In vivo modeling of polysumoylation uncovers targeting of Topoisomerase II to the nucleolus via optimal level of SUMO modification

Abstract

Conjugation of SUMO to target proteins is an essential eukaryotic regulatory pathway. Multiple potential SUMO substrates were identified among nuclear and chromatin proteins by proteomic approaches. However, the functional roles of SUMO-modified pools of individual proteins remain largely obscure, as only a small fraction of a given target is sumoylated and therefore is experimentally inaccessible. To overcome this technical difficulty in case of Topoisomerase II, we employed constitutive SUMO modification, enabling tracking of modified Top2p, not only biochemically but also cytologically and genetically. Top-oisomerase II fused to a critical number of SUMO repeats is concentrated at the specific intranuclear domain, the nucleolus, when more than four SUMO moieties are added, indicating that fused SUMO repeats are biologically active. Further analysis has established that poly-sumoylation of Top2p is required for the stable maintenance of the nucleolar organizer, linking SUMO-mediated targeting to functional maintenance of ribosomal RNA gene cluster.

Cooperation of Sumoylated Chromosomal Proteins in rDNA Maintenance

“…However, it is known that, among SUMO substrates, Top1 and Top2 topoisomerases are involved in the maintenance of tandem rDNA array stability [32]. Moreover, we have previously shown that top2ΔC and top2-SNM alleles, with either complete or partial loss of in vivo Top2p sumoylation, respectively [20],[33] (and unpublished data), result in a decrease of the rDNA copy number [11]. Consistent with the role of Top2 sumoylation in rDNA regulation, Top2ΔC ChIP from nocodazole-arrested cells (sumoylation levels of Top2p are maximal in mitosis [33]) showed a strong peak towards the end of the 35S RNA gene, which was not present in wild type (Figure 3A).…”

“…In S. cerevisiae , one prominent subnuclear domain relevant to SUMO biology is the nucleolus, as our previous results showed that the green fluorescent protein (GFP) fusion to Smt3p has a preference for nucleolar localization when deconjugation is impaired [11]. In the present study, we examine the potential cooperation of several chromosomal proteins known to be involved in the stability of ribosomal RNA genes (rDNA).…”

DNA Topoisomerase II Is a Determinant of the Tensile Properties of Yeast Centromeric Chromatin and the Tension Checkpoint

“…It is not known whether Top2 is targeted to yeast CENs in an equivalent manner. However, Top2-SUMO fusion proteins can exhibit robust cross-linking to CEN DNA (Takahashi et al, 2006) or accumulate in the nucleolus (Takahashi and Strunnikov, 2007), indicating sumoylation may also regulate yeast Top2 trafficking. In this report, we used the top2-SNM allele to specifically examine functions associated with Top2 SUMO modification, comparing top2-SNM strains to catalytically inactive top2-4 mutants.…”

SUMOylation-disrupting WAS mutation converts WASp from a transcriptional activator to a repressor of NF-κB response genes in T cells

“…At the minimum, our findings linking a selective SUMO chain defect to aberrant gene repression suggest that polySUMO modification support a specific chromatin signaling function distinct from that of the monoSUMO modification of WASp. Although the causality of this association remains speculative, emerging evidence linking poly-SUMO chain defects to impaired chromatin-to-nucleolar targeting and DNA instability, 73 altered chromatin organization and transcriptional repression, 28 and Alzheimer's disease 74 together propose a physiologically relevant role for WASp polySUMO chains in immunity.…”