Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1

Nature Structural & Molecular Biology volume 30, pages 1077–1091 (2023)Cite this article

Subjects

Abstract

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility—defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine–DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: P1(K49ac) is acquired in the spermatid nucleus in a stage-specific manner and persists in mature mouse sperm.
Fig. 2: P1(K49A) results in sperm motility defects and subfertility.
Fig. 3: P1(K49A) substitution alters sperm chromatin composition.
Fig. 4: Protamine–DNA binding ability varies with DNA length.
Fig. 5: P1(K49A) substitution alters DNA compaction and decompaction kinetics in vitro.
Fig. 6: The K49A substitution in P1 results in decreased blastocyst formation, accelerated decondensation of paternal chromatin, and altered gene expression.
Fig. 7: Defects in P1K49A/K49A males are not driven simply by changes to protamine–DNA electrostatics.

Similar content being viewed by others

Data availability

Relevant raw data are supplied in the source data file. All DNA tracking data are available at https://github.com/ReddingLab/Moritz_et_al_2021. Raw video files are available upon request. The MS proteomics data were searched against the Swissprot Mouse database and have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository109 with the dataset identifier PXD028917 for protamine PTM analysis and via MassIVE with the dataset identifier MSV000091920. All raw and processed genomic data files for single embryo sequencing and MNase-seq experiments are available under the GEO accession number GSE225271. Single-embryo RNA-seq samples were compared with previously published data across multiple embryonic stages (GSE45719). Source data are provided with this paper.

Code availability

DNA tracking code is available at https://github.com/ReddingLab/Moritz_et_al_2021.

References

  1. Tachiwana, H., Osakabe, A., Kimura, H. & Kurumizaka, H. Nucleosome formation with the testis-specific histone H3 variant, H3t, by human nucleosome assembly proteins in vitro. Nucleic Acids Res. 36, 2208–2218 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Tachiwana, H. et al. Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T. Proc. Natl Acad. Sci. USA 107, 10454–10459 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ueda, J. et al. Testis-specific histone variant H3t gene is essential for entry into spermatogenesis. Cell Rep. 18, 593–600 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Barral, S. et al. Histone variant H2A.L.2 guides transition protein-dependent protamine assembly in male germ cells. Mol. Cell 66, 89–101 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Yan, W., Ma, L., Burns, K.H. & Matzuk, M. M. HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis. Proc. Natl. Acad. Sci. USA 100, 10546–10551 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Montellier, E. et al. Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B. Genes Dev. 27, 1680–1692 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shinagawa, T. et al. Disruption of Th2a and Th2b genes causes defects in spermatogenesis. Development 142, 1287–1292 (2015).

    CAS  PubMed  Google Scholar 

  8. Meistrich, M. L., Trostle-Weige, P. K., Lin, R., Bhatnagar, Y. M. & Allis, C. D. Highly acetylated H4 is associated with histone displacement in rat spermatids. Mol. Reprod. Dev. 31, 170–181 (1992).

    Article  CAS  PubMed  Google Scholar 

  9. Shirakata, Y., Hiradate, Y., Inoue, H., Sato, E. & Tanemura, K. Histone h4 modification during mouse spermatogenesis. J. Reprod. Dev. 60, 383–387 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lu, L. Y. et al. RNF8-dependent histone modifications regulate nucleosome removal during spermatogenesis. Dev. Cell 18, 371–384 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Abe, H., Meduri, R., Li, Z., Andreassen, P. R. & Namekawa, S. H. RNF8 is not required for histone-to-protamine exchange in spermiogenesis. Biol. Reprod. 105, 1154–1159 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Sin, H.-S. et al. RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids. Genes Dev. 26, 2737–2748 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Shirley, C. R., Hayashi, S., Mounsey, S., Yanagimachi, R. & Meistrich, M. L. Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice. Biol. Reprod. 71, 1220–1229 (2004).

  14. Yu, Y. E. et al. Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice. Proc. Natl Acad. Sci. USA 97, 4683–4688 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ward, W. S. & Coffey, D. S. DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol. Reprod. 44, 569–574 (1991).

    Article  CAS  PubMed  Google Scholar 

  16. Wykes, S. M. & Krawetz, S. A. The structural organization of sperm chromatin. J. Biol. Chem. 278, 29471–29477 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Cho, C. et al. Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat. Genet. 28, 82–86 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Schneider, S. et al. Re-visiting the protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile. Sci. Rep. 6, 36764 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Takeda, N. et al. Viable offspring obtained from Prm1-deficient sperm in mice. Sci. Rep. 6, 27409 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Merges, G. E. et al. Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice. Development 149, dev200330 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Arévalo, L. et al. Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice. PLoS Genet. 18, e1010272 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Green, G. R., Balhorn, R., Poccia, D. L. & Hecht, N. B. Synthesis and processing of mammalian protamines and transition proteins. Mol. Reprod. Dev. 37, 255–263 (1994).

