Article23 April 2019Open Access Source DataTransparent process Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance Markus Höpfler Markus Höpfler orcid.org/0000-0002-2129-2220 Max Planck Institute of Biochemistry, Molecular Cell Biology, Martinsried, Germany Search for more papers by this author Maximilian J Kern Maximilian J Kern Max Planck Institute of Biochemistry, Molecular Cell Biology, Martinsried, Germany Search for more papers by this author Tobias Straub Tobias Straub orcid.org/0000-0002-0547-0453 Biomedizinisches Centrum, Core Facility Bioinformatics, Ludwig-Maximilians-Universität München, Martinsried, Germany Search for more papers by this author Roman Prytuliak Roman Prytuliak Max Planck Institute of Biochemistry, Computational Biology Group, Martinsried, Germany Search for more papers by this author Bianca H Habermann Bianca H Habermann orcid.org/0000-0002-2457-7504 Max Planck Institute of Biochemistry, Computational Biology Group, Martinsried, Germany Aix-Marseille Univ, CNRS, IBDM UMR 7288, Marseille Cedex 9, France Search for more papers by this author Boris Pfander Corresponding Author Boris Pfander [email protected] orcid.org/0000-0003-2180-5054 Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author Stefan Jentsch Stefan Jentsch Max Planck Institute of Biochemistry, Molecular Cell Biology, Martinsried, GermanyDeceased during the course of this study Search for more papers by this author Markus Höpfler Markus Höpfler orcid.org/0000-0002-2129-2220 Max Planck Institute of Biochemistry, Molecular Cell Biology, Martinsried, Germany Search for more papers by this author Maximilian J Kern Maximilian J Kern Max Planck Institute of Biochemistry, Molecular Cell Biology, Martinsried, Germany Search for more papers by this author Tobias Straub Tobias Straub orcid.org/0000-0002-0547-0453 Biomedizinisches Centrum, Core Facility Bioinformatics, Ludwig-Maximilians-Universität München, Martinsried, Germany Search for more papers by this author Roman Prytuliak Roman Prytuliak Max Planck Institute of Biochemistry, Computational Biology Group, Martinsried, Germany Search for more papers by this author Bianca H Habermann Bianca H Habermann orcid.org/0000-0002-2457-7504 Max Planck Institute of Biochemistry, Computational Biology Group, Martinsried, Germany Aix-Marseille Univ, CNRS, IBDM UMR 7288, Marseille Cedex 9, France Search for more papers by this author Boris Pfander Corresponding Author Boris Pfander [email protected] orcid.org/0000-0003-2180-5054 Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany Search for more papers by this author Stefan Jentsch Stefan Jentsch Max Planck Institute of Biochemistry, Molecular Cell Biology, Martinsried, GermanyDeceased during the course of this study Search for more papers by this author Author Information Markus Höpfler1, Maximilian J Kern1, Tobias Straub2, Roman Prytuliak3, Bianca H Habermann3,4, Boris Pfander *,5 and Stefan Jentsch1 1Max Planck Institute of Biochemistry, Molecular Cell Biology, Martinsried, Germany 2Biomedizinisches Centrum, Core Facility Bioinformatics, Ludwig-Maximilians-Universität München, Martinsried, Germany 3Max Planck Institute of Biochemistry, Computational Biology Group, Martinsried, Germany 4Aix-Marseille Univ, CNRS, IBDM UMR 7288, Marseille Cedex 9, France 5Max Planck Institute of Biochemistry, DNA Replication and Genome Integrity, Martinsried, Germany *Corresponding author. Tel: +49 89 85783050; E-mail: [email protected] The EMBO Journal (2019)38:e100368https://doi.org/10.15252/embj.2018100368 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 Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STUbLs select specific substrates among myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term "ubiquitin hotspots". Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and to be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation. Synopsis SUMO-targeted ubiquitin ligases (STUbLs), involved in nuclear quality control, genome maintenance and transcription, co-localize with ubiquitin to seven distinct genomic loci on yeast chromatin, which are determined by the transcription factor-like protein Euc1 and contribute to stress tolerance. Seven "ubiquitin hotspots" are sites of Cdc48-dependent protein turnover within the yeast genome. The previously uncharacterized protein Ymr111c/Euc1 binds a ubiquitin-hotspot DNA motif and recruits the STUbL Slx5/Slx8. SUMO-dependent and -independent interactions between Euc1 and Slx5 cooperate for a novel bipartite Slx5/Slx8 recruitment mechanism. Euc1 and ubiquitin hotspots are required for cellular stress tolerance when gene expression control and chromatin maintenance by Rpd3-HDACs or SWR1-C are impaired. Introduction SUMO-targeted ubiquitin ligases (STUbLs) modify SUMOylated proteins with ubiquitin and thereby transfer substrates from the SUMO (small ubiquitin-like modifier) to the ubiquitin pathway (Sriramachandran & Dohmen, 2014). To achieve this, STUbLs combine binding to SUMOylated proteins via SUMO-interacting motifs (SIMs) with ubiquitin ligase activity (Prudden et al, 2007; Sun et al, 2007; Uzunova et al, 2007; Xie et al, 2007). Apart from this defining feature, the STUbL enzyme family is highly heterogeneous, as is the regulation of each member, even though functional aspects appear to be conserved (Sriramachandran & Dohmen, 2014). Of note, thousands of proteins are SUMOylated in cells (Hendriks & Vertegaal, 2016), but only a handful of them were shown to be targeted by STUbLs. This raises the question of which features make a protein a STUbL substrate. A hallmark of several STUbL substrates is modification with SUMO chains (polySUMOylation) (Uzunova et al, 2007; Tatham et al, 2008), but it has also been suggested for the human STUbLs RNF4 and Arkadia/RNF111, as well as for Drosophila Degringolade/Dgrn that they might use additional SUMO-independent interactions for substrate recognition (Abed et al, 2011; Groocock et al, 2014; Kuo et al, 2014; Sun et al, 2014; Thomas et al, 2016). However, in case of the prototypical STUbL, budding yeast Slx5/Slx8, no substrate recognition elements have been characterized other than its SUMO-interacting motifs. STUbLs orchestrate many nuclear functions such as, but not limited to, DNA repair, quality control, and transcriptional regulation (Sriramachandran & Dohmen, 2014). Accordingly, most STUbL substrates are nuclear proteins. Human RNF4, for example, targets the PML (promyelocytic leukemia) protein, which is polySUMOylated in nuclear PML bodies upon arsenic exposure (Tatham et al, 2008). Other RNF4 substrates include transcription factors and proteins involved in different DNA repair pathways (for reviews, see Sriramachandran & Dohmen, 2014; Nie & Boddy, 2016). Budding yeast Slx5/Slx8 was initially identified for its role in genome stability as well, which manifests in a synthetic lethal phenotype with the DNA helicase Sgs1 (Mullen et al, 2001). Slx5/Slx8 is involved in the repositioning of DNA lesions to nuclear pore complexes (Nagai et al, 2008; Su et al, 2015; Churikov et al, 2016; Horigome et al, 2016). In line with an additional