Decision letter: PIE-1 SUMOylation promotes germline fates and piRNA-dependent silencing in C. elegans (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Germlines shape and balance heredity, integrating and regulating information from both parental and foreign sources. Insights into how germlines handle information have come from the study of factors that specify or maintain the germline fate. In early Caenorhabditis elegans embryos, the CCCH zinc finger protein PIE-1 localizes to the germline where it prevents somatic differentiation programs. Here, we show that PIE-1 also functions in the meiotic ovary where it becomes SUMOylated and engages the small ubiquitin-like modifier (SUMO)-conjugating machinery. Using whole-SUMO-proteome mass spectrometry, we identify HDAC SUMOylation as a target of PIE-1. Our analyses of genetic interactions between pie-1 and SUMO pathway mutants suggest that PIE-1 engages the SUMO machinery both to preserve the germline fate in the embryo and to promote Argonaute-mediated surveillance in the adult germline. Introduction During every life cycle, the eukaryotic germline orchestrates a remarkable set of informational tasks that shape heredity and create variation necessary for the evolution of new species. One approach for understanding the mechanisms that promote germline specification and function has been the identification of genes whose protein products localize exclusively to the germline and for which loss-of-function mutations result in absent or non-functional germ cells and gametes (Seydoux and Braun, 2006). In Caenorhabditis elegans, PIE-1 is a key regulator of germline specification (Mello et al., 1992). The C. elegans zygote, P0, undergoes a series of asymmetric divisions that generate four somatic founder cells and the germline blastomere P4. The PIE-1 protein is maternally deposited and uniformly present in the cytoplasm and nucleus of the zygote, but rapidly disappears in each somatic blastomere shortly after division (Mello et al., 1996; Reese et al., 2000; Tenenhaus et al., 1998). In pie-1 mutants, the germline lineage differentiates into extra intestinal cells causing an embryonic arrest (Mello et al., 1992). PIE-1 localizes prominently in nuclei in the early P-lineage blastomeres and persists in the primordial embryonic germ cells through much of embryogenesis (Mello et al., 1996). The presence of PIE-1 correlates with global hypo-phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II) in germline blastomeres (Seydoux and Dunn, 1997), and some studies suggest that PIE-1 may directly inhibit the CTD kinase to prevent transcriptional activation (Batchelder et al., 1999; Ghosh and Seydoux, 2008). PIE-1 is a member of the tandem CCCH zinc finger protein family (Blackshear et al., 2005). PIE-1 differs from most of its homologs in having a prominent nuclear localization. However, like the majority of its family members, PIE-1 also localizes in the cytoplasm where it is thought to bind and regulate the expression of germline mRNAs, including the nos-2 mRNA (Tenenhaus et al., 2001). Hints at the nuclear function of PIE-1 came from a yeast two-hybrid screen which identified the Krüppel-type zinc finger protein MEP-1 as a PIE-1 interacting factor (Unhavaithaya et al., 2002). MEP-1 co-purifies with LET-418, a homolog of mammalian ATP-dependent nucleosome remodeling factor Mi-2 (von Zelewsky et al., 2000), and with HDA-1, a homolog of mammalian histone deacetylase HDAC1 (Shi and Mello, 1998). Inactivation of maternal mep-1 and let-418 causes a striking developmental arrest of L1-stage larvae, whose somatic cells adopt germline-specific transcriptional programs, and assemble germline-specific peri-nuclear nuage-like structures called P granules (Unhavaithaya et al., 2002). These soma-to-germline transformations depend on the trithorax-related protein MES-4 and components of a polycomb repressive complex (PRC2) (MES-2 and MES-3) (Unhavaithaya et al., 2002), whose functions are thought to promote fertility by maintaining germline chromatin (Strome and Updike, 2015). Taken together, these previous studies on PIE-1 suggest that it functions as a master-regulator of the germline fate in C. elegans embryos, preventing somatic differentiation, while also protecting the germline chromatin