    Article  CAS  PubMed  Google Scholar 

  23. Yelick, P. C. et al. Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not. Mol. Cell. Biol. 7, 2173–2179 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Aoki, V. W. et al. Sperm protamine 1/protamine 2 ratios are related to in vitro fertilization pregnancy rates and predictive of fertilization ability. Fertil. Steril. 86, 1408–1415 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. de Mateo, S. et al. Protamine 2 precursors (Pre-P2), protamine 1 to protamine 2 ratio (P1/P2), and assisted reproduction outcome. Fertil. Steril. 91, 715–722 (2009).

    Article  PubMed  Google Scholar 

  26. Zatecka, E. et al. The effect of tetrabromobisphenol A on protamine content and DNA integrity in mouse spermatozoa. Andrology 2, 910–917 (2014).

    Article  CAS  PubMed  Google Scholar 

  27. Balhorn, R., Brewer, L. & Corzett, M. DNA condensation by protamine and arginine-rich peptides: analysis of toroid stability using single DNA molecules. Mol. Reprod. Dev. 56, 230–234 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Bench, G. S., Friz, A. M., Corzett, M. H., Morse, D. H. & Balhorn, R. DNA and total protamine masses in individual sperm from fertile mammalian subjects. Cytometry 23, 263–271 (1996).

    Article  CAS  PubMed  Google Scholar 

  29. Brewer, L. R., Corzett, M. & Balhorn, R. Protamine-induced condensation and decondensation of the same DNA molecule. Science 286, 120–123 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Brewer, L., Corzett, M., Lau, E. Y. & Balhorn, R. Dynamics of protamine 1 binding to single DNA molecules. J. Biol. Chem. 278, 42403–42408 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Prieto, M. C., Maki, A. H. & Balhorn, R. Analysis of DNA–protamine interactions by optical detection of magnetic resonance. Biochemistry 36, 11944–11951 (1997).

    Article  CAS  PubMed  Google Scholar 

  32. Krawetz, S. A. & Dixon, G. H. Sequence similarities of the protamine genes: implications for regulation and evolution. J. Mol. Evol. 27, 291–297 (1988).

    Article  CAS  PubMed  Google Scholar 

  33. Lewis, J. D., Song, Y., de Jong, M. E., Bagha, S. M. & Ausió, J. A walk though vertebrate and invertebrate protamines. Chromosoma 111, 473–482 (2003).

    Article  PubMed  Google Scholar 

  34. Wyckoff, G. J., Wang, W. & Wu, C.-I. Rapid evolution of male reproductive genes in the descent of man. Nature 403, 304–309 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Queralt, R. et al. Evolution of protamine P1 genes in mammals. J. Mol. Evol. 40, 601–607 (1995).

    Article  CAS  PubMed  Google Scholar 

  36. Rooney, A. P., Zhang, J. & Nei, M. An unusual form of purifying selection in a sperm protein. Mol. Biol. Evol. 17, 278–283 (2000).

    Article  CAS  PubMed  Google Scholar 

  37. Torgerson, D. G., Kulathinal, R. J. & Singh, R. S. Mammalian sperm proteins are rapidly evolving: evidence of positive selection in functionally diverse genes. Mol. Biol. Evol. 19, 973–980 (2002).

    Article  Google Scholar 

  38. Brunner, A. M., Nanni, P. & Mansuy, I. M. Epigenetic marking of sperm by post-translational modification of histones and protamines. Epigenetics Chromatin 7, 2 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Soler-Ventura, A. et al. Characterization of human sperm protamine proteoforms through a combination of top-down and bottom-up mass spectrometry approaches. J. Proteome Res. 19, 221–237 (2020).

    Article  CAS  PubMed  Google Scholar 

  40. Chira, F. et al. Phosphorylation of human sperm protamines HP1 and HP2: identification of phosphorylation sites. Biochim. Biophys. Acta 1203, 109–114 (1993).

    Article  Google Scholar 

  41. Itoh, K. et al. Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo. Sci. Signal 12, eaao7232 (2019).

    Article  PubMed  Google Scholar 

  42. Pirhonen, A., Linnala-Kankkunen, A. & Menpaa, P. H. P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species. Eur. J. Biochem. 223, 165–169 (1994).