major function in chromatin maintenance, several DNA-associated proteins have been described as Slx5/Slx8 substrates (Ohkuni et al, 2016; Schweiggert et al, 2016; Thu et al, 2016; Liang et al, 2017), including transcription factors (TFs) such as Mot1 (mutant variant) and Matα2 (Wang & Prelich, 2009; Xie et al, 2010). Interestingly, in the latter case, Matα2 SUMOylation is dispensable for Slx5/Slx8 targeting, but the SIMs of Slx5 and Matα2 DNA binding are required (Xie et al, 2010; Hickey et al, 2018). Matα2 ubiquitylation subsequently facilitates the recruitment of the Cdc48 complex (p97/VCP in mammalian cells) (Wilcox & Laney, 2009), a segregase that can extract ubiquitylated proteins from their local environment, such as chromatin (Rape et al, 2001; Ramadan et al, 2007; Maric et al, 2014; Franz et al, 2016). It emerges that both sequence-specific DNA-binding proteins and other chromatin-associated proteins are STUbL substrates. However, it is still unknown whether STUbLs fulfill a general role in regulating protein turnover at chromatin and to what extent other components of the ubiquitin–proteasome system (UPS), such as Cdc48, are involved. To address these questions, we obtained genome-wide binding profiles of Slx8 and ubiquitylated proteins. Notably, Slx8 localized to surprisingly few genomic sites, and the ubiquitin signal at seven of these sites was Slx5/Slx8-dependent and strongly enriched in cdc48 mutant strains. These data indicate that these "ubiquitin hotspots" are sites of STUbL- and Cdc48-dependent protein turnover on chromatin. Ubiquitin hotspots are bound by the poorly characterized transcription factor-like protein Ymr111c/Euc1, which is modified with SUMO and is a STUbL substrate. Notably, however, deletion of EUC1 does apparently not lead to an abrogation of transcription in the vicinity of ubiquitin hotspots, but rather results in strong genetic interactions with H2A.Z and Rpd3 pathways, which regulate expression of many genes. Euc1 and ubiquitin hotspots are part of an Rpd3S-dependent pathway that is required to cope with cellular stress induced by suboptimal temperature. Moreover, the analysis of the Slx5/Slx8-recruitment mechanism led to the identification of a SUMO-independent substrate-binding domain within Slx5, suggesting a new mode of substrate recognition by Slx5/Slx8. Results Slx5/Slx8 and Cdc48 control seven ubiquitin hotspots across the yeast genome To investigate Slx5/Slx8-catalyzed ubiquitylation of chromatin-associated proteins, we developed chromatin immunoprecipitation (ChIP) protocols for ubiquitylated proteins (Appendix Fig S1A) and Slx5/Slx8 in Saccharomyces cerevisiae. We used the FK2 ubiquitin antibody with broad specificity toward mono-ubiquitylated proteins and K29-, K48-, and K63-linked chain types combined with genome-wide tiling arrays (ChIP-chip, Fig 1A). We detected ubiquitin signals at open reading frames (ORFs). These signals appear to represent histone H2B mono-ubiquitylation that has been described to be particularly abundant on highly transcribed genes (Braun & Madhani, 2012), as they were either lost in cells that harbor a mutation of the main H2B ubiquitylation site (h2b-K123R) or in cells that lack Rad6, the E2 enzyme for H2B ubiquitylation (Jentsch et al, 1987; Robzyk et al, 2000) (Fig 1A and Appendix Fig S1B, Dataset EV1). However, some ubiquitin signals were H2B ubiquitylation-independent and notably enhanced in cdc48 mutants (cdc48-6, Fig 1A, Dataset EV1). Figure 1. The SUMO-targeted ubiquitin ligase Slx5/Slx8 is required for the formation of seven ubiquitin hotspots across the yeast genome Genome-wide ubiquitin binding profiles identify numerous regions of histone H2B ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ("ubiquitin hotspot", ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig S1A), and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semi-permissive temperature for ts-alleles) unless stated otherwise. Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates. Schematic representation of the 16 yeast chromosomes (I-XVI) with positions of the ub-HSs marked by red triangles. Vertical bars indicate positions of centromeres. Slx8 and Slx5 are recruited to ub-HSs. DNA from Slx8-9myc (top) or 3HA-Slx5 (bottom) ChIP experiments of the indicated strains was analyzed by quantitative real-time PCR (ChIP-qPCR) at selected ub-HSs. Data represent mean ± standard deviation (SD, n = 2). Consistent with a previous study, we did not observe any Slx8 enrichment at centromeres (van de Pasch et al, 2013); however, we note that we could not confirm the reported centromere binding of Slx5 (see also Appendix Fig S2E). K48-linked ubiquitin chains accumulate in a cdc48 mutant (cdc48-3). Ub-K48 ChIP followed by qPCR for the same loci as in (D) is depicted. See also Fig EV1C and D. Data represent means ± SD (n = 2). Slx5 and Slx8 are required for ub-HS formation. Ub-K48 ChIP-qPCR in strains lacking one subunit of the Slx5/Slx8 complex (slx5∆, slx8∆). Data represent means ± SD (n = 3). Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub-HS sites. Ub-K48 ChIP for the indicated strains grown at the semi-permissive temperature of 30°C. Data represent means ± SD (n = 2). Data information: All ChIP-qPCR data represent means ± SD from 2 to 5 independent experiments as indicated, with quantification in triplicates. Data were normalized to an unrelated control region on ChrII (see Materials and Methods). Download figure Download PowerPoint In similar experiments, Slx8 bound specifically to only few sites in the genome (Slx8-9myc, Fig 1B, Dataset EV2). Comparison of our genome-wide profiles of regions enriched for both ubiquitin- and Slx8-binding revealed a striking correlation for seven sites, which we term "ubiquitin hotspots" (ub-hotspots, ub-HS in figures, Figs 1B and C, and EV1A and B, Datasets EV1, EV2 and EV3). Besides these seven "ubiquitin hotspots", we detected only two sites of major ubiquitin accumulation in cdc48 mutants without Slx8 enrichment (ub-only-sites), and two distinct sites of major Slx8 enrichment without ubiquitin accumulation (Datasets EV1 and EV2, see also Appendix Fig S2E). Click here to expand this figure. Figure EV1. Related to Figs 1 and 2. Seven ubiquitin hotspots across the yeast genome share a Cdc48-dependent extraction mechanism and recruit Ymr111c/Euc1 A. The FK2 ubiquitin antibody and a ub-K48 specific antibody detect the same ub-HSs in genome-wide ChIP-chip experiments. 