from remodeling. However, the possible biochemical mechanisms through which this small CCCH zinc finger protein exerts its dual effects on transcription and chromatin in the germline were entirely unknown. Here, we show PIE-1 promotes the regulation of its targets at least in part through the small ubiquitin-like modifier (SUMO). By yeast two-hybrid screening, we show that PIE-1 engages the highly conserved E2 SUMO ligase UBC-9. UBC-9 enzymes catalyze the addition SUMO to lysine residues on target proteins (Capili and Lima, 2007b; Geiss-Friedlander and Melchior, 2007; Johnson, 2004). The reversible addition of SUMO (or SUMOylation) and its removal by de-SUMOylating enzymes is thought to occur on thousands of substrate proteins with diverse functions, especially nuclear functions including DNA replication, chromatin silencing, and the DNA damage response (Hendriks and Vertegaal, 2016). SUMOylation can have multiple effects and is not primarily associated with the turnover of its targets, but rather is often associated with changes in protein interactions. For example, SUMOylation of a protein can promote interactions with proteins that contain SUMO-interacting motifs (Matunis et al., 2006; Psakhye and Jentsch, 2012; Shen et al., 2006). Through a series of genetic and biochemical studies, we show that the SUMO pathway promotes the activity of PIE-1 in preserving the embryonic germline. We show that PIE-1 is itself modified by SUMO. Paradoxically, PIE-1 is not SUMOylated in early embryos, but rather in adult animals where PIE-1 was not previously known to be expressed or functional. Indeed, CRISPR-mediated GFP tagging of the endogenous pie-1 locus confirmed uniform nuclear expression of PIE-1 protein throughout the meiotic zone and in oocytes of adult hermaphrodites. Using whole proteome analysis for detecting SUMO-conjugated proteins, we identify the type 1 HDAC, HDA-1, as a protein modified by SUMO in a PIE-1-dependent manner. Surprisingly, whereas PIE-1 was originally thought to inhibit the MEP-1/Mi-2/HDA-1 complex in embryos, we show that in the germline PIE-1 acts in concert with the SUMO pathway to promote the association of MEP-1 with HDA-1, and to maintain the hypoacetylation of germline chromatin. Although pie-1 lysine 68 mutants that prevent PIE-1 SUMOylation are viable, we show that they exhibit synthetic lethality in combination with null alleles of gei-17, a Drosophila Su(var)2–10 SUMO-E3 ligase homolog (Hari et al., 2001; Mohr and Boswell, 1999; Ninova et al., 2020), and that double mutants exhibit potent desilencing of a piRNA Argonaute sensor. Our findings are consistent with a model in which PIE-1 engages SUMO to preserve the embryonic germline fate, and also to promote the assembly of a MEP-1/Mi-2/HDA-1 chromatin remodeling complex required for inherited Argonaute-mediated gene silencing in the adult hermaphrodite germline. Results PIE-1 is SUMOylated in adult germ cells To explore how PIE-1 promotes germline specification, we sought to identify protein interactors. Immunoprecipitation of PIE-1 protein from embryo extracts proved to be challenging because the protein is expressed transiently in early embryos, where it is only present in early germline cells. Moreover, PIE-1 was insoluble and unstable in worm lysates preventing the analysis of PIE-1 complexes by immunoprecipitation (Figure 1—figure supplement 1). We therefore performed a yeast two-hybrid screen to identify PIE-1 interactors (Figure 1A and Supplementary file 1; see Materials and methods). As expected, this screen identified the Krüppel-type zinc finger protein MEP-1, a known PIE-1 interactor and co-factor of the Mi-2/NuRD (nucleosome remodeling deacetylase) complex (Passannante et al., 2010; Unhavaithaya et al., 2002). In addition, this screen identified the small ubiquitin-like modifier SMO-1 (SUMO); the E2 SUMO-conjugating enzyme UBC-9 (Jones et al., 2002); and GEI-17 (Holway et al., 2005; Kim and Michael, 2008), a homolog of vertebrate PIAS2 and Drosophila Su(var)2–10, an E3 SUMO ligase (Ninova et al., 2020; Pichler et al., 2017). Figure 1 with 3 supplements see all Download asset Open asset PIE-1 is SUMOylated on K68 residue in the Caenorhabditis elegans germline. (A) Summary of PIE-1 interactors identified by yeast two-hybrid screen (see