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Noncanonical inheritance of phenotypic information by protein amyloids

Nature Cell Biology volume 26, pages 1712–1724 (2024)Cite this article

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Abstract

All known heritable phenotypic information in animals is transmitted by direct inheritance of nucleic acids, their covalent modifications or histone modifications that modulate expression of associated genomic regions. Nonetheless, numerous familial traits and disorders cannot be attributed to known heritable molecular factors. Here we identify amyloid-like protein structures that are stably inherited in wild-type animals and influence traits. Their perturbation by genetic, environmental or pharmacological treatments leads to developmental phenotypes that can be epigenetically passed onto progeny. Injection of amyloids isolated from different phenotypic backgrounds into naive animals recapitulates the associated phenotype in offspring. Genetic and proteomic analyses reveal that the 26S proteasome and its conserved regulators maintain heritable amyloids across generations, which enables proper germ cell sex differentiation. We propose that inheritance of a proteinaceous epigenetic memory coordinates developmental timing and patterning with the environment to confer adaptive fitness.

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Fig. 1: mstr genes epigenetically maintain male germ cell sex specification.
Fig. 2: mstr genes determine sex by conferring 26S proteasomal selectivity.
Fig. 3: mstr genes balance GLD-1 and TRA-1 protein levels to determine germline sex.
Fig. 4: Inheritance of amyloid bodies correlates with penetrance of mstr phenotype.
Fig. 5: Germline amyloids are sufficient to recapitulate the mstr phenotype.
Fig. 6: VIT-2 traces germline inheritance of amyloids.
Fig. 7: De novo synthesized offspring VIT-2 grows around parental VIT-2 foci.
Fig. 8: Amyloids regulate WT gametogenesis and vulval cell fate specification.

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Data availability

MS data (raw, peak lists and results files) from the TurboID and amyloid purification experiments are deposited in ProteomeXchange through partners Mass Spectrometry Interactive Virtual Environment (MassIVE) and PRIDE (MSV000088183/PXD028890 and PXD043926, respectively). Raw sequencing reads of RNA sequencing and CLIP-seq experiments are deposited in the Gene Expression Omnibus (GSE185304). Strains generated in this paper are deposited to the Caenorhabditis Genetics Centre. Reagent suppliers and catalogue numbers, antibodies and dilutions, commercial kits and oligonucleotide sequences are provided in Supplementary Table 6. The minimum dataset necessary to interpret, verify and extend the research is provided with the paper and all strains/reagents generated here are available on request from the corresponding authors. Source data are provided with this paper.

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Acknowledgements

We thank M. Schertzberg for assistance on variant calling analysis and J. Tong for discussions on proteomics; J.M. Claycomb and D.R. Kaplan for discussion, advice and mentorship throughout the project; O. Hobert for microscopy resources; G. Riddihough and A. Andersen of Life Science Editors and D. Schramek and M. Ramalho-Santos for feedback on the paper. All MS analysis was performed by SPARC BioCentre (Molecular Analysis) at the Hospital for Sick Children, Toronto, Canada. Next-generation sequencing for CLIP-seq and whole-genome sequencing was performed by the Donnelly Sequencing Centre at the University of Toronto, Canada. Next-generation sequencing for RNA-seq and Sanger sequencing was performed by the Centre for Applied Genomics, Hospital for Sick Children, Toronto, Canada. W.B.D. is the Canada Research Chair in Animal Models of Human Disease. Confocal microscopy on the Leica SP8 and Nikon A1R was performed at the Imaging Facility, Hospital for Sick Children, Toronto, Canada. Confocal microscopy on the Zeiss LSM980 was performed in O. Hobert’s laboratory at Columbia University. This work was supported by a Canadian Institutes of Health Research Project grant to W.B.D. (PJT 165837). Some strains were provided by the Caenorhabditis Genetics Centre, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440).

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Authors and Affiliations

  1. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Matthew Eroglu, Autumn Zocher, Jacob McAuley, Rachel Webster, Maggie Z. X. Xiao, Calvin Mok & W. Brent Derry

  2. Developmental and Stem Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada

    Matthew Eroglu, Autumn Zocher, Jacob McAuley, Rachel Webster, Maggie Z. X. Xiao, Bin Yu & W. Brent Derry

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Contributions

M.E. conceived of the study, designed all experiments, performed or supervised all experiments, performed data analysis and wrote the paper. A.Z. and J.M. performed amyloid isolation, TEM, some drug treatments and brood counts. R.W. performed longevity experiments. M.Z.X.X. performed EMS mutagenesis, F2 screening and quantification of suppressor brood sizes. B.Y. generated CRISPR alleles, dissected and stained gonads with PROTEOSTAT reagent. C.M. performed MIP-MAP library preparation, sequencing and data analysis. W.B.D. also conceived of the study, contributed to the paper, supervised and secured funding for the project.

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Correspondence to Matthew Eroglu or W. Brent Derry.

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Extended data

Extended Data Fig. 1 Transgenerational self-sterility of mstr mutants is caused by germline feminization.

(a) Schematic of germ cell sex differentiation in WT hermaphrodites and sterile mstr worms. L4, 4th larval stage. YA, young adult. Ad, adult. d, distal. p, proximal. (b) Penetrance of sterility in worms of the indicated genotype at the indicated generation and maintenance temperature. (c) Representative DIC micrographs of WT and sterile mstr-2 late generation worms. Dashed line, germline. Bar, 20 μm. (d) Fluorescence and DIC micrographs of WT and mstr hermaphrodites showing expression of the oocyte marker RME-2::GFP. Inset, most proximal germ cells. Dashed line, germline. Bars, 20 μm. (e) Fluorescence and DIC micrographs of somatically male mstr worms showing expression of the oocyte marker RME-2::GFP. White dashed line, germline. Green dashed line, intestine Bar, 20 μm. (f) Transcriptomes of WT worms compared to mstr-1 worms (20 °C). Left panel P values, negative binomial exact test (edgeR). Right panel, ****P < 0.0001 two-tailed Mann–Whitney test. n = 3 populations of WT or mstr worms sequenced at L4 stage. Numerical data and statistical values are available in source data.

Source data

Extended Data Fig. 2 Selective proteasomal regulation by MSTR-1 determines germ cell sex.

(a) Schematic of the forward genetic screen for identifying suppressors of mstr germline feminization. ‘sdm’, suppressors of the double mstr worms, temporary nomenclature. Bar, 1 mm. (b) Brood sizes of the top suppressors of mstr feminization. Lines, mean (± S.D.). (c) Representative mapping plots of suppressors that were mapped to regions with mutations in proteasomal subunits but not validated by CRISPR/Cas9. Mapping data for all suppressors shown in b and c are provided in Source Data. Specific information on mapped mutations is listed in Supplementary Table 1. (d) Assessment of proteasomal function determined by expression of GFP tagged with uncleavable ubiquitin, Ub(G76V)::GFP. Plots, mean (± S.D.). ****P < 0.0001, ** P < 0.01, * P < 0.05. Two-way ANOVA with Bonferroni’s correction. Whole-worm difference in WT vs mstr at 20 °C is only significant when excluding pas-1 positive control from the analysis. Whole WT worms n = 3 20 °C, 9 P0 25 °C, 6 F1 25 °C worms. Whole mstr worms n = 9 20 °C, 10 P0 25 °C, 8 F1 25 °C worms. pas-1 RNAi treated worms: n = 3. WT germlines: n = 6 20 °C, 6 P0 25 °C, 4 F1 25 °C. mstr germlines: n = 8 20 °C, 7 P0 25 °C, 10 F1 25 °C. Exact P values are provided in Source Data. (e) Worms treated with proteasome inhibitor MG132 at L3 stage. Lines, mean (± S.D.). ****P < 0.0001, * P < 0.05, ns not significant. Percentages, percent of variation attributable to stated independent variable. Two-way ANOVA with Bonferroni’s correction. WT DMSO and MG132: n = 12 worms in each P0-F5 generation and treatment, 6 F6 worms in each treatment. mstr: n = 12 mstr worms in each generation and treatment. mstr; rpt-1 on DMSO: 5 P0, 6 F1, 6 F2, 8 F3, 7 F4, 8 F5, 8 F6 worms. mstr; rpt-1 on MG132: 6 P0-F3 each, 8 F4-6 each. Exact P values are provided in Source Data. (f) Expression of autophagy genes in mstr worms. Two-way ANOVA with Bonferroni’s correction. n = 3 WT and mstr worm populations. Numerical data and statistical values are available in source data.

Source data

Extended Data Fig. 3 Transgenerational regulation of GLD-1 expression by mstr genes.

