. IRFs constitute a family of TFs (IRF-1-IRF-9) that are in control of the type I interferon (IFN) system and are involved in executing the innate and adaptive immunity associated with host resistance against pathogens, including virus infection. To promote its own survival, KSHV exploits a number of different strategies to suppress the host immune system. Recent evidence has shown that the virus triggers the SUMOylation of IRFs, leading to a targeting and blocking of the type I interferon pathway [24, 40, 41]. K-bZIP of KSHV has also been found to inhibit type I IFN signaling in a signal transducers and activators of transcription (STAT) dependent manner and in an IFN-stimulated gene factor 3 (ISGF3) independent manner . Moreover, KSHV K-bZIP inhibits IRF-3 by preventing IRF-3 from binding to target promoter, which precludes the formation of the enhanceosome. The potential SUMO-2/3 target IRFs identified here (Figure 5) provides an additional novel mechanism for globally inhibiting the activation of the host immune system.
The growing links between the viral and cellular SUMO systems makes SUMO a potential target for antiviral therapy . Identifying the preferential usage of SUMO paralogues in viruses may help to improve the specificity of any SUMO-targeted antiviral therapies. Recently, growing evidence, including ours, suggests that some herpesviruses have a preference for SUMO-2/3 [16, 55]. Significant increase in SUMO-2/3 coating across human genome, but not in SUMO-1 coating, during viral reactivation found here suggest that a new class of combine therapy targeting SUMO-2/3 may disrupt the dynamic balance of the herpesvirus latent and lytic phases. Disrupting the balance may help the clearance of the herpesvirus from the infected cells and improve current therapy.
In summary, we found that SUMO-1 and SUMO-2/3 share a highly similar binding landscape on chromatin. They are preferentially enriched in promoter regions and are associated with highly transcribed genes. Differential chromatin-binding profiles of the SUMO paralogues are able to be observed during herpesvirus reactivation. We found that SUMO-2/3 peaks significantly increased in promoter regions during viral reactivation and this was associated with the genes that do not undergo changes in transcription level. TFs identification and GO analysis suggests that SUMO-2/3 preferentially target immune pathways during viral reactivation.