Supplementary file 1 for complete list). (B and C) Domain structure of PIE-1 containing two zinc fingers (ZF1 and ZF2) and proline-rich region, and location (red bar) of a consensus small ubiquitin-like modifier (SUMO) acceptor motif (ψKXE, where ψ represents a hydrophobic amino acid, K is the acceptor lysine, and X is any amino acid) conserved in PIE-1 from other Caenorhabditis species. (D) Western blot analysis of SUMO-conjugated proteins in total worm lysates prepared with guanidine-HCl denaturing buffer or IP buffer. The resistant band (asterisk) migrates with the expected size of the E1 enzyme AOS-1, which attaches to SUMO by a thioester bond and may therefore resist SUMO proteases, which cleave isopeptide bonds. The black triangle indicates free SMO-1. (E and F) Western blot analyses of SUMOylated proteins enriched from (E) early embryo or (F) adult lysates from wild-type pie-1::flag or pie-1(K68R)::flag worms. SUMOylated proteins were enriched from worms expressing HIS10::SMO-1 by Ni-NTA chromatography. Black triangles indicate SUMOylated forms of PIE-1 or MRG-1. White triangles indicate unmodified PIE-1 or MRG-1. MRG-1 is a robustly SUMOylated protein (Supplementary files 2 and 3; Drabikowski et al., 2018; Kaminsky et al., 2009) and thus serves as a positive control. (G) Confocal images of PIE-1::GFP and mCherry::H2B in adult germline of live pie-1::gfp; pie-1p::mCherry::his-58 worms. Oocyte nuclei are indicated by white circles and numbered. Motif analysis predicted one consensus SUMO acceptor site (ψKXE; Rodriguez et al., 2001) in PIE-1 that is perfectly conserved in PIE-1 orthologs of other Caenorhabditis species (Figure 1B and C). Although covalent in nature, the addition of SUMO to substrates is rapidly reversible by SUMO protease enzymes that are active under conditions typically used to prepare lysates for immunoprecipitation studies (Di Bacco et al., 2006; Zhang et al., 2017). For example, even the preparation and centrifugation of lysates at 4°C followed by reconstitution in IP buffer with no other incubation resulted in the complete removal of SUMO from its target proteins (Figure 1D). To identify SUMO-conjugated proteins, we inserted a poly-histidine epitope into the endogenous smo-1 gene, and then used nickel (Ni) affinity chromatography under stringent denaturing conditions to enrich SUMOylated proteins (Tatham et al., 2009). Eluates from Ni-affinity chromatography were then analyzed by mass spectrometry (MS) and western blotting (Figure 1—figure supplement 2B and C and see below). In principle, it is possible to directly detect SUMOylated peptides by mass spectrometry (Impens et al., 2014; Knuesel et al., 2005). To create a cleavage signature suitable for high-throughput identification of modified peptide fragments, leucine 88 of SUMO must be substituted with lysine. Unfortunately, editing the endogenous smo-1 locus to encode the L88K mutation caused a lethal smo-1 phenotype in C. elegans (data not shown). Previous proteomic analyses of SUMOylated worm proteins have relied on smo-1 transgenes that encode HIS-tagged SUMO fused to the FLAG epitope or to GFP (Drabikowski et al., 2018; Kaminsky et al., 2009). Inserting in-frame FLAG or mCherry sequences into the endogenous smo-1 locus caused pronounced hypomorphic smo-1 phenotypes (data not shown). We therefore inserted hexahistidine (HIS6) or decahistidine (HIS10) sequences directly after the initiator methionine codon of smo-1 without any other sequences. Both HIS fusions resulted in fully viable and healthy strains indistinguishable from wild type. We chose to use the HIS10 fusion because it was robustly conjugated to target proteins (Figure 1D) and allowed better retention to the Ni-NTA resin under stringent denaturing and washing conditions (Figure 1—figure supplement 2A, B and C). The Ni-NTA resin retains non-SUMOylated protein species that are recovered even in the absence of HIS10::SUMO expression (e.g., see lanes 3 and 8 in Figure 1E and F). These non-SUMOylated proteins are not recovered via associations with SUMO-modified proteins but rather directly due to background binding of the individual denatured protein to the Ni-affinity matrix. The recovery of these background species from the matrix is not, therefore, a good measure of the actual ratio