(a) STRING interaction network of proteins detected in proximity to MSTR-1 by BioID. Red circle, highly connected interactors by heat diffusion. Green highlights, 19S proteasomal regulatory subunits; orange highlight, 20S core proteasomal subunit. (b) Schematic of the mstr-1 locus and alignment of the Alphafold structures of C. elegans MSTR-1 and human ZFAND5. Plot, brood size of the mstr-1(ok1685) allele which only ablates the A20 domain. Lines, mean (± S.D.). ns, not significant for genotype effect. Two-way ANOVA with Bonferroni’s correction. n = 31 mstr-1(ok1685) and 26 mstr-1(ok1685); mstr-2 worms. (c) Western blot of immunoprecipitated MSTR-1::FLAG. FK2, antibody used to detect mono- and poly-ubiquitin conjugated proteins. M2, FLAG antibody. Red arrowhead, band corresponding to MSTR-1. (d) Representative CLIP-seq experiment showing regions excised for library preparation and sequencing. (e) Left, MA plot of CLIP-seq counts of reads mapping to each transcript. Blue dots, significantly enriched in respective sample (P < 0.02, Benjamini–Hochberg-adjusted Wald test). Red dots, significantly enriched germ line sex determining genes (SDG) deduced from a GO enrichment analysis (GO: 0018992, FE = 6.22, Fisher’s exact P = 0.000811, Benjamini–Hochberg FDR = 0.0157). Right, comparison of statistically significant (P < 0.02) transcripts enriched in MSTR-1 CLIP-seq (X-axis) to transcripts significantly differentially expressed in mstr-1 ablated worms. n = 2 MSTR-1 pulldowns and 1 control pulldown. (f) Immunoprecipitation of GLD-1::V5 followed by Western blot using anti-ubiquitin (left) and V5 (right) antibodies. (g) GLD-1 expression in wild-type and mstr worms at late generation 20 °C. Bar, 20 μm. (h, i) Transferring worms to 25 °C leads to increased penetration of ectopic GLD-1::mScarlet expression over a small number of generations. C, P0 at 25 °C, D, F3 at 25 °C. Transgenerational ectopic expression of GLD-1 in mstr worms is rescued by the rpt-1 suppressor. Exact n values are provided in Source Data. Bar, 20 μm. Uncropped blots, numerical data and statistical values are available in source data.

Source data

Extended Data Fig. 4 mstr genes regulate epigenetic information independent of known mechanisms of inheritance.

(a) GLD-1 and TRA-1 mRNA levels (relative to WT at 20 °C) across generations and maintenance conditions in WT, mstr and mstr; rpt-1 worms. Mean ± S.E.M. (b) 26S proteasome mRNA levels (relative to WT at 20 °C) across generations and maintenance conditions in WT, mstr and mstr; rpt-1 worms. (c) Schematic of possible genetic interactions between mstr-1 and small RNA pathways implicated in epigenetic inheritance. Lower than expected fertility represents a negative genetic interaction, consistent with parallel or compensatory pathways converging on the same biological output. This can occur if gene or protein targets of the respective pathways display overlap (for example, suppression of mRNA translation versus degradation of the respective proteins achieve the same overall outcome). Higher than expected fertility represents a positive genetic interaction (masking epistasis) consistent with shared pathways or dependence. (d) Epistasis experiment between mstr-1 and hrde-1. HRDE-1 protein is depleted by auxin treatment of worms expressing endogenously auxin-inducible degron (AID) tagged HRDE-1. Lines, mean ± S.D. **** P < 0.0001 for genotype effect between HRDE-1 protein depletion versus HRDE-1 depletion in a mstr-1 null background. Two-way ANOVA with Bonferroni’s correction. n = 96 HRDE-1 depleted and 121 HRDE-1 depleted mstr-1 worms. (e) Epistasis experiments between mstr-1 and miRNAs, 22 G RNAs, 26 G RNAs or piRNAs at 20 or 25 °C. Lines, mean ± S.D. Numbers denote mean self-fertility of indicated mutant as percent of wild type. Numbers in brackets indicate expected double mutant self-fertility as percent of wild type if there is no interaction based on a multiplicative model. **** P < 0.0001, *** P < 0.001, ** P < 0.01; * P < 0.05 two-tailed Mann-Whitney test. For 20 °C n = 118 mstr-1, 58 alg-1, 58 alg-1; mstr-1, 38 alg-2, 59 alg-2; mstr-1, 49 alg-5, 50 alg-5; mstr-1, 66 drh-3, 77 drh-3; mstr-1, 58 rrf-3, 51 rrf-3; mstr-1, 30 prg-1, 16 prg-1; mstr-1 worms. For 25 °C n = 39 mstr-1, 20 alg-1, 20 alg-1; mstr-1, 20 alg-2, 20 alg-2; mstr-1, 19 alg-5, 20 alg-5; mstr-1, 20 drh-3, 20 drh-3; mstr-1, 20 rrf-3, 20 rrf-3; mstr-1 worms. Numerical data and statistical values are available in source data.