    Article  CAS  PubMed  Google Scholar 

  43. Seligman, J., Zipser, Y. & Kosower, N. S. Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa. Biol. Reprod. 71, 1009–1015 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Gou, L. T. et al. Initiation of parental genome reprogramming in fertilized oocyte by splicing kinase SRPK1-catalyzed protamine phosphorylation. Cell 180, 1212–1227 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hogarth, C. A. et al. Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446. Biol. Reprod. 88, 40 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Griswold, M. & Hogarth, C. Synchronizing spermatogenesis in the mouse. Biol. Reprod. 107, 1159–1165 (2022).

  47. Dong, Y. et al. EPC1/TIP60-mediated histone acetylation facilitates spermiogenesis in mice. Mol. Cell. Biol. 37, e00082–17 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ketchum, C. C., Larsen, C. D., McNeil, A., Meyer-Ficca, M. L. & Meyer, R. G. Early histone H4 acetylation during chromatin remodeling in equine spermatogenesis. Biol. Reprod. 98, 115–129 (2018).

    Article  PubMed  Google Scholar 

  49. Luense, L. J. et al. Gcn5-mediated histone acetylation governs nucleosome dynamics in spermiogenesis. Dev. Cell 51, 745–758 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Shiota, H. et al. Nut directs p300-dependent, genome-wide H4 hyperacetylation in male germ cells. Cell Rep. 24, 3477–3487 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Chereji, R. V., Bryson, T. D. & Henikoff, S. Quantitative MNase-seq accurately maps nucleosome occupancy levels. Genome Biol. 20, 198 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Hammoud, S. S. et al. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460, 473–478 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hisano, M. et al. Genome-wide chromatin analysis in mature mouse and human spermatozoa. Nat. Protoc. 8, 2449–2470 (2013).

    Article  CAS  PubMed  Google Scholar 

  54. Brykczynska, U. et al. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat. Struct. Mol. Biol. 17, 679–687 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Yin, Q. et. al. Revisiting chromatin packaging in mouse sperm. Preprint at bioRxiv https://doi.org/10.1101/2022.12.26.521943 (2022).

  56. Casadio, F. et al. H3R42me2a is a histone modification with positive transcriptional effects. Proc. Natl Acad. Sci. USA 110, 14894–14899 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Balhorn, R. The protamine family of sperm nuclear proteins. Genome Biol. 8, 227 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Aoki, E. & Schultz, R. M. DNA replication in the 1-cell mouse embryo: stimulatory effect of histone acetylation. Zygote 7, 165–172 (1999).

    Article  CAS  PubMed  Google Scholar 

  59. Palmerola, K. L. et al. Replication stress impairs chromosome segregation and preimplantation development in human embryos. Cell 185, 2988–3007 (2022).

    Article  CAS  PubMed  Google Scholar 

  60. Bryant, H. E. et al. PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination. EMBO J. 28, 2601–2615 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Maya-Mendoza, A. et al. High speed of fork progression induces DNA replication stress and genomic instability. Nature 559, 279–284 (2018).

    Article  CAS  PubMed  Google Scholar 

  62. Kai, M., Boddy, M. N., Russell, P. & Wang, T. S.-F. Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress. Genes Dev. 19, 919–932 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Brooks, K. E. et al. Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development. Development 149, dev198341 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Aoki, F., Worrad, D. M. & Schultz, R. M. Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev. Biol. 181, 296–307 (1997).

    Article  CAS  PubMed  Google Scholar 

  65. Falco, G. et al. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells. Dev. Biol. 307, 539–550 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Grow, E. J. et al. p53 convergently activates Dux/DUX4 in embryonic stem cells and in facioscapulohumeral muscular dystrophy cell models. Nat. Genet. 53, 1207–1220 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Hendrickson, P. G. et al. Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons. Nat. Genet. 49, 925–934 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. De Iaco, A. et al. DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nat. Genet. 49, 941–945 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  69. De Iaco, A., Verp, S., Offner, S., Grun, D. & Trono, D. DUX is a non-essential synchronizer of zygotic genome activation. Development 147, dev177725 (2020).

    PubMed  Google Scholar 

  70. Gao, Y. et al. Protein expression landscape of mouse embryos during pre-implantation development. Cell Rep. 21, 3957–3969 (2017).

    Article  CAS  PubMed  Google Scholar 

  71. Boussouar, F. et al. A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis. Andrology 2, 351–359 (2014).

    Article  CAS  PubMed  Google Scholar 

  72. Gaucher, J. et al. Bromodomain-dependent stage-specific male genome programming by Brdt. EMBO J. 31, 3809–3820 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Mylonis, I. et al. Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis. J. Biol. Chem. 279, 11626–11631 (2004).

