Genetics: Early Online, published on February 2, 2016 as 10.1534/genetics.116.187252 SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus in Saccharomyces cerevisiae Jennifer Gillies*, Christopher M. Hickey*, Dan Su*1, Zhiping Wu†, Junmin Peng†, and Mark Hochstrasser*2 *Yale University Department of Molecular Biophysics & Biochemistry 266 Whitney Avenue, New Haven, CT 06520-8114 †Departments of Structural Biology and Developmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN 38105 Running Title: SUMO affects rDNA protein binding. Key words: SUMO, Ulp2, rDNA, RENT complex, Fob1 1Current address: Protein Sciences Corp., 1000 Research Parkway, Meriden CT 06450 2To whom correspondence should be addressed: Ph: (203) 432-5101; Fax: (203) 432-5175; E-mail: [email protected] Copyright 2016. Summary By identifying cellular targets of Ulp2, a yeast SUMO protease, we discovered a role for SUMO-protein modification in ribosomal DNA (rDNA) regulation. Net1, Tof2, and Fob1, all rDNA chromatin-associated proteins, were found to be sumoylated and Ulp2 substrates in vivo. All three proteins lose rDNA binding in ULP2-deficient cells. Slx5/Slx8, a heterodimeric SUMO- targeted ubiquitin ligase, contributes to the impaired rDNA binding by these proteins in cells lacking Ulp2. Genetic interactions between ulp2Δ and either loss or overexpression of rDNA regulatory factors were also observed, underscoring the importance of Ulp2 in the proper function of the rDNA locus. Abstract In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae SUMO proteases, and investigate the function of SUMO modification of these proteins. Poly- SUMO conjugates from ulp2Δ and ulp2Δ slx5Δ cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2Δ, slx5Δ and ulp2Δ slx5Δ cells. The increase in sumoylation of Net1 and Tof2 in ulp2Δ, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2. Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2Δ cells and both factors largely regain this association in ulp2Δ slx5Δ. A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in 2 wild-type cells, and its modification levels increase specifically in ulp2Δ cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2Δ cells, which is also rescued by elimination of Slx5. Additionally, overexpression of Sir2, another RFB-associated factor, suppresses the growth defect of ulp2Δ cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2Δ cellular defects. 3 Introduction Post-translational protein modifications provide a common mechanism for regulating protein-protein interactions, protein localization, and protein degradation. The SUMO (small ubiquitin-related modifier) protein family modifies target proteins through covalent attachment to lysine side chains [1]. The function of substrate “sumoylation” varies with the protein that is sumoylated, but a common role is to modulate the assembly of large protein complexes either by creating additional binding sites or by blocking existing sites [2]. Protein sumoylation occurs through an enzymatic cascade similar to ubiquitylation [1]. In S. cerevisiae, a single gene, SMT3, codes for SUMO, and as in most species, SUMO ligation is essential for viability. Removal of SUMO from target proteins is also an important regulatory step. S. cerevisiae has two SUMO proteases, Ulp1 and Ulp2 [3]. Most Ulp1 is bound to the nuclear pore complex (NPC), and the enzyme is essential for cell-cycle progression. NPC tethering regulates Ulp1 activity by restricting its access to sumoylated proteins [4, 5]. Ulp1 also processes the SUMO precursor by removing its last three residues, thereby exposing the C-terminal Gly-Gly motif required for SUMO conjugation. Ulp1 is essential for growth even if cells are provided with mature SUMO, indicating a critical function for its protein desumoylation activity. Ulp2 is less active than Ulp1 in vitro, and is not required for viability, although cells lacking Ulp2 are growth defective and sensitive to a wide range of stresses. Ulp2 localizes throughout the nucleus, with a slight concentration in the nucleolus [6]. It has long, poorly conserved regions flanking the catalytic domain, which are of ill-defined function other than a pair of nuclear- localization signals (NLSs) in the N-terminal domain [7]. Large amounts of high molecular weight (HMW) SUMO conjugates, which accumulate in the stacker of SDS-PAGE gels, are observed in ulp2 cells. Correspondingly, Ulp2 has a preference in vitro for cleaving between SUMO 4 monomers in polySUMO chains [3, 8]. The identities of most of the HMW-SUMO conjugates are unknown, as is their contribution to the growth defects of ulp2Δ cells [9]. Slx5/Slx8 is a heterodimeric ubiquitin ligase (E3) that can recognize SUMO-protein conjugates and is therefore referred to as a SUMO-targeted ubiquitin ligase (STUbL) [10-13]. The Slx5 subunit interacts directly with SUMO and polySUMO chains through four tandem SUMO- Interacting Motifs (SIMs). Slx8 has a single apparent SIM [10, 14, 15]. Deleting the SLX5 gene suppresses ulp2Δ defects but exacerbates those of the ulp1ts mutant, consistent with distinct cellular roles of the two SUMO proteases [14, 16]. Slx5/Slx8 has multiple functions and targets (not all requiring prior SUMO ligation, [11]), including roles in DNA replication and repair [17]. The STUbL localizes at various sites within the nucleus as well, such as DNA replication forks and the NPC [18-20]. The SUMO pathway has been linked to the nucleolus, the cellular hub for the initial stages of ribosome assembly [6, 21-24]. To maintain robust ribosome production, cells have numerous rDNA copies, even though repeated DNA is prone to aberrant homologous recombination. S. cerevisiae has between 100-200 copies of the rDNA repeat, which consists of the 35S precursor rRNA gene, the 5S rRNA gene and two non-transcribed spacers [25]. Excessive homologous recombination of the rDNA leads to an increased number of rDNA repeats, which can produce extra-chromosomal rDNA circles, potentially promoting senescence [26]. Suppression of homologous rDNA recombination relies on a variety of proteins, and many post-translational modifications of nucleolar proteins have been implicated in rDNA silencing including acetylation, phosphorylation, methylation, ubiquitylation, and sumoylation [25, 27-30]. In this study, we have identified Net1 and Tof2 as proteins that become highly sumoylated in ulp2Δ cells. These paralogous scaffolding proteins are part of a complex rDNA regulatory 5 system (see Figure 8). Net1, along with Sir2 and Cdc14, form the RENT (Regulator of Nucleolar Silencing and Telophase Exit) complex [25]. Nucleolar silencing by the RENT complex prevents homologous recombination of the rDNA repeats and represses RNA polymerase II transcription within the region. Deleting NET1 causes nucleolar proteins, such as Nop1, to mislocalize throughout the nucleus [31, 32]. Sir2 is a histone deacetylase that de-acetylates histones H3 and H4 in the silent genomic regions in yeast, which include rDNA [33]. Sir2 localizes to the rDNA by binding Net1, which also activates Sir2. Cdc14, the final member of the RENT complex, is a phosphatase responsible for dephosphorylating specific cell-cycle regulators in a coordinated fashion [34, 35]. Cdc14 is sequestered in the nucleolus and its activity is suppressed when bound to Net1 in the cell cycle stages leading up to early anaphase [36]. Cdc14 is released from the nucleolus in a two-step process during mitosis, and once released, it dephosphorylates Cdk1 targets, leading to exit from mitosis [36]. Net1 localizes to the rDNA through binding to Fob1 and RNA polymerase I (RNAPI) [37]. Fob1 is a replication fork-blocking protein that binds specifically to the replication-fork barrier (RFB) sequence in the non-transcribed regions of rDNA repeats [38]. Tof2 also binds to Fob1 and Cdc14, but not to Sir2 or RNAPI [37]. Furthermore, Tof2 interacts with the cohibin complex, Lrs4-Csm1, which in turn binds to and localizes cohesin and condensin complexes [39, 40]. Cohibin additionally associates with Src1-Nur1, thereby tethering the rDNA to the inner nuclear membrane [37, 41]. Particularly germane to the current work, cohibin also binds to and localizes Ulp2 to the nucleolus [6], placing Ulp2 physically adjacent to Net1 and Tof2, consistent with previous ChIP analysis of Ulp2 within the rDNA repeats [21]. Tof2 has been shown to be sumoylated and the level of the sumoylation increases when cells are treated with MMS, a DNA damaging agent [6, 42-45]. 6 Protein complex formation on the rDNA is hierarchical (see Figure 8). Fob1 is required for both Net1 and Tof2 to bind to the RFB [37]. RNAPI localizes Net1 to the transcription initiation region (TIR) [37]. Net1 is in turn necessary for Sir2 and Cdc14 nucleolar localization