of SUMO-modified and -unmodified species in the lysate. The unavoidable recovery of unmodified species also impairs the sensitivity of MS as a means for identifying SUMO-modified proteins. Given the very high rate of false-negative detection in SUMO MS studies, we chose to apply the lowest possible arbitrary cut off of 1 spectral count enrichment over two controls—untagged SMO-1 and depletion of HIS10::SMO-1 by smo-1(RNAi)—when generating our list of candidate SUMO conjugates. This analysis identified 977 candidate SUMO (Supplementary file 2; see Supplementary file 3, Figure 1—figure supplement 2D, and Discussion section for comparison with previous studies). SUMOylated MRG-1, for example, was strongly enriched by Ni-affinity chromatography as detected by MS and western blot analyses (Supplementary file 2 and Figure 1E and F). Importantly, failure to detect a protein by SUMO proteomics should not be construed as evidence the protein is not modified or regulated by SUMO. For each candidate protein of interest, co-valent modification by SUMO should be assessed directly with the more sensitive approach of Ni-affinity chromatography and western blotting. PIE-1 did not pass the arbitrary cut-off we applied to our MS data (Supplementary file 2). We therefore carefully monitored PIE-1 SUMOylation by western blotting. To do so, we inserted a sequence encoding the FLAG epitope into the endogenous pie-1 gene. The PIE-1::FLAG protein was expressed at similar levels in wild-type and his10::smo-1 worms (Figure 1E and F), and these worms were fully viable. Ni-affinity chromatography of embryo lysates failed to enrich a modified form of PIE-1::FLAG (Figure 1E). However, Ni-affinity chromatography of adult lysates enriched a slowly migrating form of PIE-1::FLAG protein that was ~12 kD larger than unmodified PIE-1::FLAG (compare lanes 14 and 19, Figure 1F). The modified PIE-1::FLAG band was absent when we mutated the presumptive SUMO acceptor site lysine 68 to arginine in PIE-1::FLAG (K68R) (Figure 1F, lane 20). Thus, lysine 68 is required for PIE-1 SUMOylation. We were surprised to detect SUMOylated PIE-1 in adult hermaphrodites but not in embryos. Previous studies had only detected PIE-1 protein within embryonic germ cells and proximal oocytes of adult hermaphrodites (Reese et al., 2000; Tenenhaus et al., 1998). We therefore monitored PIE-1::GFP expressed from the endogenous pie-1 locus (Kim et al., 2014). As expected, in embryonic germ cells, PIE-1::GFP localized to nuclei, cytoplasm, and cytoplasmic P granules (i.e., P-lineage; Figure 1—figure supplement 3; Mello et al., 1996; Reese et al., 2000; Tenenhaus et al., 1998). In the adult pachytene germline, we found that PIE-1::GFP co-localized with chromosomes (as monitored by co-localization with mCherry histone H2B) and PIE-1 protein levels appeared to gradually increase during oocyte maturation (Figure 1G and Figure 1—figure supplement 3). These findings suggest that SUMOylation of PIE-1 occurs within a heretofore unexplored zone of PIE-1 expression within the maternal germline. PIE-1 SUMOylation and SUMO pathway factors function together to promote fertility and embryonic development To investigate the functional consequences of the K68R lesion, we performed a genetic complementation test with a previously described null allele, pie-1(zu154). In early embryos, maternal PIE-1 promotes the germ cell fate of the P2 blastomere and prevents P2 from adopting the endoderm and mesoderm fates of its somatic sister blastomere, EMS (Mello et al., 1992). Hermaphrodite worms homozygous for the loss-of-function pie-1(zu154) allele are fertile, but 100% of their embryos arrest development with extra pharyngeal and intestinal cells (Mello et al., 1992). By contrast, we found that most homozygous PIE-1(K68R) animals produce viable and fertile progeny but exhibit reduced fertility and increased embryonic lethality (Figure 2A and B), suggesting that the K68R lesion causes a partial loss of PIE-1 function. Consistent with this idea, further lowering pie-1 activity by placing the K68R allele over pie-1(zu154) dramatically enhanced the deficits in fertility (Figure 2A) and in embryo viability (Figure 2B). For example, about 11% of embryos produced by trans-heterozygotes failed to hatch (Figure 2B). When