    Article  CAS  PubMed  Google Scholar 

  74. Wilhelm, T. et al. Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells. Proc. Natl Acad. Sci. USA 111, 763–768 (2014).

    Article  CAS  PubMed  Google Scholar 

  75. Nakatani, T. et al. DNA replication fork speed underlies cell fate changes and promotes reprogramming. Nat. Genet. 54, 318–327 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Liu, B. & Grosshans, J. in Zygotic Genome Activation. Methods in Molecular Biology Vol. 1605 (ed. Lee, K.) 11–30 (Humana Press, 2017).

  77. Blythe, S. A. & Wieschaus, E. F. Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition. Cell 160, 1169–1181 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Zeng, F., Baldwin, D. A. & Schultz, R. M. Transcript profiling during preimplantation mouse development. Dev. Biol. 272, 483–496 (2004).

    Article  CAS  PubMed  Google Scholar 

  79. Xue, Z. et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature 500, 593–597 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Ihara, M. et al. Paternal poly (ADP-ribose) metabolism modulates retention of inheritable sperm histones and early embryonic gene expression. PLoS Genet. 10, e1004317 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  81. Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  82. Liu, L., Aston, K. I. & Carrell, D. T. Protamine extraction and analysis of human sperm protamine 1/protamine 2 ratio using acid gel electrophoresis. Methods Mol. Biol. 927, 445–450 (2013).

    Article  CAS  PubMed  Google Scholar 

  83. Beausoleil, S. A., Villen, J., Gerber, S. A., Rush, J. & Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24, 1285–1292 (2006).

    Article  CAS  PubMed  Google Scholar 

  84. Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  85. Nakata, H., Wakayama, T., Takai, Y. & Iseki, S. Quantitative analysis of the cellular composition in seminiferous tubules in normal and genetically modified infertile mice. J. Histochem. Cytochem. 63, 99–113 (2015).

    Article  CAS  PubMed  Google Scholar 

  86. Herrmann, C., Avgousti, D. & Weitzman, M. Differential salt fractionation of nuclei to analyze chromatin-associated proteins from cultured mammalian cells. Bio. Protoc. 7, e2175 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  87. Garcia, B. A., Shabanowitz, J. & Hunt, D. F. Characterization of histones and their post-translational modifications by mass spectrometry. Curr. Opin. Chem. Biol. 11, 66–73 (2007).

    Article  CAS  PubMed  Google Scholar 

  88. Garcia, B. A. et al. Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat. Protoc. 2, 933–938 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. MacLean, B. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Camarillo, J. M. et al. Coupling fluorescence-activated cell sorting and targeted analysis of histone modification profiles in primary human leukocytes. J. Am. Soc. Mass. Spectrom. 30, 2526–2534 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Giorgini, F., Davies, H. G. & Braun, R. E. Translational repression by MSY4 inhibits spermatid differentiation in mice. Development 129, 3669–3679 (2002).

    Article  CAS  PubMed  Google Scholar 

  92. de Yebra, L. & Oliva, R. Rapid analysis of mammalian sperm nuclear proteins. Anal. Biochem. 209, 201–203 (1993).

    Article  PubMed  Google Scholar 

  93. Erkek, S. et al. Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa. Nat. Struct. Mol. Biol. 20, 868–875 (2013).

    Article  CAS  PubMed  Google Scholar 

  94. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  96. Zhang, Y. et al. Model-based Analysis of ChIP–seq (MACS). Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  97. Quinlan, A. R. BEDTools: The Swiss‐Army Tool for Genome Feature Analysis. Curr. Protoc. Bioinformatics 47, 11.12.1-34 (2014).

  98. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Wickham, H. ggplot2 (Springer Cham, 2016).

  100. Gallardo, I. F. et al. High-throughput universal DNA curtain arrays for single-molecule fluorescence imaging. Langmuir 31, 10310–10317 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Larson, A. G. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Yoshida, N. & Perry, A. C. Piezo-actuated mouse intracytoplasmic sperm injection (ICSI). Nat. Protoc. 2, 296–304 (2007).

    Article  CAS  PubMed  Google Scholar 

  103. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10 (2011).

    Article  Google Scholar 

  104. Andrews, S. FastQC: A quality control tool for high throughput sequence data. (2010).

  105. Wingett, S. W. & Andrews, S. FastQ Screen: a tool for multi-genome mapping and quality control. F1000Res. 7, 1338 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  106. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  107. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf. 12, 323 (2011).

    Article  CAS  Google Scholar 

  108. Deng, Q., Ramsköld, D., Reinius, B. & Sandberg, R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343, 193–196 (2014).