16-kb windows from genome-wide ChIP-chip data using either the ubiquitin (FK2) or ub-K48 (clone Apu2) antibodies are shown. For comparison, data for ubiquitin (FK2) ChIP in WT, cdc48-6, cdc48-3 are reproduced from Fig 1B and for h2b-K123R partially (ub-HS4) from Fig 1A. Data represent means from two independent replicates. B. Table summarizing the ub-HSs identified in Fig 1B. Stretches were defined by significantly enriched regions in ub-K48 ChIP-chip in cdc48-6 (all except ub-HS2) or Slx8-9myc-enriched regions in the cdc48-3 background (ub-HS2). C, D. Ubiquitin signals increase around 5–10-fold in both cdc48-6 and cdc48-3 mutants with ubiquitin (FK2) and ub-K48-chain-specific antibodies. ChIP-qPCR experiment using ubiquitin (FK2) (C) and ub-K48 (D) antibodies and indicated strains. Data represent means ± SD (n = 3 for (C), n = 4 for (D)). E. ufd1-2, ubx4∆, and ubx5∆ show an increase of ubiquitin conjugates at all ub-HSs similar to cdc48-6. Genome-wide ChIP-chip data using the ub-K48 or a non-specific IgG control antibody for the indicated strains. Data for WT and cdc48-6 reproduced from (A) for comparison. Data represent means from two independent replicates. F. A single point mutation within the ub-HS-motif abolishes ubiquitin enrichment. A G>T mutation was introduced in one of the conserved TTGTT repeats of ub-HS4 F7 (bottom scheme) and integrated at the LEU2 locus as described in Fig 2A. ChIP-qPCR for ub-K48 demonstrated that the ubiquitin enrichment is lost upon mutation of the ub-HS-motif (ub-HS4 F7-mut). Experiments were performed in cdc48-6 strains. Data represent means ± SD (n = 5). G. Table summarizing the confirmed hits from two independent Y1H screens as described in Fig 2D. Protein start sites are indicated. aa: amino acid, N.D.: not determined. H. Endogenous Euc1 does not bind the mutated ub-HS4-motif. ChIP with an Euc1-specific antibody was performed in strains with ub-HS4 F7 or ub-HS4 F7-mut integrated at the LEU2-locus as described in (F). Experiments were performed in cdc48-6 strains. Data represent means ± SD (n = 2). Download figure Download PowerPoint Next, we determined the accumulation of specific ubiquitin chain types at ub-hotspots. Using a ubiquitin K48 chain-specific antibody (clone Apu2, "ub-K48"), we detected the same ub-hotspots as with the FK2 antibody (Fig EV1A, Dataset EV3). Moreover, strains harboring different mutant alleles of the CDC48 gene showed an increase of ubiquitin-ChIP signals from around 5–10-fold (WT) to 15–50-fold (cdc48-6, cdc48-3) enrichment over background, with comparable results for both antibodies (Fig EV1C and D). Since the ub-K48 antibody was more specific for the Slx8-bound and Cdc48-controlled ub-hotspots, we used this antibody for the rest of our study. Consistent with recruitment of the Slx5/Slx8 heterodimer to chromatin, we found that both subunits were enriched to similar levels at selected ub-hotspots, and observed a moderate increase in Slx5/Slx8 binding in cdc48-3 mutant cells (Fig 1D). While the ubiquitin signals did not correlate with Slx5/Slx8 enrichment levels at the tested sites (compare Fig 1D and E), they dropped to background levels in slx5Δ and slx8Δ cells (Fig 1F), indicating that Slx5/Slx8 is the relevant ubiquitin E3 ligase at these sites. Cdc48 targeting is usually facilitated by cofactors that mediate substrate specificity (Buchberger et al, 2015). Consistent with previous results for other chromatin-bound substrates (Verma et al, 2011), we found that specifically the Cdc48Ufd1-Npl4 complex removes ubiquitylated proteins from ub-hotspots (Figs 1G and EV1E), assisted by additional Ubx4 and Ubx5 cofactors (Fig EV1E and Appendix Fig S1C, Dataset EV3). In contrast, impairment of Cdc48 substrate delivery to the proteasome (rad23∆ dsk2∆; Richly et al, 2005) or proteasome assembly (ump1∆; Ramos et al, 1998) did not cause accumulation of ubiquitin conjugates at ub-hotspots (Appendix Fig S1D). Taken together, our analysis of genome-wide ubiquitin and Slx8 ChIP data reveals seven ubiquitin hotspots that share similar features: (i) strong accumulation of ubiquitin in cdc48 and associated cofactor mutants, (ii) recruitment of Slx5 and Slx8, and (iii) dependence on the functional Slx5/Slx8 dimer for ubiquitylation. A sequence motif within ub-hotspots is bound by Ymr111c/Euc1 Interestingly, all ub-hotspots lie within intergenic regions, do not seem to be associated with any annotated features within the yeast genome, and appear to be distributed among the sixteen yeast chromosomes (Fig 1C). We could also not identify any shared pathway or function of the adjacent genes (Dataset EV4). To investigate whether any sequence features define ub-hotspots, we cloned a 1,038-bp region of ub-HS4 on chromosome XIII (ChrXIII) and inserted it into the LEU2 locus on ChrIII (ectopic ub-HS4, Fig 2A). This fragment was sufficient to drive formation of an ectopic ub-hotspot at the new position (Appendix Fig S2A), suggesting a role for specific DNA sequences. Indeed, we were able to map a minimal 39-bp fragment required for ub-hotspot formation (ub-HS4 F7, Appendix Fig S2A, Fig 2A–C). Figure 2. A sequence motif within ub-hotspots is bound by Ymr111c/Euc1 A. Schematic of the ub-HS-motif mapping strategy. A 1,038-bp stretch of ub-HS4 (blue line) was cloned and integrated at the LEU2 locus (gray) using the integrative YIplac128 vector (green). Initial mapping led to the identification of fragment F1 (Appendix Fig S2A), which was further truncated for fine-mapping ((B), F5–F8). qPCR primers were designed to bind within the YI128 backbone. B. A 39-bp fragment of ub-HS4 is sufficient to drive ectopic ub-HS formation. Fragments of ub-HS4 were integrated ectopically, and ub-K48 ChIP-qPCR was performed for the endogenous ub-HS5 and the ectopic ub-HS4 fragments as depicted in (A). contr.: control, empty YIplac128 vector was integrated at LEU2. Experiments were performed in cdc48-6 background. Data represent means ± SD (n = 2). C. Experimental mapping and bioinformatic prediction identify a similar ub-HS-motif. Comparison between the experimentally mapped ub-HS4 F7 (B) and the consensus motif of all ub-HSs identified by the MEME software. D. A yeast one-hybrid screening strategy to identify proteins binding to the ub-HS-motif. Three copies of the ub-HS4-motif (F7) were cloned upstream of a minimal promoter followed by a HIS3 reporter gene and integrated at the URA3 locus. A yeast cDNA library with N-terminally fused Gal4 activation domain (AD) was used to screen for survival on media lacking histidine and multiple plasmids coding for 4 different genes were recovered (bottom). See also Fig EV1G. E. Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20 mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days. F. Euc1 is required for the formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains, and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4). G, H. Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of K48-linked ubiquitin chains (G) or Euc1 (H) were obtained in ChIP-chip experiments as described in Fig 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292–462. Data represent means from two independent replicates. Download figure Download PowerPoint We also used the MEME suite to predict sequence motifs within the ub-hotspots (Bailey et al, 2009). Consistent with our experimental mapping, a sequence motif of 36 bp was identified, which largely overlapped with the experimentally mapped 39-bp fragment (Fig 2C). This "ub-HS-motif" was found in all the ub-hotspots in at least one copy (Appendix Fig S2B), suggesting that a sequence-specific DNA-binding protein might localize to ub-hotspots. Supporting this notion, a single point mutation within one of the central, conserved TTGTT repeats led to a complete loss of ubiquitin from the ectopic ub-hotspot (ub-HS4 F7-mut, Fig EV1F). To identify proteins binding to the ub-HS-motif, we applied an unbiased yeast one-hybrid (Y1H) screening strategy (Fig 2D). In two independent screens in either a WT or a ubx5Δ strain, which shows enriched ubiquitin at ub-hotspots (Fig EV1E), we identified several clones of four different genes: SMT3 (encoding for SUMO), SLX5, and the uncharacterized YMR111C/EUC1 and YFR006W (Figs 2D and EV1G). Identification of SUMO suggests a SUMOylation event upstream of Slx5/Slx8 recruitment, while isolation of SLX5 clones confirms our ChIP data (Fig 1D). We confirmed the recruitment of Ymr111c/Euc1, SUMO, and Yfr006w with Gal4 activation domain (AD) fusion fragments (Fig 2E and Appendix Fig S2C). Importantly, deletion of YMR111C/EUC1 led to a complete loss of ubiquitin-ChIP signals at ub-hotspots, while deletion of YFR006W had no effect (Fig 2F and G, Dataset EV3). Therefore, we here name YMR111C as "EUC1" (Enriches Ubiquitin on Chromatin 1). Consistent with a key role of Euc1 in the formation of ub-hotspots, we found that activation of the HIS3 reporter by AD-SUMO was Euc1-dependent (Appendix Fig S2C), suggesting that Euc1 binding occurs before SUMO binding or a SUMOylation event. In line with this, Euc1 could not bind the mutated ub-HS4 sequence (Appendix Fig S2D). We raised an antibody against Euc1 to test its association with the endogenous ub-hotspot sites in ChIP-chip experiments (Fig 2H, Dataset EV5). As expected from the Y1H assays, Euc1 strongly accumulated at ub-hotspots in WT but not in euc1∆ cells (Fig 2H), nor at the mutated ectopic ub-HS4 sequence (Fig EV1H). Notably, ub-hotspots are the major sites of Euc1 binding in the entire genome, with only three additional sites of Euc1 accumulation (two of which also enrich Slx8 and contain the ub-HS-motif, Appendix Fig S2E). These data indicate that Euc1 specifically localizes to ub-HS-motif sites and is required for the formation of the ub-hotspots. The transcription factor-like Euc1 shows transactivation in reporter gene assays Euc1 harbors a predicted coiled-coil (CC) domain in its N-terminal part and a GCR1 domain at its C-terminus (Fig 3A). GCR1 domains have been shown to confer sequence-specific DNA binding in Gcr1 and related transcription factors (TFs) (Huie et al, 1992; Hohmann, 2002). A distantly related GCR1 domain protein that also binds DNA is Cbf2, which is part of the CBF3 complex and establishes kinetochore attachment with centromeres (Espelin et al, 2003). Protein structure prediction suggested a myb-like DNA-binding fold within the GCR1 domain (Appendix Fig S3A) (Biedenkapp et al, 1988; Kelley et al, 2015) and our mapping results confirm that the complete GCR1 domain and C-terminus are essential for Euc1 association with the ub-HS-motif (Appendix Fig S3B). Introduction of two point mutations to the predicted DNA-binding loop (W333A, R334A, euc1-DBD*) resulted in complete loss of association with ub-hotspots in Y1H assays (Appendix Fig S3C) and Euc1 ChIP experiments (Appendix Fig S3D and E), suggesting that Euc1 directly binds the ub-HS-motif. Concomitantly, ubiquitin enrichment at ub-hotspots was also lost in euc1-DBD* cells (Appendix Fig S3E). Figure 3. The transcription factor-like Euc1 shows transactivation in reporter gene assays Euc1 domain structure is reminiscent of transcription factors. Top: Schematic representation of Euc1, with a predicted coiled-coil domain (CC) and GCR1 domain indicated. Predicted DNA binding of the C-terminal part was confirmed in Y1H experiments (Appendix Fig S3A–C). Bottom: Euc1-like protein sequences from closely related Saccharomyces species were aligned, and a phylogenetic tree was generated using Clustal Omega. Jalview was used to graphically display the degree of sequence conservation (see also Appendix Fig S4A). The ub-HS-motif is conserved in closely related yeast species. The 7 yeast Multiz Alignment & Conservation tool of the UCSC Genome Browser was used to retrieve alignments of sequences corresponding to ub-HS-motifs from other Saccharomyces species. Dot (.) indicates a conserved base. Euc1 can induce transactivation via its N-terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig 2D, and serial dilutions were spotted on control or selective media to test HIS3 activation. Cells were grown at 30°C for 3 days. Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC-Leu), harvested in logarithmic growth phase, and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT–qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). P = 2.43 × 10−5 (Student's t-test). Download figure Download PowerPoint Phylogenetic analysis revealed EUC1-like genes in several other yeast species, with most pronounced sequence homology in CC- and GCR1 domains (Fig 3A, Appendix Fig S4A). Conversely, ub-hotspot sequences could be identified in those Saccharomyces species, where corresponding intergenic regions could be aligned to S. cerevisiae (Fig 3B, Appendix Fig S4B). We note that residues most conserved in the different hotspot motifs also appeared most highly conserved in related yeasts, hinting at a similar function of Euc1 proteins at these sites. To test whether Euc1 itself could also function as a transcriptional activator, we deleted endogenous EUC1 in the ub-HS4-HIS3 ubx5∆ Y1H reporter strain, which reduced background activation
Abstract The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis and DNA damage repair, and its de-regulation is linked to neuro-muscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. FAM104 proteins bind p97 directly via a novel, alpha-helical motif and associate with the p97- UFD1-NPL4 complex in cells. FAM104 proteins localize to the nucleus and promote both the nuclear import and chromatin binding of p97. Loss of FAM104 proteins results in slow growth and hypersensitivity to p97 inhibition in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.
The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.
Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions. Editor's evaluation This article reports on hitherto unrecognized adaptors of p97/VCP, which is a multifunctional ATPase that unwinds diverse protein substrates subserving important roles in cell physiology. The adaptors in question, members of the FAM104 family, direct p97 to the nucleus to enable unwinding events in that location. The findings, which are supported by solid experimental observations, are valuable and will inform the work of the sizable community that studies various aspects of p97/VCP. https://doi.org/10.7554/eLife.92409.sa0 Decision letter eLife's review process Introduction The abundant, highly conserved AAA+-type ATPase p97 (also known as VCP in mammals and Cdc48 in plants and lower eukaryotes) plays a central role in the maintenance of protein and organelle homeostasis as well as genome integrity (reviewed in Ahlstedt et al., 2022; Franz et al., 2016a; Papadopoulos and Meyer, 2017). The best-characterized function of p97 is the segregation of ubiquitin-modified proteins in various protein quality control pathways for