Source data

Extended Data Fig. 5 Autofluorescent bodies accumulate in WT and mstr worms over generations at 25 °C.

(a) Confocal microscopy of green germline autofluorescence in WT worms at the indicated generation at 25 °C. Dashed line, germlines. Bars, 20 µm. (b) Same as (a) but with mstr worms. Bars, 20 µm. (c) Quantification of green autofluorescence in WT and mstr germlines of worms maintained at 25 °C. Left panels, signal along germline (mean ± S.E.M.). Right panel, total signal per gonad. * P < 0.05, ** P 0.01, *** P < 0.001; two-way ANOVA with Holm-Šidák correction. n = 9 WT P0, 13 WT F1, 7 WT F10, 6 mstr P0, 6 mstr F1 worms. Box, 25th-75th percentiles; whiskers, min to max; centre line, median. Numerical data and statistical values are available in source data.

Source data

Extended Data Fig. 6 Green autofluorescence in the germline significantly overlaps with physiological amyloid bodies.

(a) Confocal microscopy of live worms showing green autofluorescent puncta in the germline are distinct from P-granules (germ granules). Dashed line, germline. Bars, 20 µm. (b) Confocal microscopy of live worms showing green autofluorescent puncta displays poor overlap with mitochondria stained with mitotracker deep red (MTDR). Dashed line, germline. Bars, 20 µm. (c) Confocal microscopy of dissected and fixed gonads of WT and mstr worms (dashed line) at the indicated temperature and generation stained with the amyloid detection dye Proteostat. Bars, 20 µm. (d) Confocal microscopy of live mstr worms stained with the amyloid detection dye Thioflavin T, which shows significant overlap with green autofluorescence in the germline (dashed line), notably among brighter punctate bodies. Bars, 20 µm. (e) Confocal microscopy of dissected and fixed gonads of WT and mstr worms stained with the amyloid detection dye Amytracker 680, which shows significant overlap with green autofluorescence, notably among brighter punctate bodies. Bars, 20 µm.

Extended Data Fig. 7 Approach for purification of the amyloids.

(a) Schematic summarizing the modified method by which amyloid proteins were isolated from WT and mstr worms. (b) Transmission electron microscopy (TEM) of purified amyloids displaying fibrillar and other structures. Bars are defined in the figure panel. (c) Purified amyloids stain with the amyloid detection dyes Proteostat and Thioflavin T and are autofluorescent. Bars, 20 µm.

Extended Data Fig. 8 Characterization of the amyloid composition reveals traceable markers.

(a) STRING interaction network of amyloid (left) and soluble amyloid-associated proteins (right) identified by mass spectrometry. Bright red, vitellogenins; light red, heat diffusion on vitellogenin nodes; dark green, 26S proteasomal subunits; light green, heat diffusion on 26S proteasomal nodes. RPN-1 (yellow) was identified from heat diffusion of vitellogenins, indicating high connectivity. n = 2 WT amyloid isolates and 1 corresponding run of the soluble co-isolate fraction. (b) Left, overlap of mRNAs enriched by MSTR-1 CLIP-seq and amyloid proteins isolated. Only 6 proteins would be expected to overlap by chance among two similarly sized groups of randomly selected proteins. Right, RNAi knockdown of 3 of the overlapping hits yields a modest rescue of the mstr phenotype at P0. ** P < 0.01, * P < 0.05; one-way ANOVA with Dunnett’s correction. n = 57 control, 57 ketn-1, 57 imph-1, and 19 nep-17 RNAi treated worms. (c) Western blot of WT and mstr amyloids stained with the protein detection reagent SYPRO Ruby. (d) Fraction of protein sequence covered by at least one peptide (mappability) across all proteins detected in the mass spectrometry of isolated amyloids or the soluble co-isolate. Right plots, mean sequence coverage across all proteins and total detected spectra in each run. **** P < 0.0001. Two-tailed t-test. n = 825 proteins detected across 2 WT and 2 mstr amyloid isolates and 1 corresponding run of the soluble co-isolates. (e) Transmission electron microscopy of WT and mstr female yolk granules in fixed worm sections. Red arrowheads, fibril-like structures. Bar, 50 nm. (f) Expression of IMPH-1::mEos in WT and mstr distal germlines and its colocalization with Amytracker 680 in the indicated backgrounds. Bar, 20 µm. (g) Top, staining of VIT-2::GFP expressing worms with the amyloid detection dye Amytracker 680. Bottom, staining of VIT-2::RFP with amyloid detection dye Proteostat. Dashed line, proximal oocytes. Bars, 20 µm. (h) Overlap of green autofluorescence with VIT-2::RFP in the germline. VIT-2 increasingly associates with autofluorescent amyloid-like bodies during oocyte maturation. Dashed line, germline. Solid line, embryo. Plot, mean ± S.D. of Pearson’s correlation coefficients measured in indicated location of individual worms, P < 0.0001 for increasing overlap and linear trend from distal to -1 oocyte, one-way ANOVA with Dunnett correction and post-test for linear trend. Bars, 20 µm. n = 6 worms. Uncropped blots, numerical data and statistical values are available in source data.