    Article  CAS  PubMed  Google Scholar 

  109. Perez-Riverol, Y. et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank members of the Hammoud lab for scientific discussions and comments on the manuscript; Y.-C. Hu and members of the Cincinnati Children’s Hospital Transgenic Animal and Genome Editing Core Facility; T. Saunders and members of the University of Michigan Transgenic Animal Model Core; members of the Bardwell lab for assistance with fluorescence anisotropy; H. Malik, R. Schultz, and A. Peters for scientific and experimental discussion; and H. Schorle for providing essential reagents and for scientific and experimental discussion. Portions of Figs. 2, 4, and 7 were created with BioRender.com. This research was supported by National Institute of Health (NIH) grants 1R21HD090371-01A1 (S.S.H.), 1DP2HD091949-01 (S.S.H.), R01 HD104680 01 (S.S.H.), 1R35GM147477-01 (S.R.), UCSF Program for Breakthrough Biomedical Research provided by the Sandler Foundation (S.R.), 5K12 HD065257-07 (S.B.S.), 1R03HD10150101A1 (S.B.S.), R01-AG050509 (J.N.), R01-GM120094 (J.N.), R35GM137832 (K.R.), training grants NSF 1256260 DGE (L.M.), Rackham Predoctoral Fellowship (L.M.), T32GM007315 (L.M.), an American Cancer Society Research Scholar grant RSG-17-037-01-DMC (J.N.), an American Heart Association predoctoral fellowship award ID: 830111 (R.A.), and Open Philanthropy Grant 2019-199327 (5384) (S.S.H.).

Author information

Author notes

  1. These authors contributed equally: Lindsay Moritz, Samantha B. Schon.

  2. These authors jointly supervised this work: Sy Redding, Saher Sue Hammoud.

Authors and Affiliations

  1. Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI, USA

    Lindsay Moritz & Saher Sue Hammoud

  2. Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA

    Lindsay Moritz, Mashiat Rabbani, Caleb Sultan, Jourdan Clements, Alan P. Boyle, Jun Z. Li & Saher Sue Hammoud

  3. Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA

    Samantha B. Schon & Saher Sue Hammoud

  4. Department of Obstetrics, Gynecology and Reproductive Sciences, Magee Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Yi Sheng & Kyle E. Orwig

  5. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Ritvija Agrawal & Jayakrishnan Nandakumar

  6. Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, MA, USA

    Juniper Glass-Klaiber & Sy Redding

  7. Departments of Chemistry, Molecular Biosciences, and the National Resource for Translational and Developmental Proteomics, Northwestern University, Evanston, IL, USA

    Jeannie M. Camarillo & Neil L. Kelleher

  8. Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA

    Michael R. Baldwin & Patrick J. O’Brien

  9. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

    Adam G. Diehl, Alan P. Boyle & Jun Z. Li

  10. Department of Biology, Brandeis University, Waltham, MA, USA

    Kaushik Ragunathan

  11. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    Yueh-Chiang Hu

  12. Department of Urology, University of Michigan, Ann Arbor, MI, USA

    Saher Sue Hammoud

Authors
  1. Lindsay Moritz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samantha B. Schon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mashiat Rabbani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yi Sheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ritvija Agrawal
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Juniper Glass-Klaiber
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Caleb Sultan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jeannie M. Camarillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jourdan Clements
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Michael R. Baldwin
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Adam G. Diehl
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Alan P. Boyle
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Patrick J. O’Brien
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Kaushik Ragunathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Yueh-Chiang Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Neil L. Kelleher
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Jayakrishnan Nandakumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Jun Z. Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Kyle E. Orwig
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Sy Redding
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Saher Sue Hammoud
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.S.H., L.M., and S.B.S. contributed to overall project design. L.M., S.B.S., M.R., J.G.-K., C.S., and J.C. performed experiments. Y.S. performed ICSI experiments. R.A. assisted with purification of protamines using chromatography for in vitro biochemistry. J.M.C. performed MS experiments with N.L.K.’s oversight. M.R.B. performed analysis of fluorescence anisotropy data with oversight from P.J.O. A.G.D. and A.P.B. aided in MNase-seq analysis of sperm. S.R. performed DNA curtain experiments. Y.-C.H. generated P1(K49A) mice. J.Z.L. analyzed single embryo RNA-seq data. General project insight was provided by K.R., J.N., J.Z.L., K.E.O., S.R., and S.S.H. L.M. and S.S.H. wrote the manuscript, with input from S.R. All authors provided comments on the manuscript.