their subsequent degradation by the 26S proteasome (Brandman et al., 2012; Dantuma and Hoppe, 2012; Tanaka et al., 2010; Ye et al., 2001). p97 is also involved in the lysosomal degradation of endocytic cargo (Ritz et al., 2011) and in the selective autophagy of protein aggregates, stress granules, as well as damaged mitochondria and lysosomes (Buchan et al., 2013; Ju et al., 2008; Kim et al., 2013; Papadopoulos et al., 2017; Tanaka et al., 2010; Turakhiya et al., 2018). Moreover, p97 possesses a number of crucial nuclear functions, for instance, in DNA replication and DNA damage repair (Acs et al., 2011; Davis et al., 2012; Franz et al., 2011; Maric et al., 2014; Meerang et al., 2011; Moreno et al., 2014; Mosbech et al., 2012; Mouysset et al., 2008; Raman et al., 2011). Importantly, mutational perturbation of p97 function causes the neuromuscular degenerative disease multisystem proteinopathy 1 (MSP1) (Johnson et al., 2010; Pfeffer et al., 2022; Watts et al., 2004), and several cancers as well as viral and bacterial pathogens rely on p97 activity, making p97 an attractive target for therapeutic intervention (Das and Dudley, 2021; Deshaies, 2014; Humphreys et al., 2009; Huryn et al., 2020). The molecular basis underlying the diverse cellular functions of p97 is the partial or complete unfolding of substrate proteins by threading through the ring-shaped p97 homohexamer in an ATP hydrolysis-driven process (Blythe et al., 2017; Bodnar and Rapoport, 2017; Buchberger, 2022). Since p97 itself lacks appreciable specificity for its physiological substrates, a large number of cofactor proteins control substrate binding, subcellular localization, and oligomeric state of p97 (reviewed in Buchberger et al., 2015). Some of these cofactors bind to p97 in a mutually exclusive manner and define functionally distinct, major p97 complexes: a heterodimer of UFD1 (also known as UFD1L) and NPL4 (also known as NPLOC4) recruits ubiquitylated substrates for p97-dependent unfolding and subsequent proteasomal degradation or processing (Bays et al., 2001; Jarosch et al., 2002; Rape et al., 2001; Ye et al., 2001), whereas UBXN6 (also known as UBXD1) controls p97 functions in lysosomal and autophagic degradation pathways (Papadopoulos et al., 2017; Ritz et al., 2011) and SEP domain-containing cofactors such as p47 (also known as NSFL1C) and UBXN2B (also known as p37) mediate the maturation of protein phosphatase 1 complexes (Weith et al., 2018). At least in the case of p97-UFD1-NPL4, auxiliary cofactors from the UBA-UBX family can fine-tune the subcellular localization and/or substrate specificity of a major p97 complex (Buchberger et al., 2015; Schuberth and Buchberger, 2008). For example, the yeast cofactor Ubx2 recruits Cdc48-Ufd1-Npl4 to the endoplasmic reticulum (ER) and mitochondria to promote ER- and outer mitochondrial membrane-associated protein degradation, respectively (Mårtensson et al., 2019; Neuber et al., 2005; Schuberth and Buchberger, 2005). Similarly, the metazoan cofactors UBXN7 and FAF1 direct p97-UFD1-NPL4 to nuclear chromatin to promote the removal of ubiquitylated substrates such as CDT1 and the CMG helicase (Franz et al., 2016b; Fujisawa et al., 2022; Kochenova et al., 2022; Tarcan et al., 2022). The majority of cofactors interact with p97 through a small number of defined domains/motifs, including the UBX(-like) domain, the PUB and PUL domains, the SHP box, the VCP interacting motif (VIM), and the VCP binding motif (VBM) (reviewed in Buchberger et al., 2015). However, some p97 interactors do not possess any of these canonical binding motifs, suggesting that the current inventory of p97 cofactors is incomplete and, hence, that the full scope of the p97–cofactor network is far from being understood. Examples illustrating this point include ZFAND1 (yeast Cuz1) and GIGYF1/2 (yeast Smy2), which were recently shown to regulate p97 functions in stress granule clearance (Turakhiya et al., 2018) and the transcription stress response (Lehner et al., 2022), respectively. Here, we report the identification of the previously uncharacterized, evolutionarily conserved FAM104 protein family as p97 cofactors. We show that the human FAM104 family members VCF1 and VCF2 bind p97 directly via a non-canonical helical motif and that they associate with different p97 complexes in cells. We demonstrate that VCF1/2 promote the nuclear localization of p97 and that their loss causes impaired growth and hypersensitivity to chemical inhibition of p97. Results FAM104 proteins are a conserved family of p97 interactors In our ongoing efforts to identify p97 binding partners, we isolated multiple clones encoding the hitherto uncharacterized proteins FAM104A (also known as FLJ14775) and FAM104B (also known as FLJ20434, CXorf44) in a yeast two-hybrid screen of a human testis cDNA library (Supplementary file 1). In the following, we will use the new official names VCF1 and VCF2 for human FAM104A and FAM104B, respectively, and the term FAM104 when addressing the entire protein family. VCF1 and 2 are both expressed in various isoforms originating from alternative transcript variants (Figure 1A). For VCF1, the two-hybrid hits matched isoforms 1, 2, and 5, whereas isoforms 3 and 4, which share just the N-terminal 73 residues with isoforms 1/2, were not isolated. For VCF2, the two-hybrid hits matched isoforms 3 and 4/5/6, respectively, which differ by the insertion of a single valine residue after residue 40. Compared to isoform 4, isoforms 5 and 6 of VCF2 possess slightly different N termini. Isoforms 1 and 2, which possess 30 divergent C-terminal residues, and isoform 7, which is truncated after 46 residues, were not isolated. Of note, those isoforms of VCF1 and 2 that were isolated in the two-hybrid screen possess significant sequence homology, suggesting that they are evolutionarily related. Indeed, FAM104-like proteins are present in vertebrates as well as in many invertebrates, including insects, octopuses, and echinodermata, whereas the occurrence of two distinct, VCF1- and VCF2-like homologs appears to be restricted to mammals (Figure 1B; Figure 1—figure supplement 1A). Basically, FAM104 family members possess a predicted mono- or bipartite classical nuclear localization signal (cNLS) at or close to the N terminus and a C-terminal, highly conserved helical region, which are separated by largely unstructured stretches of variable length and lower sequence conservation (Figure 1A and B and Figure 1—figure supplement 1B). Figure 1 with 1 supplement see all Download asset Open asset VCF1/2 bind to p97. (A) Schematic overview of human VCF1 and VCF2 isoforms isolated in a yeast two-hybrid screen. Relevant amino acid residue numbers are shown, and conserved N- and C-terminal sequence motifs are indicated by purple and orange boxes, respectively. Sequence identity outside these boxes is indicated by different shades of gray (light, medium, dark). Internal deletions in VCF1 isoform 2 and VCF2 isoform 4 are indicated by thin lines. (B) Multiple-sequence alignment showing representative members of the FAM104 family. Regions with predicted alpha-helical