we examined a subset of the dead embryos by light microscopy, we found that 64% (14/22) arrested development with supernumerary intestinal cells, a hallmark of pie-1 loss of function (Mello et al., 1992). Thus, PIE-1(K68R) exhibits a partial loss of PIE-1 function. Figure 2 Download asset Open asset Genetic interactions between pie-1 and SUMO pathway. (A) Brood size analysis and (B) embryonic lethality of wild-type (N2), pie-1(ne4303[K68R]), pie-1(zu154)/qC-1, and pie-1(ne4303[K68R])/pie-1(zu154). Statistical significance was determined by Wilcoxon-Mann-Whitney test: *p≤0.05; **p≤0.01; ****p≤0.0001. (C) Tests of genetic interactions between pie-1 and SUMO pathway mutants. Bar graphs show the percentage of dead embryos (gray) and percentage of dead embryos with extra intestine (yellow) among ‘n’ embryos scored. (D) Partial sequence alignment of UBC enzymes, including C. elegans UBC-9. Residues conserved in all UBC proteins are shown in red. Temperature-sensitive (ts) alleles of yeast Cdc34 result from mutations in highly conserved residues (blue boxes). Mutating the proline resides (P69S and P73S) resulted in non-conditional lethality in C. elegans. A G56R mutation in C. elegans UBC-9 caused a ts phenotype. (E) Location of the G56R mutation introduced into the endogenous ubc-9 gene by CRISPR genome editing. (F) Genetic interaction between pie-1 and ubc-9(ne4446[G56R]) allele at 25°C. Bar graphs show the percentage of embryos with extra intestine among ‘n’ embryos scored. Figure 2—source data 1 Brood size and embryonic lethality. https://cdn.elifesciences.org/articles/63300/elife-63300-fig2-data1-v1.xlsx Download elife-63300-fig2-data1-v1.xlsx We reasoned that if the loss of SUMOylation is responsible for the hypomorphic phenotype of PIE-1(K68R) animals, then compromising the SUMO machinery might cause similar synthetic genetic interactions. To test this idea, we depleted the activity of each SUMO pathway gene by RNAi beginning at the L4 larval stage in wild-type and pie-1(zu154)/+ heterozygotes. RNAi at the L4 stage allows the worms to produce fertilized embryos depleted of SUMO activity. When exposed to control RNAi, both wild-type and pie-1(zu154) heterozygous worms produce 100% viable embryos (Figure 2C). RNAi targeting SUMO pathway factors (smo-1, gei-17, or ubc-9) caused wild-type and pie-1(zu154) heterozygous worms to dead embryos. only of embryos in the wild-type background extra about of embryos by the pie-1(zu154) heterozygous worms arrested with extra intestinal cells (Figure 2C). The between and pie-1(zu154) was whereas in wild-type worms caused only embryonic in worms caused of embryos to arrest development and of these produced extra intestinal cells (Figure 2C). In an to create (ts) alleles of endogenous we used genome editing to mutations to ts alleles of yeast (Figure and and 1996; et al., One allele, which a G56R amino resulted in worms that were viable and fertile at the of but when to the of 25°C. of the phenotypes of this allele can be by worms to the (data not shown). When to beginning at the L4 ubc-9(ne4446[G56R]) worms into fertile that produced 100% dead embryos with but of ubc-9(ne4446[G56R]) embryos arrested with extra intestinal cells, consistent with previous studies that in and embryos, the P2 blastomere cell lineage a pie-1 phenotype et al., 2016). the of arrested embryos with a phenotype was when G56R were to in a heterozygous pie-1(zu154)/+ background (Figure the that SUMO promotes the activity of PIE-1 in protecting the embryonic germline. is SUMOylated in the adult The that PIE-1 with and GEI-17 in the yeast two-hybrid (Figure 1A and Supplementary file the that PIE-1 the SUMO machinery to regulate its so, then one to the functions of PIE-1 is to for proteins that are modified by SUMO in a PIE-1-dependent manner. To do we used RNAi to PIE-1 from a of adult his10::smo-1 As we used RNAi targeting smo-1 and an RNAi The depletion of PIE-1 and SMO-1 was monitored by western blot (Figure Ni-affinity chromatography followed by MS proteins that were reduced to a similar in lysates from both and animals in Figure and Supplementary file this of PIE-1-dependent SUMO targets, our because a previous study identified the nucleosome remodeling and deacetylase including and its binding and MEP-1, as targets