Source data

Extended Data Fig. 9 Inheritance of injected labelled amyloids and detection of amyloid species across broad tissues.

(a) Isolated WT amyloids were labelled with an Alexa Fluor 647-NHS Ester, covalently marking amines (that is, proteins), and were then injected into gonads of WT worms where they are deposited into oocytes. Dashed line, germline. Bar, 20 µm. (b) Labelled amyloids are passed on through fertilization and detectable in fertilized oocytes. Dashed line, germline. Bar, 20 µm. (c) Labelled amyloids are stable in offspring where they can be detected in various tissues including in germlines, the intestine and head during L1 and L2 stages. Dashed lines: left, whole L1 stage worm; centre two, L2 germline; right, L2 head. Bars, 20 µm. (d) The labelled amyloids are stable into L3 germlines, the stage at which germ cell begin meiosis and differentiate to gametes. They are also still stable in the head, intestine, and body wall. Dashed line, germline. Bars, 20 µm. (e) Range of vulval phenotypes observed in F2 WT worms shifted to 20 °C after >30 generations of maintenance at 25 °C. n = 1724 worms observed over three separate experiments. Bar, 100 µm. (f) Substantial presence of punctate green autofluorescence and similar appearing Thioflavin T-stained bodies can be detected throughout development of wild type worms. Bars, 20 µm. (g) MSTR-1 is broadly expressed throughout development and displays modest nuclear localization within cells. Bar, 100 µm. (h) Green autofluorescent bodies in a fixed adult worm are costained by Amytracker 680, consistent with broader physiological prevalence of amyloid-like bodies in WT C. elegans. Specifically, gut granules, structures in the coelom, body wall and head, as well as punctate structures in embryos are strongly stained by Amytracker. However, significant permeability of Amytracker into the germline required extrusion and permeabilization of gonads, as displayed in Extended Data Fig. 6. Bars, 20 µm.

Supplementary information

Supplementary Information

Supplementary Fig. 1. V5 antibody validation for detection of GLD-1::V5 (relevant for Fig. 3b and Extended Data Fig. 3f). Supplementary Fig. 2. Effect of EGCG oxidation on rescue of the mstr phenotype (relevant for Fig. 5a). Points, selfing brood size of individual worms. One-way ANOVA with Bonferroni’s correction. n = 113 DMSO-, 89 unoxidized EGCG- and 99 oxidized EGCG-treated worms.

Reporting Summary

Peer Review File

Supplementary Tables 1–6

Supplementary Table 1: strains used in the study, including suppressors of mstr double mutants. Supplementary Table 2: BioID of MSTR-1. Quantitative values are NTPI. Supplementary Table 3: CLIP-seq of MSTR-1. DESeq2 output file. Statistical values, Benjamini–Hochberg-adjusted Wald test (DESeq2). Supplementary Table 4: RNA-seq of mstr-1 mutants. Statistical values, negative binomial exact test (edgeR). Supplementary Table 5: composition of the amyloids by MS. Values are the number of total peptides mapping to listed protein. Domain enrichment (SMART and InterPro) as well as overlapping hits with the CLIP-seq experiment are also listed. Supplementary Table 6: catalogue of reagents, antibodies, commercial kits and oligonucleotides used in the study.

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Source Data Unprocessed Blots

Unprocessed western blots for Figs. 2 and 3 and Extended Data Figs. 3 and 8.

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Eroglu, M., Zocher, A., McAuley, J. et al. Noncanonical inheritance of phenotypic information by protein amyloids. Nat Cell Biol 26, 1712–1724 (2024). https://doi.org/10.1038/s41556-024-01494-9

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