secondary structure are indicated at the top by 'H'. The most highly conserved residues in the C-terminal sequence motif are boxed in red. Other conserved residues are boxed in black or gray, according to the degree of conservation. Numbers in square brackets indicate the length of insertions. For human VCF1 and VCF2, the sequences of isoforms 2 and 4, respectively, are shown, and numbers in round brackets indicate a 21-residue insertion present in isoforms 1 and 5 of VCF1 and a 1-residue insertion present in isoform 3 of VCF2, respectively. (C) Yeast two-hybrid analysis. Yeast PJ69-4a reporter cells transformed with the indicated combinations of bait (BD-) and prey (AD-) plasmids were spotted onto agar plates containing synthetic complete medium lacking uracil and leucine (control) or uracil, leucine, and histidine (-His). Growth was monitored after 3 d. (D) Glutathione sepharose pulldown assay using wild-type p97 and GST fusions of the indicated VCF1/2 proteins. Binding of p97 was analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. (E, F) Glutathione sepharose pulldown assays as in (D), using the indicated p97 variants and GST fusions of VCF1 isoform 5 (E) and VCF2 isoform 3 (F), respectively. Arrowheads mark the position of the p97 N domain. Figure 1—source data 1 Related to Figure 1D–F. https://cdn.elifesciences.org/articles/92409/elife-92409-fig1-data1-v2.zip Download elife-92409-fig1-data1-v2.zip To validate the results of the two-hybrid screen, we tested isoforms 1, 2, and 5 of VCF1 and isoform 3 of VCF2 in directed yeast two-hybrid assays and confirmed that all four full-length proteins interact with p97 (Figure 1C). Next, we performed glutathione sepharose pulldown experiments with purified GST fusions of these four VCF1/2 proteins and found that they were all able to efficiently bind recombinant p97 (Figure 1D). In order to determine the VCF1/2 binding region of p97, we performed pulldown assays with VCF1 isoform 5 and VCF2 isoform 3 using different truncated variants of p97 (Figure 1E and F). The ND1 variant of p97 lacking the D2 ATPase domain as well as the N domain alone bound efficiently to VCF1 and 2, whereas the ΔN variant lacking the N domain did not. Taken together, these results show that FAM104 proteins are a new family of evolutionarily conserved p97 interactors, and that the N domain is necessary and sufficient for the direct binding of p97 to VCF1/2. FAM104 proteins bind to p97 via their C-terminal helix As VCF1/2 do not possess any of the canonical p97 binding motifs found in other cofactors, we next sought to map their binding site for p97. The shortest VCF1 fragment isolated in the two-hybrid screen starts at glycine residue G131 of isoform 1 (equivalent to G110 and G64 of isoforms 2 and 5, respectively). Since the C-terminal alpha-helical region shows the highest sequence conservation among FAM104 family members (Figure 1B) and is missing in those isoforms of VCF1/2 that were not isolated in the two-hybrid screen, we truncated this region from the C-terminus to test its importance for p97 binding (Figure 2A). While deletion of the four C-terminal residues did not affect the two-hybrid interaction between VCF1 isoform 5 and p97, larger deletions of 7–26 residues completely abolished p97 binding (Figure 2B), even though the truncated proteins were expressed to similar levels in the reporter yeast strain (Figure 2C). Consistent with this, deletion of the C-terminal 26 residues impaired the ability of all four VCF1/2 isoforms to bind p97 in a pulldown experiment (Figure 2D), indicating that the C-terminal region contains the p97 binding site. We therefore tested whether a peptide spanning the conserved alpha-helical and flanking residues (residues C180 to G203 in VCF1 isoform 1) can bind to p97. To that end, we immobilized the N-terminally biotinylated peptide on streptavidin sepharose beads and performed a pulldown assay with p97 (Figure 2E). The peptide pulled down full-length p97 as well as the ND1 and N domain variants, demonstrating that the C-terminal conserved region of FAM104 family proteins is not only necessary, but also sufficient for p97 binding. We also noted some residual binding of the p97 ΔN variant, suggesting that the VCF1-derived peptide has some weak affinity for a p97 region(s) outside the N domain. Figure 2 with 1 supplement see all Download asset Open asset FAM104 proteins bind to p97 via their C-terminal helix. (A) Schematic overview of the C-terminal truncations of VCF1 isoform 5 used for yeast two-hybrid analysis. Labeling as in Figure 1A. (B) Yeast two-hybrid analysis of p97 binding to the C-terminally truncated VCF1 isoform 5 variants shown in (A). (C) Expression levels of the two-hybrid fusion proteins in (B) were analyzed by western blot (WB) using antibodies against p97 and the Gal4 transactivation domain (Gal4-TA). The asterisk in the p97 blot marks a cross-reactivity with endogenous Cdc48. (D) Glutathione sepharose pulldown assay using wild-type p97 and GST fusions of the indicated full-length or C-terminally truncated VCF1/2 proteins. (E) Streptavidin sepharose pulldown assay using the biotinylated peptide CQGLYFHINQTLREAHFHSLQHRG spanning the conserved C-terminal alpha-helix and flanking residues of VCF1 (residues C180–G203 in isoform 1) and the indicated p97 variants. p97 binding to the immobilized peptide was analyzed by SDS-PAGE, followed by Coomassie brilliant blue staining. Arrowheads mark the position of the p97 N domain. (F, G) AlphaFold Multimer model of the C-terminal alpha-helix of VCF1 (turquoise) bound to the N domain of p97. (F) Overview showing binding to the subdomain cleft of the N domain. (G) Close-up view showing the interaction of the four most highly conserved residues with the N domain (green). Residue numbers refer to isoform 1 of VCF1. (H) Glutathione sepharose pulldown assay using wild-type p97 and GST fusions of the indicated full-length (wildtype, NL->AA, NL->RR) or C-terminally truncated (Cdel26) variants of VCF1 isoform 5. NL->AA, N188A/L191A double mutant; NL->RR, N188R/L191R double mutant. Figure 2—source data 1 Related to Figure 2C. https://cdn.elifesciences.org/articles/92409/elife-92409-fig2-data1-v2.zip Download elife-92409-fig2-data1-v2.zip Figure 2—source data 2 Related to Figure 2D, E, and H. https://cdn.elifesciences.org/articles/92409/elife-92409-fig2-data2-v2.zip Download elife-92409-fig2-data2-v2.zip Using AlphaFold Multimer, we next generated a structural model of the C-terminal region of VCF1 bound to the N domain of p97 (Figure 2FG). The central part of the C-terminal region was predicted as an alpha-helix that binds to the subdomain cleft of the p97 N domain (Figure 2F), which is the major binding site for other N domain binding motifs including the UBX(-like) domain and the VIM and VBM linear motifs (Buchberger et al., 2015). Closer inspection of the modeled interface revealed that the four most strongly conserved residues of the helix, Y184, N188, L191, and H195, all contact the N domain (Figure 2G; Figure 1B). The predicted interactions are centered around L191, which occupies a