The Ulp2 SUMO protease promotes transcription elongation through regulation of histone sumoylation
2019; Springer Nature; Volume: 38; Issue: 16 Linguagem: Inglês
10.15252/embj.2019102003
ISSN1460-2075
AutoresHong‐Yeoul Ryu, Dan Su, Nicole R Wilson‐Eisele, Dejian Zhao, Francesc López‐Giráldez, Mark Hochstrasser,
Tópico(s)Cancer-related gene regulation
ResumoArticle17 July 2019free access Source DataTransparent process The Ulp2 SUMO protease promotes transcription elongation through regulation of histone sumoylation Hong-Yeoul Ryu orcid.org/0000-0002-3367-9887 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Dan Su Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Nicole R Wilson-Eisele Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Dejian Zhao Yale Center for Genome Analysis, Yale University, New Haven, CT, USA Search for more papers by this author Francesc López-Giráldez Yale Center for Genome Analysis, Yale University, New Haven, CT, USA Search for more papers by this author Mark Hochstrasser Corresponding Author [email protected] orcid.org/0000-0002-1131-5484 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Hong-Yeoul Ryu orcid.org/0000-0002-3367-9887 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Dan Su Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Nicole R Wilson-Eisele Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Dejian Zhao Yale Center for Genome Analysis, Yale University, New Haven, CT, USA Search for more papers by this author Francesc López-Giráldez Yale Center for Genome Analysis, Yale University, New Haven, CT, USA Search for more papers by this author Mark Hochstrasser Corresponding Author [email protected] orcid.org/0000-0002-1131-5484 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Search for more papers by this author Author Information Hong-Yeoul Ryu1, Dan Su1,†, Nicole R Wilson-Eisele1,†, Dejian Zhao2, Francesc López-Giráldez2 and Mark Hochstrasser *,1 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA 2Yale Center for Genome Analysis, Yale University, New Haven, CT, USA †Present address: Protein Science Corp., Meriden, CT, USA †Present address: Max Planck Institute of Biochemistry, Martinsried, Germany *Corresponding author. Tel: +1 203 432 5101; E-mail: [email protected] EMBO J (2019)38:e102003https://doi.org/10.15252/embj.2019102003 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Many eukaryotic proteins are regulated by modification with the ubiquitin-like protein small ubiquitin-like modifier (SUMO). This linkage is reversed by SUMO proteases, of which there are two in Saccharomyces cerevisiae, Ulp1 and Ulp2. SUMO-protein conjugation regulates transcription, but the roles of SUMO proteases in transcription remain unclear. We report that Ulp2 is recruited to transcriptionally active genes to control local polysumoylation. Mutant ulp2 cells show impaired association of RNA polymerase II (RNAPII) with, and diminished expression of, constitutively active genes and the inducible CUP1 gene. Ulp2 loss sensitizes cells to 6-azauracil, a hallmark of transcriptional elongation defects. We also describe a novel chromatin regulatory mechanism whereby histone-H2B ubiquitylation stimulates histone sumoylation, which in turn appears to inhibit nucleosome association of the Ctk1 kinase. Ctk1 phosphorylates serine-2 (S2) in the RNAPII C-terminal domain (CTD) and promotes transcript elongation. Removal of both ubiquitin and SUMO from histones is needed to overcome the impediment to S2 phosphorylation. These results suggest sequential ubiquitin-histone and SUMO-histone modifications recruit Ulp2, which removes polySUMO chains and promotes RNAPII transcription elongation. Synopsis SUMO-protein modifications are known to modulate transcription, but the dynamics of these modifications and the roles of SUMO proteases remain unclear. This work reveals that the yeast Ulp2 SUMO protease promotes transcription by coordinating histone sumoylation and serine-2 phosphorylation of the RNA polymerase II (RNAPII) CTD. Ulp2 is preferentially recruited to highly transcribed genes and ribosomal