of PIE-1 regulation (Unhavaithaya et al., 2002). Figure 3 Download asset Open asset PIE-1 SUMOylation promotes SUMOylation in the adult germline. (A) Western blot levels of SUMOylation in HIS10::SMO-1 worms with control or (B) the levels of SUMOylated proteins in worms and worms Eluates from affinity chromatography of and lysates were analyzed by mass The of the between spectral in control and was for each proteins whose spectral were reduced in and on the proteins whose spectral increased in to control. indicate the of a between the changes in and worms. A list of PIE-1-dependent SUMO targets is in Supplementary file and Western blot analyses of SUMOylated (C) or MEP-1 (D) enriched from embryo or adult (blue lysates of or smo-1 mutants. Ni-NTA are by Black triangles indicate SUMOylated white triangles indicate unmodified proteins. Figure data 1 the levels of SUMOylated proteins in with in Download To directly explore the that PIE-1 promotes the SUMOylation of and MEP-1, we used Ni-affinity chromatography followed by western blotting. To SUMOylation of MEP-1, we used CRISPR to tandem and sequences into the mep-1 For we used a previously et al., Western blot analyses of from Ni-affinity chromatography that slowly migrating of (Figure lanes and were present in adult his10::smo-1 but absent from embryo lysates (Figure lane By contrast, slowly migrating MEP-1 were present in both embryos and (Figure lanes and These modified and MEP-1 were ~12 kD larger than unmodified and MEP-1, were only enriched by Ni-affinity chromatography of his10::smo-1 and were not detected in lysates prepared from worms (Figure and lane suggesting that the larger result from SUMOylation. SUMOylated was not detected in extracts from (Figure lane and appeared reduced in adult extracts from homozygous animals expressing PIE-1(K68R) (Figure lanes and In contrast, MEP-1 SUMOylation was by genetic of pie-1 (Figure lanes and Thus, SUMOylation of occurs only in and to least pie-1 activity and PIE-1 SUMOylation. PIE-1 SUMOylation promotes of an adult germline complex To PIE-1 SUMOylation the interaction of with other complex we from early embryo and adult lysates a protein et al., and detected or by western In embryo where PIE-1 and are not modified by MEP-1 with both and (Figure lanes and These interactions were not by the PIE-1(K68R) mutation (Figure lane In adult MEP-1 robustly with LET-418, but only with (Figure lanes and a background band present in control from animals without (see Figure lanes and this interaction between and MEP-1 was required both smo-1 and pie-1 activity (Figure lanes and and was reduced in PIE-1(K68R) mutants (Figure lane Thus, SUMOylation of PIE-1 promotes a to detect but interaction between and MEP-1 in the adult germline, but is not required for their much more robustly detected interaction in embryos. Given the of the SUMO it is that if the interaction between and MEP-1 in the presence of then our IP the in levels of the protein complex (see Figure Download asset Open asset PIE-1 SUMOylation is required for the assembly of complex in the adult germline. Western blot analyses of proteins that with from embryo or adult lysates (blue of or smo-1 worms. was with GFP (see and were with HDA-1, LET-418, or of the the reduced interaction of with MEP-1 in pie-1 and smo-1 mutants. PIE-1 histone and germline gene expression PIE-1 promotes the assembly of a functional complex in the adult germline, then we global levels of to be increased in pie-1 Indeed, increased levels of histone lysine in pie-1 to wild-type (Figure and B), especially in the and in To more strongly PIE-1 we an in-frame (see and these animals to from the stage PIE-1 expression in the adult germline (Figure supplement and caused a pie-1 embryonic lethal phenotype. adult worms uniformly high levels of throughout the germline (Figure Moreover, levels were in oocytes of worms than in oocytes of worms (Figure These findings suggest that PIE-1(K68R) is not for its to promote of in the adult germline. Figure with 2 supplements see all Download asset Open asset PIE-1 histone and genes in the adult germline. (A) of and in adult of wild-type pie-1(ne4303[K68R]), and animals Oocyte nuclei are indicated with white (B) of in oocytes to was by The of the total cell is on the was a test: