Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans

Abstract

In the fetal mouse testis, PIWI-interacting RNAs (piRNAs) guide PIWI proteins to silence transposons but, after birth, most post-pubertal pachytene piRNAs map to the genome uniquely and are thought to regulate genes required for male fertility. In the human male, the developmental classes, precise genomic origins and transcriptional regulation of postnatal piRNAs remain undefined. Here, we demarcate the genes and transcripts that produce postnatal piRNAs in human juvenile and adult testes. As in the mouse, human A-MYB drives transcription of both pachytene piRNA precursor transcripts and messenger RNAs encoding piRNA biogenesis factors. Although human piRNA genes are syntenic to those in other placental mammals, their sequences are poorly conserved. In fact, pachytene piRNA loci are rapidly diverging even among modern humans. Our findings suggest that, during mammalian evolution, pachytene piRNA genes are under few selective constraints. We speculate that pachytene piRNA diversity may provide a hitherto unrecognized driver of reproductive isolation.

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Fig. 1: Three classes of human postnatal piRNA genes.
Fig. 2: Three groups of adult samples.
Fig. 3: Dysregulated expression of A-MYB and HIWI in group 3 adult testis.
Fig. 4: Feedforward regulation of piRNA production by A-MYB is conserved in macaque.
Fig. 5: Comparative genomic analysis of human piRNA genes.
Fig. 6: Sequence variation for different genomic features within the human population.

Data availability

Sequencing data are available from the National Center for Biotechnology Information Sequence Read Archive using accession number PRJNA506245 and from the Gene Expression Omnibus using accession number GSE135791.

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Acknowledgements

We thank D. Conte and members of the Zamore Laboratory for discussions and comments on the manuscript, and K. Orwig for providing testis specimens. The silhouettes have not been altered in any way. This work was supported in part by National Institutes of Health grants (nos. R37GM062862 to P.D.Z. and P01HD078253 to Z.W. and P.D.Z.).

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Contributions

D.M.Ö., Z.W. and P.D.Z. conceived and designed the experiments. D.M.Ö., H.M., I.G., C.C., K.C. and P.-H.W. performed the experiments. Y.T., D.M.Ö., Y.K., K.F. and A.K. analysed the sequencing data. D.M.Ö., Z.W. and P.D.Z. wrote the manuscript.

Corresponding authors

Correspondence to Zhiping Weng or Phillip D. Zamore.

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Extended data

Extended Data Fig. 1 Strategy for defining human post-natal piRNA genes.

a, Strategy for transcriptome assembly, identification of piRNA precursor transcripts, and annotation of genomic loci. Pink rectangles: input for analyses; gray rectangles: bioinformatic tools. Green hexagons: output from analyses. b,c, Histograms showing the change in the abundance of piRNAs produced from piRNA genes between juvenile and adult samples. The analysis was conducted twice: once with the set of samples used to define the piRNA genes (b) and then with an independent set of samples to validate the annotations (c). d, Genomic positions of 182 human piRNA-producing loci on the 22 autosomal chromosomes. Two pre-pachytene piRNA genes were identified on the X chromosome.

Extended Data Fig. 2 Examples of human post-natal piRNA genes.

a, An exemplary pre-pachytene piRNA gene. b, Uni- and bi-directional pachytene piRNA genes. Human piRNA cluster boundaries annotated in Ref. 10 (dark green) and Ref. 11 (purple).

Extended Data Fig. 3 Analyses of transcription start sites (TSS) and transcript 3′ ends for human piRNA genes.

a, Heatmaps displaying the H3K4me3 ChIP-seq signal around the transcription start sites of piRNA-producing genes from adult testis and the results of Cap-seq and PAS-seq around TSS and transcript 3′ ends for piRNA-producing genes from adult testes. Kidney serves as a negative control. Data are reported as uniquely mapping reads per million reads (RPM). b, Metagene plot of Cap-seq and PAS-seq signals 5 kb upstream of the TSS and 5 kb downstream of the transcript 3′ ends of pre-pachytene and pachytene piRNA genes for juvenile and adult testis samples. Graphs report trimmed mean (that is, lowest and highest 5% removed). c, piRNA density per 1 kb within exons and introns of the pre-pachytene and pachytene piRNA genes defined by our analysis.

Extended Data Fig. 4 Three classes of human post-natal piRNA genes.

a, Cumulative distributions of the percentage of 25–31 nt long piRNAs explained by the length of annotated genomic sequence for the piRNA-producing loci defined here or previously10,11,12. b, Percentage of transposon sequences present in piRNA-producing, protein-coding, and lncRNA genes. Gray line indicates the transposons content of the entire human genome. c, Percentage of different classes of transposons within the piRNA-producing genes.

Extended Data Fig. 5 Characterization of the mouse repro9 mutation within exon 6 of A-Myb.

a, The repro9 mutation creates a stronger 5′ splice site within exon 6 of A-Myb, leading to a truncated mRNA. Splice site strength was determined using MaxEntScan13. b, Heatmaps of the relative abundance of mouse piRNAs mapping to previously defined piRNA genes14. piRNA abundance was normalized to the total number of mapped reads. Spg: spermatogonia; SpI: primary spermatocytes.

Extended Data Fig. 6 Three groups of adult testes defined by length distribution of total piRNAs and A-MYB and HIWI expression.

a, The abundance of piRNAs was normalized to the total number of genome-mapping reads. b,c, Relative protein abundance of A-MYB (b) and HIWI (c) in adult testis samples. ACTIN serves as a loading control, while mouse A-Mybrepro9 and Miwi−/− mutant testis lysates provide negative controls. Each lane contained 75 µg protein of testis lysate.

Extended Data Fig. 7 Molecular characterization of group 3 testes.

a, piRNA length distribution, hematoxylin and eosin (H&E) stained testis sections, and immunohistochemical detection of A-MYB and HIWI for representative samples from groups 1, 2, and 3. b, Scatter plot of steady-state transcript abundance of transcripts in group 1 versus group 3 testes. Each dot represents mean abundance of an mRNA. c, Gene ontology analysis of mRNAs detected in group 3 samples and whose abundance changed >3-fold (FDR <0.05) compared to group 1.

Extended Data Fig. 8 Three classes of human post-natal piRNA genes expressed by group 1, group 2, and juvenile testis samples.

a, Histogram shows the change in the abundance of piRNAs produced from piRNA genes between juvenile and healthy adult testis samples (groups 1 and 2). b, MA plot showing change in mean piRNA abundance comparing healthy adult (groups 1 and 2) to juvenile testis samples for 182 annotated piRNA-producing loci.

Extended Data Fig. 9 Comparative analysis of human piRNA-producing genes.

a, Long RNA and piRNA abundance for different genomic features including piRNA-producing genes; protein-coding genes; lincRNA genes; and 22,604 randomly selected, 10 kb, non-transcribed genomic regions. b, DNA sequence conservation of different genomic features for 46 eutherian mammals calculated using PhyloP15. The 22,604 randomly selected, 10 kb, non-transcribed genomic regions, which do not produce piRNAs, provide a background control.

Extended Data Fig. 10 mRNA abundance of transcripts from orthologous genes that produce pre-pachytene piRNAs in humans and evolutionary classes of human pachytene piRNA genes.

a, Transcript abundance in representative primate, rodent, marsupial, and bird species for mRNAs that produce piRNAs in placental mammals and for mRNAs expressed in other species but that make piRNAs only in humans. Expression data for species other than human testis was obtained from EMBL-EMI Expression Atlas. b, An exemplary pre-pachytene piRNA gene that is syntenic across other species, but piRNA source in placental mammals only, in primates only, in humans only. c, Abundance of piRNAs from the syntenic locations for human pachytene piRNA loci in other animals. Credit: silhouettes from http://phylopic.org. Rat by Rebecca Groom; https://creativecommons.org/licenses/by/3.0/. Opossum by Sarah Werning; https://creativecommons.org/licenses/by/3.0/.

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Özata, D.M., Yu, T., Mou, H. et al. Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans. Nat Ecol Evol 4, 156–168 (2020). https://doi.org/10.1038/s41559-019-1065-1

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