protein genes to control local (poly)sumoylation. Loss of ULP2 leads to impaired association of RNAPII with, and diminished expression of, both constitutive genes and the inducible CUP1 gene. Histone H2B ubiquitylation promotes histone sumoylation, which in turn appears to inhibit nucleosome binding of the CTD serine-2 kinase, Ctk1. Removal of ubiquitin and SUMO from histones by Ubp8 and by Ulp2, respectively, is required to facilitate Ctk1-nucleosome association and subsequent RNAPII elongation. Introduction The small ubiquitin-like modifier (SUMO) protein is an evolutionarily conserved post-translational modifier modulating proteins in diverse regulatory pathways (Flotho & Melchior, 2013). Mature SUMO is conjugated via its C-terminal Gly-Gly motif to substrate lysine side chains. Conjugation occurs through an enzyme cascade involving the E1 SUMO-activating enzyme (Aos1-Uba2), the E2 SUMO-conjugating enzyme E2 (Ubc9), and one of a small number of E3 SUMO ligases (Hendriks & Vertegaal, 2016). Sumoylation is readily reversed by SUMO proteases; nine SUMO proteases have been described in humans and two, Ulp1 and Ulp2, in Saccharomyces cerevisiae (Hickey et al, 2012). While there are numerous reports of functions and substrates of Ulp1, most cellular roles of Ulp2 remain unclear. Ulp2 has particularly high activity toward polySUMO chains (Bylebyl et al, 2003), and it preferentially localizes to the nucleus (Li & Hochstrasser, 2000). Its loss results in a pleiotropic mutant phenotype including defects in cell growth; sensitivity to heat, DNA damage, or aberrant spindle formation; multi-chromosome aneuploidy; and upregulated expression of ribosomal proteins (Li & Hochstrasser, 2000; Ryu et al, 2016). SUMO modification serves both positive and negative roles in transcription in yeast as well as mammals (Chymkowitch et al, 2015b). For example, SUMO conjugation to many gene-specific transcription factors, including C/EBP, c-Jun, ELK-1, and the TFIID subunit TAF5, suppresses the transcription of their target genes (Muller et al, 2000; Kim et al, 2002; Yang et al, 2003; Boyer-Guittaut et al, 2005). Another demonstrated mechanism for SUMO-dependent repression is sumoylation of the transcriptional coactivator p300; this modification promotes p300 binding to histone deacetylase complexes (HDACs), which locally deactivate chromatin (Girdwood et al, 2003). In some cases, however, transcription factor sumoylation stimulates transcription (Lyst & Stancheva, 2007; Guo & Sharrocks, 2009). For example, sumoylation of ZNF76, a repressor targeting the TATA-binding protein (TBP), inhibits ZNF76 repression activity (Zheng & Yang, 2004). Intriguingly, the histone methyltransferase SETDB1 is differentially modulated by sumoylation of distinct cofactors (Lyst et al, 2006; Ivanov et al, 2007). Such conjugation can either promote gene expression (Lyst et al, 2006) or repress it (Ivanov et al, 2007). In S. cerevisiae, SUMO is usually localized at constitutively transcribed genes and promotes their transcription as well as the activation of inducible genes (Rosonina et al, 2010). In a genome-wide study, SUMO was found to be enriched at genes encoding tRNA and ribosomal protein (RP) genes, and sumoylation of the Rap1 transcriptional regulator promotes recruitment of the basal transcription machinery to these genes (Chymkowitch et al, 2015a. Interestingly, SUMO plays an important role not only in transcriptional activation by the Gcn4 transcription factor but also in subsequent transcriptional deactivation through a combination of SUMO-dependent clearance of Gcn4 and stabilization of Tup1 corepressor binding at promoters (Ng et al, 2015). The yeast SUMO proteases also can modulate transcription. Ulp1 localized at nuclear pore complexes accelerates GAL1 de-repression kinetics (Texari et al, 2013), while association of Ulp2 with RP and snoRNA genes limits accumulation of SUMO at these loci (Ryu et al, 2018). The amino- and carboxy-terminal tails of histones are major targets for multiple post-translational modifications including acetylation, phosphorylation, methylation, and