predominantly hydrophobic pocket in the p97 subdomain cleft. Adjacent residues contribute to a hydrogen bonding network with p97, while Y184 is predicted to exhibit an orthogonal pi-stacking with Y138 of p97. To test the importance of this interface, we mutated the two central residues N188 and L191 to either alanine or arginine. We reasoned that the NL->AA double exchange should eliminate key contacts with the N domain, whereas the NL->RR double exchange should introduce steric clashes. Intriguingly, both double mutations abolished the interaction of VCF1 with p97 as efficiently as the deletion of the entire C-terminal region (Figure 2H). Consistent with a conserved essential role of residues N188 and L191 for p97 binding, an AlphaFold Multimer model of the Drosophila melanogaster homologs of VCF1/2 (CG14229) and p97 (TER94) showed a highly similar binding interface with basically identical positions of the equivalent residues N90 and L93 (Figure 2—figure supplement 1). Together, our data identify the C-terminal alpha-helix of FAM104 proteins as a novel p97 binding motif and show that the strictly conserved residues N188 and L191 are of central importance for the interaction. VCF1/2 bind to several p97 complexes in cells To investigate whether FAM104 proteins associate with p97 in cells, we ectopically expressed N-terminally FLAG epitope-tagged VCF1/2 in HEK293T cells and performed anti-FLAG immunoprecipitations (Figure 3A). Endogenous p97 was efficiently co-precipitated with all four wild-type VCF1/2 proteins tested, despite the fact that VCF1 isoform 5 was less and VCF2 isoform 3 was much less well expressed than VCF1 isoforms 1 and 2, and that the levels of soluble p97 in the input were strongly reduced upon ectopic expression of VCF1 isoforms 1 and 2. Importantly, UFD1 and NPL4 were co-precipitated at levels roughly proportional to those of p97, strongly suggesting that VCP1/2 form complexes with p97-UFD1-NPL4. This conclusion is further supported by the finding that VCF1 Cdel26 variants lacking the C-terminal helix failed to co-precipitate not only p97, as expected, but also UFD1 and NPL4. (The expression level of the VCF2 isoform 3 Cdel26 variant was below the detection limit, precluding its analysis in this experiment.) We also detected a robust co-precipitation of the SEP domain cofactor UBXN2B with the wild-type, but not Cdel26 VCF1/2 proteins (Figure 3A and Figure 3—figure supplement 1A and B), indicating the existence of ternary p97-UBXN2B-VCF1/2 complexes and thereby confirming and extending recent data from high-throughput interaction studies (Huttlin et al., 2017; Luck et al., 2020). By contrast, we were unable to confirm the recently reported co-immunoprecipitation of the related SEP domain cofactor p47 with VCF1 (Figure 3—figure supplement 1A; Raman et al., 2015), even though our data do not exclude the possibility of a transient, conditional or weak interaction with p47. Together, our data show that FAM104 proteins can associate via their C-terminal helix with distinct cellular p97 complexes including p97-UFD1-NPL4 and p97-UBXN2B, but not with all p97 complexes. Figure 3 with 1 supplement see all Download asset Open asset VCF1/2 form complexes with p97 and several p97 cofactors in cells. (A) HEK293T cells ectopically expressing the indicated N-terminally FLAG-epitope-tagged wildtype or C-terminally truncated (Cdel26) VCF1/2 proteins were subjected to anti-FLAG immunoprecipitation (IP). Input and IP samples were immunoblotted for the FLAG epitope tag, p97, and the indicated p97 cofactors. The central empty lane had been loaded with a marker. The asterisks label marker bands cross-reactive with the FLAG and FAF1 antibody, respectively. (B) Glutathione sepharose pulldown assay, using the indicated p97 cofactors and GST-VCF1 isoform 1; U-N, UFD1-NPL4. (C) Glutathione sepharose pulldown assay, using FAF1 and GST fusions of the indicated VCF1/2 proteins. Upper panel: 2,2,2-trichloroethanol-stained gel; lower panel: immunoblot with FAF1 antibody. Figure 3—source data 1 Related to Figure 3A. https://cdn.elifesciences.org/articles/92409/elife-92409-fig3-data1-v2.zip Download elife-92409-fig3-data1-v2.zip Figure 3—source data 2 Related to Figure 3B. https://cdn.elifesciences.org/articles/92409/elife-92409-fig3-data2-v2.zip Download elife-92409-fig3-data2-v2.zip Figure 3—source data 3 Related to Figure 3C. https://cdn.elifesciences.org/articles/92409/elife-92409-fig3-data3-v2.zip Download elife-92409-fig3-data3-v2.zip We also determined the impact of ectopically expressing wild-type VCF1/2 on p97–cofactor complexes by immunoprecipitating endogenous p97 (Figure 3—figure supplement 1C). Intriguingly, the strongly expressed VCF1 isoforms 1 and 2 efficiently outcompeted the major cofactors UFD1-NPL4 and p47 as well as high molecular weight ubiquitin species presumably representing polyubiquitylated substrate proteins. By contrast, the more weakly expressed VCF1 isoform 5 and VCF2 isoform 3 did not interfere with binding of UFD1-NPL4 and p47 and even appeared to increase the association of ubiquitylated substrates with p97. These results suggest that FAM104 proteins bind very tightly to p97 and that their effect on cofactor and substrate binding depends on their expression level. Interestingly, FAF1 and UBXN7, two auxiliary cofactors important for nuclear functions of p97-UFD1-NPL4, were also co-precipitated with VCF1/2 (Figure 3A). Whereas FAF1 bound to isoforms 1 and 2 of VCF1, UBXN7 bound to all four VCF1/2 isoforms. Of note, the amounts of co-precipitated FAF1 and UBXN7 did not correlate with those of p97, UFD1, and NPL4, suggesting that the interaction of FAF1 and UBXN7 with VCF1/2 may not strictly depend on the p97-UFD1-NPL4 complex. To directly address this possibility, we performed pulldown experiments with purified p97 cofactors and found that FAF1, but not p47, UFD1-NPL4, or UBXN7, bound to VCF1 isoforms 1 and 2, whereas no binding of FAF1 to VCF1 isoform 5 and VCF2 isoform 3 was detected (Figure 3B and C). These results suggest that the N-terminal extension shared by VCF1 isoforms 1 and 2 mediates a direct interaction with FAF1. In summary, our data indicate a complex interplay of VCF1/2 and other cofactors upon binding to p97 in living cells. Ectopic expression of VCF1/2 increases nuclear p97 levels All FAM104 proteins possess a bona fide cNLS at or close to the N terminus (Figure 1A and B and Figure 1—figure supplement 1B and C). To analyze their subcellular localization, we ectopically expressed N-terminally FLAG epitope-tagged VCF1/2 in HeLa cells and performed confocal immunofluorescence microscopy using anti-FLAG antibodies (Figure 4A). Consistent with the very high score calculated by cNLS mapper (Kosugi et al., 2009), all four VCF1/2 proteins strongly accumulated in the nucleus. Intriguingly, endogenous p97 co-accumulated with VCF1/2 in the nuclei of transfected cells, as evident from the comparison with neighboring non-transfected cells or with the vector control (Figure 4A). We next explored the effect of deleting the C-terminal helix or the cNLS of VCF1 using C-terminally truncated isoforms 1 and 2 as well as cNLS-deleted isoforms 1, 2, and 5, respectively (Figure 4B). The corresponding variants of the other VCF1/2 isoforms under study were poorly expressed below the detection limit of the immunofluorescence experiments, precluding their analysis. The C-terminally truncated VCF1 Cdel26 variants localized to the nucleus but failed to effectuate a significant nuclear accumulation of p97, as expected from the loss of their p97 binding site. The cNLS-deleted VCF1 variants showed a nuclear and cytoplasmic distribution, in accordance with their small size of less than 40 kDa, and a much less pronounced nuclear accumulation of p97 (see Figure 4—figure supplement 1A for higher intensity images of full-length versus cNLS-deleted VCF1 isoform 1). Figure 4 with 1 supplement see all Download asset Open asset Ectopic expression of VCF1/2 increases nuclear p97 levels. (A) HeLa cells ectopically expressing full-length, N-terminally FLAG epitope-tagged VCF isoforms 1, 2, and 5, VCF2 isoform 3, and empty vector control, respectively, were analyzed by confocal immunofluorescence microscopy using antibodies against endogenous p97 and the FLAG epitope. Scale bars, 50 µm. All images were taken with identical acquisition settings and processed identically. (B) HeLa cells ectopically expressing N-terminally FLAG epitope-tagged VCF1 isoforms 1 and 2 lacking the C-terminal conserved helix (Cdel26) or the classical nuclear localization signal (cNLS) (delNLS), and VCF1 isoform 5 lacking the cNLS, were analyzed as in (A). Scale bars, 50 µm. (C) Quantification of the ratio of nuclear to cytoplasmic p97 signals in panels (A) and (B). Except for the vector control where all imaged cells were included (>80 cells per replicate and condition), only transfected cells (as judged by the FLAG channel) were included in the quantification. Shown is the mean ± SD; n = 2 biological replicates with 15–30 transfected cells per replicate and condition; one-way ANOVA. *p<0.05; **p<0.01; ***p<0.001; the differences between the Cdel26 and delNLS constructs and the vector control are all not significant (p>0.87). (D) Quantification of the ratio of nuclear to cytoplasmic FLAG signals in panels (A) and (B) was performed as described in (C). Figure 4—source data 1 Related to Figure 4C and D. https://cdn.elifesciences.org/articles/92409/elife-92409-fig4-data1-v2.zip Download elife-92409-fig4-data1-v2.zip To quantify the microscopy results, we used CellProfiler (McQuin et al., 2018) for segmentation of the images into nuclei and cytoplasm (see Figure 4—figure supplement 1B for an example) and determined the ratio of nuclear to cytoplasmic p97 signal in control-transfected versus VCF1/2-transfected cells (Figure 4C). The ratio increased strongly from 2.08 ± 0.01 for control-transfected cells to up to 15.46 ± 3.21 for cells transfected with wild-type VCF1 isoform 1, reflecting the robust nuclear accumulation of p97 in the presence of VCF1/2. By contrast, no significant nuclear accumulation of p97 was observed for cells transfected with the Cdel26 or the cNLS-deleted VCF1 variants. Quantification of the FLAG signals showed that the wild-type and Cdel26 VCF1/2 proteins strongly accumulated in the nucleus, whereas the cNLS-deleted variants were detected in both cytoplasm and nucleus (Figure 4D). Together, these results suggest that VCF1/2 target p97 via a piggy-back mechanism to the nucleus. Ectopic expression of VCF1 isoforms 1 and 2 induces chromatin binding of p97 As the overexpression of VCF1 isoforms 1 and 2 had caused a noticeable reduction in the p97 input levels in the immunoprecipitation experiments described above (Figure 3A and Figure 3—figure supplement 1A and C), we hypothesized that a subpopulation of p97 may have become insoluble under these conditions. To test this possibility, we performed a biochemical fractionation of HEK293T cells ectopically expressing VCF1 isoform 1 or 2 (Figure 5A). Both isoforms strongly accumulated in the soluble nucleoplasmic and insoluble chromatin fractions and were only weakly detected in the cytoplasmic fraction, in full agreement with the immunofluorescence experiments. Intriguingly, the overexpression of both VCF1 isoforms did not affect the total amount of p97, as judged by the signal in SDS-denatured total cell extracts, but caused a strong increase of p97 in the chromatin fraction at the expense of the cytoplasmic pool. Because the other two VCF1/2 isoforms under study were only weakly expressed, their potential to cause similar effects could not be addressed in these experiments. To confirm that the reduced solubility of p97 is indeed caused by its association with chromatin, we used an alternative fractionation protocol including a benzonase treatment step to solubilize chromatin-bound proteins (Figure 5B). Comparing cells overexpressing VCF1 isoform 1 with control cells, we found that the majority of p97 was released into the solubilized chromatin fraction upon benzonase treatment, similar to VCF1 isoform 1 itself and the chromatin-associated proteins MYC, MCM7, and Ub-H2B. Figure 5 with 1 supplement see all Download asset Open asset Ectopic expression of VCF1 isoforms 1 and 2 promotes the association of p97 with chromatin. (A) HEK293T cells were transfected with plasmids encoding the indicated N-terminally FLAG-epitope-tagged VCF1 proteins or with empty vector (control). Total protein extracts were prepared by direct boiling part of the cells in SDS-PAGE sample buffer (SDS). The remaining cells were processed to cytoplasmic, soluble nuclear (nucleoplasmic), and chromatin fractions as indicated. Tubulin and ubiquitylated histone H2B (Ub-H2B) served as markers for the cytoplasmic and chromatin fractions, respectively, whereas the Coomassie staining of the membrane (total protein stain) served as loading control. (B) Fractionation of lysates from HEK293T cells ectopically expressing VCF1 isoform 1 or control cells using an alternative protocol including benzonase treatment that a
Abstract RNA polymerase II (RNAPII) is the workhorse of eukaryotic transcription and produces messenger RNAs and small nuclear RNAs. Stalling of RNAPII caused by transcription obstacles such as DNA damage threatens functional gene expression and is linked to transcription-coupled DNA repair. To restore transcription, persistently stalled RNAPII can be disassembled and removed from chromatin. This process involves several ubiquitin ligases that have been implicated in RNAPII ubiquitylation and proteasomal degradation. Transcription by RNAPII is heavily controlled by phosphorylation of the C-terminal domain of its largest subunit Rpb1. Here, we show that the elongating form of Rpb1, marked by S2 phosphorylation, is specifically controlled upon UV-induced DNA damage. Regulation of S2-phosphorylated Rpb1 is mediated by SUMOylation, the SUMO-targeted ubiquitin ligase Slx5-Slx8, the Cdc48 segregase as well as the proteasome. Our data suggest an RNAPII control pathway with striking parallels to known disassembly mechanisms acting on defective RNA polymerase III.
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