Corresponding author

Correspondence to Saher Sue Hammoud.

Ethics declarations

Competing interests

The authors have no competing interests.

Peer review

Peer review information

: Nature Structural & Molecular Biology thanks Arne Gennerich, Clinton Lau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Carolina Perdigoto and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The P1 K49 residue is highly conserved across the mouse lineage and the custom antibody against P1 K49ac is specific.

(a) Alignment of P1 amino acid sequences across multiple mouse species illustrates conservation of P1. (b) Immunoblot of acid extracted protein lysates from mature sperm illustrates a clear band for P1 K49ac that is competed off only in the presence of a specific peptide containing acetylated P1 at K49 (top blot using a non-specific, unrelated peptide and bottom blot using a P1 non-acetylated peptide). Shown are representative immunoblots and similar results were obtained from n = 3 independent experiments. (c) Acid urea immunoblot of protein lysates from P1+/+, P1K49A/+, and P1K49A/K49A sperm probed for P1 K49ac illustrates specificity of the antibody. Shown is a representative blot and similar results were obtained from n = 2 experiments. (d) Quantification of synchronization efficiency in testes collected 23- and 24 days-post retinoic acid (RA) injection illustrates successful synchronization and enrichment of stage VIII-X elongating spermatids. Similar results were obtained from n = 4 independent experiments. (e) Total epididymal sperm count (left) and progressive sperm motility after 1 hour (right) for P1+/+ and P1V5/+ males (n = 5 P1+/+ males and n = 4 P1V5/+ males). Statistical test was performed using an unpaired, two-tailed t-test, p = 0.4032 for sperm count and p = 0.8787 for sperm motility. Center line represents the mean and error bars represent standard deviation. Each dot represents a measurement from a single animal. (f) Immunofluorescence of adult P1+/+ or P1V5/+ testes cross sections illustrates specificity of staining for the V5 tag. Scale bars: 20 μm. Shown are representative images and similar results were obtained from n = 3 independent experiments. (g) Immunoblots of subcellular fractions of elongating spermatids from synchronized testes lysates (days 23 and 24 post RA) for total P1 (V5-P1) and P1 K49ac. MNase, 0.5 M NaCl, 1 M NaCl, 2 M NaCl, and pellet represent nuclear fractions of increasing inaccessibility. Shown are representative immunoblots and similar results were obtained from n = 4 independent experiments. (h) Immunofluorescence of synchronized testes cross-sections days 23 and 24 post RA. Scale bars: 20 μm. Shown are representative images and similar results were obtained from n = 4 independent experiments.

Source data

Extended Data Fig. 2 P1 K49A substitution results in sperm motility defects and subfertility.

(a) List of potential off-targets and corresponding sequencing results verify no off-target modifications generated by CRISPR/Cas9 editing. (b) Testes/body weight ratio of P1+/+, P1K49A/+, and P1K49A/K49A males (n = 4 per genotype) suggests no loss of germ cell populations due to P1 K49A substitution. Each dot represents a measurement from a single animal. Statistical test was performed using a one-way ANOVA and adjusted for multiple comparisons. Center line represents the mean and error bars represent standard deviation. (c) Periodic acid Schiff (PAS)-stained adult testes cross sections highlights normal testis morphology in P1K49A/K49A males. Scale bars: 50 μm. Shown are representative images and similar results were obtained from n = 2 males. (d) Acid urea immunoblot of acid-extracted testes from P1+/+, P1K49A/+, and P1K49A/K49A males shows comparable expression of P1 across all genotypes. Shown are representative immunoblots and similar results were obtained from n = 2 independent experiments.

Source data

Extended Data Fig. 3 P1 K49A substitution results in abnormal histone retention and altered histone PTMs in mature sperm.

