PIE-1 SUMOylation promotes germline fates and piRNA-dependent silencing in C. elegans

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.

Keywords: C. elegans; NuRD complex; SUMO pathway; genetics; genomics; germline chromatin; germline fate.

Figure 1.. 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.

Figure 1—figure supplement 1.. PIE-1 is insoluble and unstable.

(A) Analysis of lysis buffers (upper) used to test PIE-1 solubility in lysates (lower). PIE-1 is insoluble in most buffers, whereas PRG-1 and tubulin are soluble. S, supernatant; P, pellet. (B) Silver stain gel of GFP immunoprecipitation experiment from wild-type or pie-1::gfp worms. Cleavage product of PIE-1 is indicated with black arrow. (C) Sequence of the expected PIE-1::GFP fusion protein showing the location of the cleavage site at S327 identified by analysis (red arrow), which removes the last eight residues of PIE-1 and GFP.

Figure 1—figure supplement 2.. Enrichment of SUMOylated proteins from worms expressing HIS-tagged SMO-1.

(A and B) Western blots comparing Ni-affinity enrichment of SUMOylated proteins from (A) his6::smo-1 or (B) his10::smo-1 worms. (C) Silver staining gel (left) and western blot (right) showing the results of Ni-affinity chromatography (binding, washes, and elution) and enrichment of SUMOylated proteins. (D) Venn diagram showing the overlap of Caenorhabditis elegans SUMO targets identified in this study and by Kaminsky et al., 2009 and Drabikowski et al., 2018. Our list of potential SUMOylated proteins included approximately half of the 146 proteins identified by Kaminsky et al., and one-quarter of the 873 proteins identified by Drabikowski et al. Nearly 70% of the proteins we identified were not identified by these studies. Only 29 proteins (3%) were identified by all three studies. These findings suggest that the full SUMO proteome in C. elegans is far from complete.

Figure 1—figure supplement 3.. PIE-1 expression in the adult germline and early embryos.

Confocal images of PIE-1::GFP or PIE-1(K68R)::GFP fluorescence in adult germline (top) and in the germ cells (P1, P2, and P3) of early embryos.

Figure 2.. 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 3.. PIE-1 SUMOylation promotes HDA-1 SUMOylation in the adult germline.

(A) Western blot showing relative levels of SUMOylation in HIS10::SMO-1 worms treated with control (L4440), pie-1(RNAi), or smo-1(RNAi). (B) Scatter plot comparing the levels of SUMOylated proteins in pie-1(RNAi) worms (x axis) and smo-1(RNAi) worms (y axis). Eluates from affinity chromatography of control, pie-1(RNAi), and smo-1(RNAi) lysates were analyzed by mass spectrometry. The log of the difference between spectral counts in control and mutant was plotted for each protein. Positive values represent proteins whose spectral counts were reduced in pie-(RNAi) and smo-1(RNAi). Negative values on the x axis represent proteins whose spectral counts increased in pie-1(RNAi) compared to control. Dashed lines indicate the position of a 1.5-fold difference between the changes in smo-1(RNAi) and pie-1(RNAi) worms. A full list of PIE-1-dependent SUMO targets is provided in Supplementary file 4. (C and D) Western blot analyses of SUMOylated HDA-1 (C) or MEP-1 (D) enriched from embryo (yellow background) or adult (blue background) lysates of wild-type, pie-1, or smo-1 mutants. Ni-NTA pull-downs are outlined by dashed red boxes. Black triangles indicate SUMOylated proteins; white triangles indicate unmodified proteins.

Figure 4.. PIE-1 SUMOylation is required for the assembly of MEP-1/HDA-1 complex in the adult germline.

Western blot analyses of proteins that immunoprecipitate with MEP-1::GTF from embryo (yellow background) or adult lysates (blue background) of wild-type, pie-1, or smo-1 mutant worms. MEP-1::GTF was immunoprecipitated with GFP nanobody (see 'Materials and methods'). Blots were probed with HDA-1, LET-418, or anti-FLAG (MEP-1::GTF) antibodies. Longer exposure of the HDA-1 blots shows the reduced interaction of HDA-1 with MEP-1 in pie-1 and smo-1 mutants.

Figure 5.. PIE-1 regulates histone H3K9Ac and spermatogenic genes in the adult germline.

(A) Immunofluorescence micrographs of H3K9Ac and DAPI staining in adult gonad of wild-type (wt), pie-1(ne4303[K68R]), and pie-1::degron::gfp animals (100 µM auxin exposure). Oocyte nuclei are indicated with white dashed circle. (B) Quantification of immunofluorescence intensity in oocytes (−1 to −5). H3K9Ac signal was measured by ImageJ. The mean of the correlated total cell fluorescence (CTCF)/area ± SEM is plotted on the y axis. Significance was measured using a Tukey’s test: ****p<0.0001; *p<0.05. (C and D) Scatter plots comparing mRNA-seq reads in (C) pie-1(ne4303[K68R]) or (D) pie-1::degron::gfp to those in wt. Blue dashed lines indicate twofold increased or decreased in the mutant. Genes were categorized as spermatogenic, oogenic, neutral, or other, as defined by Ortiz et al., 2014. A value of 0.1 was assigned to undetected genes, thus genes with an x value of ‘−1’ were not detected in wt. (E) Bar graph showing fractions of upregulated genes involved in spermatogenesis, oogenesis, neutral, or other categories. Genes expressed in wt gonads were used as a reference (10743 genes) (Kim et al., 2021). The number of upregulated genes in each mutant is labeled at the top. (F) Venn diagram showing overlap of genes upregulated in pie-1::degron::gfp, hda-1[KKRR], and mep-1::degron. The hda-1[KKRR] and mep-1::degron data are from Kim et al., 2021.

Figure 5—figure supplement 1.. Auxin-induced depletion of PIE-1::DEGRON::GFP.

Differential interference contrast (DIC) (top) and epifluorescence images of live adult worms expressing PIE-1::DEGRON::GFP (middle) and TIR1::mRuby (bottom) in the absence (A) or presence (B) of 100 µM auxin. Intestinal autofluorescence is observed in the green and red channels.

Figure 5—figure supplement 2.. Transposons upregulated in pie-1::degron::gfp.

Volcano plot of transposon expression in pie-1::degron::gfp. The x axis shows the fold change in pie-1::degron::gfp vs. wild type, and vertical dashed lines indicate twofold change. The y axis is the adjusted p-value from DESeq2, and the horizontal dashed line indicates a p-value of 0.05. If available, transposon family names are shown with sequence names.

Figure 6.. PIE-1 and GEI-17 function together to promote piRNA-mediated silencing.

(A) Epifluorescence images (upper panels) of piRNA sensor expression in dissected gonads from wild-type and rde-3(ne3370) worms. The lower panels show differential interference contrast images of the gonads in the upper panels. (B) Synergistic effects of desilencing piRNA sensors in pie-1[K68R]; gei-17 double mutants. The desilenced piRNA sensor (gfp::csr-1) was scored in the indicated alleles. gei-17(null) alleles were generated by CRISPR editing.