ubiquitylation. Additionally, many reports suggest that chromatin structure or gene expression is regulated by crosstalk among different histone modifications (Berger, 2002). A good example of such crosstalk is a trans-tail modification pathway in which histone H2B monoubiquitylation is linked to histone H3 methylation in promoting gene transcription and telomeric silencing (Henry & Berger, 2002; Sun & Allis, 2002). All four core histones and the histone H2A variant H2A.Z are also all subject to SUMO modification in S. cerevisiae (Nathan et al, 2006; Kalocsay et al, 2009), whereas only sumoylated histones H3 and H4 have been identified in mammalian cells to date (Shiio & Eisenman, 2003; Hendriks et al, 2014). A small number of SUMO-modification sites have been identified in yeast histones, but there appear to be many such sites within each histone (Nathan et al, 2006). In human cells, histone H4 sumoylation recruits the histone deacetylase HDAC1 and heterochromatin protein 1 (HP1), thereby attenuating transcription (Shiio & Eisenman, 2003). SUMO-conjugated histones may reduce transcriptional activity by opposing activating histone marks such as acetylation or ubiquitylation (Nathan et al, 2006). However, whether histone sumoylation contributes more generally to transcription regulation is unclear. The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII), Rpo21/Rpb1, is composed of heptapeptide repeats (YSPTSPS) that are subject to extensive post-translational modifications, including phosphorylation, glycosylation, proline isomerization, acetylation, methylation, and ubiquitylation (Egloff et al, 2012). The best studied of these modifications is phosphorylation of CTD serine 5 (S5-P) and serine 2 (S2-P), which occurs primarily at the initiation and elongation stages, respectively, of the transcription cycle. S5 phosphorylation is catalyzed by the TFIIH-associated kinase Kin28 and promotes recruitment of mRNA-capping enzyme and the nuclear cap-binding complex to nascent transcripts. Subsequently, the cyclin-dependent kinase Ctk1 (pTEFb or CDK9 in metazoans) phosphorylates S2, which couples the later stages of transcriptional elongation to mRNA 3′-end processing (Ahn et al, 2004). In addition, the Bur1 kinase phosphorylates S2 at the 5′-end of genes and stimulates Ctk1 activity (Murray et al, 2001). These CTD phosphorylations are also associated with cotranscriptional histone modifications and chromatin remodeling (Srivastava & Ahn, 2015). CTD modifications control the recruitment of regulatory factors to chromatin, and reciprocally, histone modifications modulate CTD-cofactor associations. For instance, S5-P promotes cotranscriptional recruitment, via the PAF elongation complex, of the Rad6 E2 enzyme responsible for H2B ubiquitylation and the H3K4 methyltransferase complex (SET1/COMPASS; Ng et al, 2003; Wood et al, 2003; Xiao et al, 2005). In another example, deletion of CTK1 prevents H3K36 trimethylation by inhibiting the recruitment of the Set2 H3K36 methyltransferase (Wyce et al, 2007). Here, we report that Ulp2 is preferentially recruited to actively transcribed genes, and its loss impedes association of RNAPII, resulting in decreased gene expression. Whereas the N-terminal domain of Ulp2 is sufficient to promote Ulp2 chromatin localization, its C-terminal domain is important for efficient transcriptional elongation and Ulp2 interaction with nucleosomes. A genetic screen revealed a synthetic lethal interaction between mutations in ULP2 and the RAD6- and BRE1-encoded H2B ubiquitylation enzymes. Notably, histone sumoylation and chromatin localization of Ulp2 both depend on H2B ubiquitylation, and this Ulp2 localization is required for subsequent histone desumoylation. Persistent polySUMO conjugation to H2B or loss of ULP2 inhibits recruitment of the Ctk1 kinase to constitutively active genes, limiting CTD-S2 phosphorylation. A similar block to Ctk1 recruitment correlates with the persistent H2B ubiquitylation observed in cells lacking the Ubp8 ubiquitin protease. Taken together, these data suggest that Ulp2 acts as a general transcription factor to control RNAPII recruitment and SUMO levels, thereby promoting gene transcription. Furthermore, our results indicate a new crosstalk pathway in which ubiquitin and SUMO are sequentially conjugated to histones during the transcription cycle. These transient histone modifications are coupled with Ctk1-mediated RNAPII S2-P modification, facilitating later transcription elongation steps. The Ulp2 SUMO protease therefore appears to promote transcription by coordinating histone-SUMO and RNAPII CTD-S2-P modifications. Results Ulp2 localizes to actively transcribed genes Although there have been attempts to explore the relationship between Ulp2 and transcription, these studies included no evidence for direct involvement of the SUMO protease in gene expression. To investigate this further, we first grouped previously published RNA-seq data comparing wild-type (WT) and ulp2Δ strains (Ryu et al, 2018) into three categories: high, medium, or low expression levels (Fig 1A and B). Because expression of RPs is a well-known target of the SUMO pathway (Chymkowitch et al, 2015a; Ryu et al, 2018), we excluded from our re-analysis the 130 RP genes with significantly changed values in ulp2Δ strains. We found that loss of ULP2 impaired transcription of 91.1% of highly expressed genes (Fig 1B). Also, 69.9% of genes with moderate expression were significantly down-regulated in ulp2Δ cells. Expression levels of three representative genes—PMA1, ADH1, and PYK1—that have relatively high constitutive transcription rates were measured by quantitative RT–PCR (qRT–PCR) in cells lacking ULP2 (Fig 1C). We confirmed down-regulation of their expression in the ulp2Δ strain. Therefore, these results suggest that loss of Ulp2 preferentially diminishes transcription of relatively highly expressed genes. Figure 1. Ulp2 is recruited to actively transcribed genes Transcriptome re-analysis of ulp2Δ strains compared with WT (MHY1379) from previous RNA-seq experiments (Ryu et al, 2018). Two-dimensional agglomerative hierarchical clustering shows 2,903 significantly up- or down-regulated genes (P < 0.05) in triplicate RNA samples. Red and blue indicate up- and down-regulation of genes, respectively, and their intensity represents the relative gene expression changes. Summary of transcriptome data in (A). White and gray colors indicate up- and down-regulated genes, respectively, in ulp2Δ cells. The pie graph shows the percentages of significantly changed genes, except for 130 ribosomal protein genes (RPs), and are classified by FPKM (Fragments Per Kilobase of Million reads mapped) values in the bar graph. qRT–PCR analysis of the highly transcribed PMA1, ADH1, and PYK1 genes in ulp2Δ cells. Expression was measured relative to WT cells, and data were normalized to SPT15 expression. Error bars indicate the standard deviations (SDs) from three independent RNA preparations. FPKMs of each gene in WT are shown in the bottom graph. Schematic diagram of PMA1, ADH1, PYK1, and CUP1 genes. The TATA/promoter (Pro) and open reading frame (ORF) are represented by black and white boxes, respectively. Bars with numbers below the genes show the relative positions of the PCR products used in the ChIP and qRT–PCR analyses and are used for identification in all later figures. ChIP analysis using IgG-Sepharose or anti-Flag-agarose beads in strains expressing TAP-tagged Paf1, Ubc9, or Ulp1 or Flag-tagged Ulp2. An untagged strain (MHY500) was used as a negative control for immunoprecipitation of Ulp2-Flag (Fig EV1A). The qPCR signals of the indicated genes were quantitated and normalized to an internal background control and the input DNA. The primer pairs used are indicated in (D). Quantification (described in Materials and Methods) presented as fold over background; a value of 1 indicates no signal detected above background signal at a nontranscribed locus, as marked with the horizontal line. Error bars indicate SDs calculated from three independent chromatin preparations. Occupancy of Rpb3 and Ulp2 at CUP1 gene was determined by ChIP in a strain expressing Flag-tagged Ulp2 using anti-Rpb3 antibody and anti-Flag agarose beads as shown in (E). “Mock” indicates use of protein G beads without added antibody. For CUP1 induction, cells were harvested at the indicated time points after adding CuSO4. “Pro” and “ORF” represent the positions of PCR fragments 1 and 2 from the CUP1 gene as described in (D). Black bar indicates a value of 1, the background signal. Error bars, SD from four independent experiments. A percentage graph of association of Ulp2-Flag with genes classified by FPKM in the file Dataset EV1. RPs and ND indicate ribosomal protein genes and genes not detected genes in our earlier RNA-seq experiments, respectively. The numbers above the graph indicate the total number of yeast genes in each expression category. Average plot of Ulp2-Flag occupancy at transcribed genes. The values of the y-axis indicate rescaled fold enrichment, setting the maximum occupancy to 1, and the minimum occupancy to 0, and the x-axis indicates normalized distance from transcription start sites (TSS) and transcription end sites (TES). The dotted lines indicate the TSS and TES. The ChIP-seq data were obtained from duplicate experiments. Data information: Asterisks indicate statistically significant differences of Paf1-TAP and Ubc9-TAP with Ulp1-TAP and Ulp2-Flag with no tag (Fig EV1A) in (E) and significant differences between uninduced and induced cells in (F) using a two-tailed Student's t-test (*P < 0.05; **P < 0.01). See Dataset EV2 for qPCR raw data. Download figure Download PowerPoint To determine whether the Ulp2 enzyme is recruited to such genes, chromatin immunoprecipitation (ChIP) analysis was carried out on PMA1, ADH1, and PYK1 (Fig 1D and E). We used Paf1, a subunit of the PAF complex, as a positive control because it is known to associate strongly with gene coding regions (Kim et al, 2004). ChIP results with the SUMO-conjugating enzyme Ubc9 indicated weak association with both promoter and open reading frame (ORF) sequences in these genes, while the Ulp1 SUMO protease was not significantly enriched at these sites. Interestingly, Flag-tagged Ulp2 occupied the length of these three test genes, with substantially higher association within the ORF in PMA1 and PYK1. These results suggest SUMO-conjugating (Ubc9) and SUMO-deconjugating (Ulp2) enzymes contribute directly to both transcription initiation and elongation. The CUP1 gene was then examined to determine whether Ulp2 is recruited to genes in a transcription-dependent manner. CUP1 encodes the copper-binding metallothionein protein (Hottiger et al, 1994). Its transcription is induced 10- to 20-fold within 5 min of addition of 1 mM copper, and then it down-regulates its own expression, resulting in a return to basal transcript levels by ~30 min of sustained copper exposure (Hamer et al, 1985; Pena et al, 1998). Unlike mock immunoprecipitation with only protein G beads (Fig 1F, top), we observed that occupancy by Rpb3, a subunit of RNAPII, at promoter and ORF regions of CUP1 increased by ~30-fold and ~120-fold, respectively, within 5 min of exposure to copper and subsequently dropped to uninduced levels by 15–30 min (Fig 1F, middle). Ulp2 also showed a strong and rapid increase in association with CUP1, increasing ~15-fold at the ORF region within 5 min of copper addition, and then decreased in parallel with the drop in RNAPII association (Fig 1F, bottom). To map Ulp2 binding sites on a genome-wide scale, we carried out ChIP-seq experiments (Fig 1G) and compared genome-wide association of Ulp2 with the RNA-seq data in Fig 1B. Consistent with our previous report (Ryu et al, 2018), we found that Ulp2 was enriched at most ribosomal protein gene loci. Furthermore, we observed a greater percentage of genes that were highly expressed associated with Ulp2, consistent with a potential role for Ulp2 in transcription. When Ulp2 binding sites derived from the ChIP-seq data were mapped relative to transcribed regions throughout the genome (from transcription start site to transcription end site), we saw a clear enrichment in these regions (Fig 1H), consistent with the results in Figs 1E and EV1B. Although Ulp2 has previously been shown to associate specifically with rDNA, RP, and snoRNA genes (Liang et al, 2017; Ryu et al, 2018), these observations provide the first direct evidence for the transcription-linked association of Ulp2 with actively transcribed genes in vivo. Click here to expand this figure. Figure EV1. Genome-wide analysis of Ulp2-Flag binding sites in chromatin ChIP analysis using anti-Flag agarose in an untagged strain (MHY500), as described in Fig 1E. Black bar indicates value of 1, which is the background signal. Error bars indicate SDs calculated from three independent experiments. See Dataset EV2 for qPCR raw data. Representative data from ChIP-seq analysis of Ulp2-Flag. The y-axis shows fold enrichment normalized to the input DNA. Arrows and boxes with gene names indicate locations of ORFs. FPKMs of each gene in WT are shown in the bottom graph. Gene Ontology (GO) enrichment analysis of the genes displayed in Fig 1G. Bar diagrams indicate the fold enrichment of categories of biological process to the genome using GO data from PANTHER. The genes used in GO biological process are listed in the file Dataset EV1. Download figure Download PowerPoint Ulp2 is involved in transcription elongation Our RNA-seq, ChIP, and ChIP-seq analyses in Fig 1 demonstrated that Ulp2-chromatin association correlates with transcriptional activation. To explore further how Ulp2 might participate in transcription, we first tested the sensitivity of ulp2Δ cells to 6-azauracil (6-AU), a general indicator for involvement in transcription elongation (Conaway et al, 2000). Cells lacking RAD6 were used as a positive control for 6-AU sensitivity (Xiao et al, 2005). On the control SD-Ura plate, the ubc9ts and ulp1ts mutants showed modest growth defects at 30°C, whereas cell growth was more strongly impaired in the ulp2Δ strain (Fig 2A, upper panels), consistent with previous reports (Li & Hochstrasser, 2000). Notably, we observed an extremely strong sensitivity to 6-AU in ubc9ts but not ulp1ts cells. Because the ubc9 mutant reduces RNAPII occupancy and association of SUMO with constitutive genes (Rosonina et al, 2010), these defects may sensitize cells to 6-AU. By contrast, our ChIP results in Fig 1E and previous microscopic examination (Li & Hochstrasser, 2003) did not reveal any evidence of chromatin binding by Ulp1 in vivo, and ulp1ts cells grew well on 6-AU plates (Fig 2A), suggesting Ulp1 is not directly involved in the transcription elongation process. Figure 2. Ulp2 promotes transcription elongation and RNAPII localization A. Sensitivity of the indicated mutants to 6-AU. All strains carried a URA3 plasmid, pRS316, and were spotted on SD-Ura with or without 6-AU (100 μg/ml); plates were incubated for 2-4 days at 30°C. ULP2 and ulp2(C624A) represent ulp2Δ::HIS3 cells containing either pRS314-ULP2-FLAG or pRS314-ulp2(C624A)-FLAG. B, C. ChIP analyses using anti-Rpb3 (B) and anti-SUMO (C) antibodies in ulp2Δ cells as in Fig 1F. Error bars indicate the SD from three independent experiments. D. qRT–PCR analysis of CUP1 mRNA levels in ulp2Δ cells. Data were normalized to ACT1 mRNA levels. CUP1 gene induction was performed as in Fig 1F. Error bars represent the SD from three RNA samples. E, F. ChIP assays using anti-Rpb3 (E) and anti-SUMO (F) antibodies in ulp2Δ cells as done in Fig 1E. “Pro” denotes the #1 PCR product of PMA1, ADH1, and PYK1 genes described in Fig 1D, while “ORF” indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from four (E) and three (F) independent assays. Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t-test (*P < 0.05; **P < 0.01). See Dataset EV2 for qPCR raw data. Download figure Download PowerPoint Cells lacking ULP2 also showed increased sensitivity to 6-AU. Because both SUMO-conjugating and SUMO-deconjugating enzymes had clear defects in cell growth on 6-AU plates, our data suggest that properly maintained levels of SUMO conjugation at actively transcribed genes are required for efficient transcription elongation. To determine whether Ulp2 catalytic activity is required for this, the same 6-AU plate assay was employed in ulp2Δ cells carrying plasmids encoding either WT ULP2 or the inactive ulp2-C624A mutant, in which the catalytic Cys residue is replaced with Ala (Li & Hochstrasser, 2000; Fig 2A, bottom panels). As with ulp2Δ cells, ulp2-C624A cells could barely grow on 6-AU, suggesting that the SUMO protease activity of Ulp2 is crucial for regulating transcriptional elongation. Ulp2 controls RNAPII and SUMO occupancy during transcription Since our data indicated that Ulp2-chromatin binding correlates closely with transcription levels, we monitored recruitment of RNAPII and SUMO during CUP1 gene activation in ulp2Δ cells (Fig 2B and C). A dramatic increase of Rpb3 recruitment is observed in WT cells at 5–10 min following CuSO4 addition. By contrast, Rpb3 binding to the promoter and ORF of CUP1 was greatly reduced by loss of ULP2 (Fig 2B). Concomitantly, the deletion of ULP2 led to a rapid and abnormally high accumulation of SUMO at both the promoter and ORF within 5 min of induction (Fig 2C). SUMO levels gradually dropped with continuous exposure to copper. Since a change in SUMO levels at CUP1 was observed in ulp2Δ but not WT cells, local sumoylation must normally be tightly regulated by Ulp2 protease during transcription. In line with the Rpb3 ChIP results, levels of CUP1 mRNA increased strikingly at 10 min following the addition of copper to WT cells, but this was strongly attenuated in ulp2Δ cells (Fig 2D). Similarly, Rpb3 occupancy was reduced and, conversely, SUMO levels were enhanced in ulp2Δ at the constitutive test genes PMA1, ADH1, and PYK1 (Fig 2E and F). Taken together, the results indicate that localization of RNAPII and SUMO is tightly regulated by Ulp2 on transcribed genes with significant impact on transcription. The C-terminal domain of Ulp2 is important for transcription elongation We previously found that Ulp2 has a poorly conserved N-terminal domain (NTD) adjacent to the catalytic domain, but the NTD is nonetheless necessary and sufficient for nuclear localization and is required for most Ulp2 functions (Kroetz et al, 2009). By contrast, deletion of the C-terminal domain (CTD) has more modest effects on cell growth, and the C-terminally truncated protein still localizes to the nucleus. To determine the potential contribution of the NTD and CTD of Ulp2 to transcription elongation, we examined the 6-AU sensitivity of the ULP2 strain and mutants expressing Ulp2ΔN, Ulp2ΔC, and the NTD of Ulp2, all from the native chromosomal locus (Fig 3A). Expression of the Ulp2 derivatives was undiminished relative to full-length Ulp2 (Fig 3B). The ulp2ΔN strain and cells expressing just the NTD showed severe growth defects on SD-Ura, and they were almost inviable on the plate with 6-AU, suggesting that both nuclear localization and the catalytic domain of Ulp2 are likely required to overcome the stress caused by exposure to this drug. Remarkably, whereas ulp2ΔC cells grew only slightly slower than congenic WT cells on SD-Ura, they displayed very strong sensitivity to 6-AU. The CTD of Ulp2 was known to be required for efficient depolymerization of large polySUMO conjugates (Kroetz et al, 2009) and for nucleolar substrate targeting (de Albuquerque et al, 2018); these new data reveal a potential function of the CTD in transcription elongation. Figure 3. Ulp2 C-terminal domain is required for efficient association with nucleosomes Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed as in Fig 2A. The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD). Immunoblot assay of the Ulp2-Myc derivatives expressed in the strains used in (A). ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes used in Fig 2E. W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives—specifically, full-length, NTD (1-403), and CTD (667–1,034)—were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments. Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains from (A) transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting
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