(a) Immunoblotting of sperm protein extracts reveals an abnormal retention of histones in P1K49A/K49A sperm. Blots were loaded by total input sperm number. Exact sperm numbers for the various antibodies provided in Methods section. (b) Quantification of immunoblots showing fold change of histone retention in P1K49A/K49A males. Data were collected from sperm from a total of n = 3 independent males per genotype. Across the 3 biological replicates, a total of n = 12 technical replicates were performed for H3, n = 9 technical replicates for H2B, and n = 7 technical replicates for H4. Each dot represents a single technical replicate measurement. Center line represents the mean and error bars represent standard deviation. (c) Immunoblots of protein lysates from P1+/+ and P1K49A/K49A elongating spermatid-enriched testes lysate illustrates no difference in ac-H4, TNP2, or TNP1 levels. Shown are representative immunoblots and similar results were obtained from n = 2 independent experiments. (d) Quantification of abundance of histone H4 K5/K8/K12/K16 acetylation retained in P1+/+ and P1K49A/K49A sperm. Each dot represents measurement from a single technical replicate (n = 3 technical replicates per genotype). Each biological sample (n = 1 per genotype) was prepared from a pool of sperm from n = 5 males per genotype. Center line represents the mean and error bars represent standard deviation. (e) Immunofluorescence staining of adult P1+/+ or P1K49A/K49A testes cross sections stained for TNP1. Scale bars: 20 μm. Shown are representative images and similar results were obtained from n = 3 independent males. (f) Pearson correlation and hierarchical clustering shows high correlation between replicates and between WT and mutant datasets. (g) Number of peaks identified in each replicate dataset. (h) Genome-wide distribution of MNase-seq reads with respect to transcriptional start sites (TSS) of coding genes from mm10 reference genome. The region in the map is centered at the TSS and spans 2.5 kb on both sides of the TSS. Average profiles across gene regions ±2.5 kb for MNase-seq reads are shown on top.

Source data

Extended Data Fig. 4 P1 K49A sperm exhibit less fixed histone retention patterns and altered histone PTMs.

(a–d) Genome browser tracks of nucleosomes enriched at developmental loci and imprinted genes. (e, f) Quantification of abundance of various histone H3 PTMs retained in P1+/+ and P1K49A/K49A sperm. Each dot represents measurement from a single technical replicate (n = 3 technical replicates per genotype) and each biological sample (n = 1 per genotype) was prepared from a pool of sperm from n = 5 males per genotype. Center line represents the mean and error bars represent standard deviation.

Extended Data Fig. 5 Purified protamines are free of contaminating histones and their binding to DNA is unaffected by incubation time.

(a) Immunoblot of input (prior to size exclusion chromatography) protein, purified P1 (WT or K49A), and purified P2 (WT or pro P2) illustrating efficient separation of P1 and P2 from each other and absence of histones in the final purified protein. Shown are representative immunoblots and similar results were obtained from n = 3 independent experiments. (b) Coomassie-stained SDS-PAGE gel of purified proteins illustrating high purity and equivalent concentrations. Shown is a representative gel and similar results were obtained from n = 4 independent experiments. (c) Proteinase K treatment of EMSA reactions after 1 hour of equilibration with DNA. (d,e) Quantification of binding affinities of WT P1 (d) and P1 K49A (e) after 10 minutes, 1 hour, or 4 hours of equilibration with DNA. Data are presented as an average of n = 4 technical replicates across n = 3 biologically independent samples for WT P1 10 minutes, n = 8 technical replicates across n = 3 biologically independent samples for WT P1 1 hour, n = 6 technical replicates across n = 3 biologically independent samples for WT P1 4 hours, n = 4 technical replicates across n = 3 biologically independent samples for P1 K49A 10 minutes, n = 9 technical replicates across n = 3 biologically independent samples for P1 K49A 1 hour, and n = 4 technical replicates across n = 3 biologically independent samples for P1 K49A 4 hours. Error bars represent standard deviation. (f, g) Quantification of binding affinities of WT P2 (f) and pro P2 (g) after 10 minutes, 1 hour, or 4 hours of equilibration with DNA. Data are presented as an average of n = 4 technical replicates across n = 3 biologically independent samples for WT P2 10 minutes, n = 9 technical replicates across n = 3 biologically independent samples for WT P2 1 hour, n = 4 technical replicates across n = 3 biologically independent samples for WT P2 4 hours, n = 3 technical replicates across n = 3 biologically independent samples for pro P2 10 minutes, n = 8 technical replicates across n = 3 biologically independent samples for pro P2 1 hour, and n = 4 technical replicates across n = 3 biologically independent samples for pro P2 4 hours. Error bars represent standard deviation. (h) Quantification of the binding affinities of P1 (either WT or K49A) and P2 (either WT or pro P2) mixed at a 1:2 ratio to a linear ~300 bp DNA fragment. Data are presented as an average of n = 4 technical replicates across n = 2 biologically independent samples for WT P1 + WT P2, n = 4 technical replicates across n = 2 biologically independent samples for WT P1 + pro P2, n = 3 technical replicates across n = 2 biologically independent samples for P1 K49A + WT P2, and n = 4 technical replicates across n = 2 biologically independent samples for P1 K49A + pro P2. Error bars represent standard deviation. (i) Representative EMSAs of titrations of increasing amounts of indicated P1 and P2 mixed at a 1:2 ratio. Data are presented as an average of n = 4 technical replicates for all protein combinations except P1 K49A + WT P2 (n = 3 technical replicates) across n = 2 biologically independent samples. Error bars represent standard deviation.

Source data

Extended Data Fig. 6 P1 K49A substitution alters DNA compaction and decompaction kinetics in vitro.

(a) Representative kymographs of WT P2 induced DNA compaction at increasing protein concentrations. (b) Representative kymographs of pro P2 induced DNA compaction at increasing protein concentrations. (c) Average DNA compaction by WT P2 at increasing concentrations. Error bars represent standard deviation (n = 71 traces for 200 nM, n = 63 for 225 nM, n = 95 for 250 nM, and n = 108 for 275 nM). (d) Average DNA compaction by pro P2 at increasing concentrations. Error bars represent standard deviation (n = 74 traces for 150 nM, n = 54 for 175 nM, n = 62 for 200 nM, n = 64 for 225 nM, and n = 65 for 250 nM). (e) Traces of individually tracked DNA molecules over time at low or high concentration of either WT P2 (left panels) or pro P2 (right panels) illustrating cooperative behavior. (f) Decompaction of DNA initially compacted by WT P2 and pro P2 over time illustrates differences in decompaction rates. Data were collected from n = 3 independent experiments (n = 3 flow cells per each independent experiment) for each protein. A total of n = 66 single DNA molecules were measured for WT P2 and n = 99 single DNA molecules were measured for pro P2. Error bars represent SEM.

Extended Data Fig. 7 P1 K49A substitution results in premature decompaction of paternal chromatin, altered DNA replication kinetics, and stalling at the zygote stage.

(a) Cartoon representation of the four main stages of DNA replication in the mouse embryo as defined by Aoki and Schultz. (b) Immunofluorescence of zygotes collected 8.5 hpf and stained for BrdU. Representative images from each category are shown for both genotypes. Male and female pronuclei were identified based on proximity to the polar body (female being closer). Scale bars: 10 μm. Shown are representative images and similar results were obtained from n = 3 independent experiments. (c) Percent of WT and mutant embryos belonging to each category of DNA replication as defined in panel a. (d) Representative mutant embryo exhibiting altered DNA replication kinetics belonging to the ‘other’ category. Scale bar: 20 μm. Shown are representative images and similar results were obtained from n = 3 independent experiments. (e) Total fluorescence intensity measurements of BrdU per embryo indicates normal progression of DNA replication through early replication, but a stalling in late replication. Intensity measurements were taken from a total of n = 5 early replicating P1+/+ embryos, n = 7 early replicating P1K49A/K49A embryos, n = 11 late replicating P1+/+ embryos, and n = 21 late replicating P1K49A/K49A embryos. Statistical tests were performed using an unpaired, two-tailed t-test, p = 0.0325 for late replication. Center line represents the median. (f) Proportion of WT and mutant embryos collected at 30 hours post ICSI injection containing micro or multiple nuclei. (g) Total Zscan4 fluorescence intensity per blastomere for WT and mutant 2 cell embryos collected 26-30 hours post fertilization highlights a decrease in Zscan4 protein in mutant embryos. Intensity measurements were taken from a total of n = 38 P1+/+ blastomeres and a total of n = 10 P1K49A/K49A blastomeres. Statistical test was performed using an unpaired, two-tailed t-test, p = 0.0339. Center line represents the median.

Source data

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Tables 1–9

Source data

Source Data Fig. 1

Unprocessed western blots

Source Data Fig. 2

Statistical Source Data

Source Data Fig. 3

Unprocessed gel and western blots

Source Data Fig. 3

Statistical Source Data

Source Data Fig. 4

Unprocessed gels

Source Data Fig. 4

Statistical Source Data

Source Data Fig. 6

Statistical Source Data

Source Data Fig. 7

Unprocessed gel and western blots

Source Data Fig. 7

Statistical Source Data

Source Data Extended Data Fig. 1

Unprocessed western blots

Source Data Extended Data Fig. 1

Statistical Source Data

Source Data Extended Data Fig. 2

Unprocessed western blots

Source Data Extended Data Fig. 2

Statistical Source Data

Source Data Extended Data Fig. 3

Unprocessed western blots

Source Data Extended Data Fig. 3

Statistical Source Data

Source Data Extended Data Fig. 5

Unprocessed western blots

Source Data Extended Data Fig. 5

Statistical Source Data

Source Data Extended Data Fig. 7

Statistical Source Data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moritz, L., Schon, S.B., Rabbani, M. et al. Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1. Nat Struct Mol Biol 30, 1077–1091 (2023). https://doi.org/10.1038/s41594-023-01033-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41594-023-01033-4

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing