Endogenous retroviruses drive species-specific germline transcriptomes in mammals

Abstract

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.

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Fig. 1: Dynamic expression of repetitive elements during mouse spermatogenesis.
Fig. 2: Identification of enhancer-like ERVs in meiosis.
Fig. 3: Enhancer-like ERVs provide binding motifs for critical TFs.
Fig. 4: A-MYB acts on ERV enhancers to drive the expression of adjacent genes.
Fig. 5: ERV enhancers function to activate adjacent germline genes.
Fig. 6: Genes adjacent to rodent enhancer-like ERVKs are less conserved across species.
Fig. 7: Enhancer-like human ERVKs and ERV1s are associated with meiotic gene expression.

Data availability

The H3K27ac ChIP-seq data reported in this study are described in the accompanying study by Maezawa et al.30 and are deposited to the Gene Expression Omnibus (GEO) under accession code GSE130652. H3K27ac native ChIP-seq data in WT and A-myb mutant PSs and RNA-seq data in CRISPRa ESCs are deposited under accession code GSE142173. All other next-generation sequencing datasets used in this study are publicly available and referenced in Supplementary Data 77,9,24,28,37,43,75,76,77,78,79,80. Source data are provided with this paper.

Code availability

Source code for all software and tools used in this study, with documentation, examples and additional information, is available at following URLs: https://github.com/GenomeImmunobiology/Sakashita_et_al_2020 (best match TE annotation set), https://github.com/alexdobin/STAR (STAR RNA-seq aligner), http://crispor.tefor.net (CRISPOR), http://daehwankimlab.github.io/hisat2 (HISAT2), https://ccb.jhu.edu/software/stringtie (StringTie), https://htseq.readthedocs.io/en/master (HTSeq), https://bedtools.readthedocs.io/en/latest/content/installation.html (BEDTools), https://bioconductor.org/packages/release/bioc/html/DESeq2.html (DESeq2), https://david.ncifcrf.gov/summary.jsp (DAVID), https://software.broadinstitute.org/morpheus (Morpheus), https://software.broadinstitute.org/software/igv/igvtools (IGVTools), http://bowtie-bio.sourceforge.net/bowtie2 (bowtie2), https://www.bioinformatics.babraham.ac.uk/projects/seqmonk (SeqMonk), https://github.com/taoliu/MACS (MACS), https://github.com/shenlab-sinai/ngsplot (ngsplot), https://cran.r-project.org/web/packages/gplots/index.html (gplots), https://github.com/tidyverse/ggplot2 (ggplot2), https://cran.r-project.org/web/packages/chromoMap/vignettes/chromoMap.html#install-chromomap (chromoMap), http://homer.ucsd.edu/homer (HOMER), ·http://great.stanford.edu/public/html (GREAT), https://useast.ensembl.org/info/data/biomart/index.html (BioMart), https://imagej.net/Fiji/Downloads (Fiji, ImageJ), https://systems.crump.ucla.edu/hypergeometric/ (Hypergeometric P value calculator).

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Acknowledgements

We thank M. Weirauch and members of the Namekawa laboratory for discussion and helpful comments regarding the manuscript, the CCHMC Research Flow Cytometry Core for sharing FACS equipment (supported by NIH S10OD023410), X. Li at the University of Rochester Medical Center for sharing A-myb mutant mice, the laboratory of B. Bernstein at Massachusetts General Hospital for providing human testis H3K27ac ChIP-seq data (ENCSR136ZQZ, ENCODE) and the Transgenic Animal and Genome Editing Core at CCHMC for generating the Zfy2 enhancer-deletion mice. We acknowledge the following funding sources: Lalor Foundation Postdoctoral Fellowship and JSPS Overseas Research Fellowship to A.S.; the Research Project Grant by the Azabu University Research Services Division, Ministry of Education, Culture, Sports, Science and Technology (MEXT) Supported Program for the Private University Research Branding Project (2016–2019), a Grant-in-Aid for Research Activity Start-up (19K21196) and the Uehara Memorial Foundation Research Incentive Grant (2018) to S.M.; an Albert J. Ryan Fellowship to K.G.A.; National Institute of Health (NIH) grant DP2 GM119134 to A.B.; a March of Dimes Prematurity Research Centre Collaborative Grant (#22-FY14-470) to M.P.; NIH R01 GM122776 grant to S.H.N.

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Authors

Contributions

The manuscript was written by A.S., K.G.A. and S.H.N., with critical feedback from all other authors. A.S. and S.H.N. designed the study. S.M. performed crosslinking ChIP-seq experiments and A.S. performed native ChIP-seq experiments. A.S. analyzed A-myb mutant mice with the help of K.T. A.S. and K.T. performed CRISPRa experiments. A.S. performed immunostaining and dual-luciferase reporter assays. Y.-C.H. supervised the generation of the Zfy2 enhancer-deletion mice. A.S., K.G.A., M.Y., S.K., N.F.P., A.B., M.P. and S.H.N. designed and interpreted the computational analyses. A.S. performed the majority of computational analyses. S.H.N. supervised the project.

Corresponding author

Correspondence to Satoshi H. Namekawa.

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Competing interests

A.B. is a cofounder of Datirium, LLC.

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Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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

Extended data

Extended Data Fig. 1 Analysis of repetitive element expression during mouse spermatogenesis.

a, The RNA-seq pipeline for comprehensive quantification of TE copies. The flowchart indicates the various RNA-seq and data analysis processes that comprise the pipeline. Round-corner rectangles, input files; rectangles, output files; diamond, branch condition. The specific tools used are highlighted in red. b, The proportion of expressed and unexpressed copies of repetitive elements in each class during spermatogenesis. Of note, nearly half of rRNA genes are expressed in spermatogenic differentiation following the KIT+ spermatogonia stage.

Extended Data Fig. 2 ATAC-seq read enrichment at representative enhancer-like ERV loci and 5,000 randomly selected repetitive element loci.

a, Heatmap depicts RPKM-normalized ATAC-seq reads at enhancer-like RLTR10 and RMER17 loci (n = 694), and 5,000 randomly selected repetitive element loci in representative stages of spermatogenesis. b, Top: Venn diagram shows the intersection between total copy numbers of MMERVK10C loci (green) and total copy numbers of all RLTR10C loci (pink). Bottom: Venn diagram shows the intersection between total copy numbers of MMERVK10C loci (green) and total copy numbers of enhancer-like RLTR10C loci (red).

Extended Data Fig. 3 H3K4me3 enrichment at enhancer-like ERVs loci.

a, Average tag density plots and heatmaps show H3K27ac and H3K4me3 enrichments around enhancer-like ERVs (±1 kb around ±5 kb of ERVs) in PS. b, Scatter plot depicts H3K4me3 enrichments at enhancer-like ERV loci in PS. X-axis indicates relative distance of enhancer-like ERV loci from TSS of nearest genes. Y-axis indicates relative H3K4me3 enrichments at individual enhancer-like ERV loci. Red line shows a regression line.

Extended Data Fig. 4 The genomic features of enhancer-like ERVs in meiosis.

a, Representative track views show H3K27ac ChIP-seq, ATAC-seq, RNA-seq, and A-MYB ChIP-seq signals on chromosome X. The red highlight indicates an enhancer-like ERV locus. b, Pie charts indicate the distributions of enhancer-like ERVs on autosomes and sex chromosome. c, Top: Bar chart depicts the numbers of enhancer-like ERVs on each chromosome. Bottom: Chromosome map shows the distribution of enhancer-like ERVs throughout the mouse genome. Values for H3K27ac enrichment represent log2 fold enrichment of H3K27ac signal relative to input. d, Box-and-whisker plots show relative H3K27ac enrichment at enhancer-like ERV loci on autosomes and sex chromosomes. Values: log2 fold enrichment of H3K27ac signal relative to input. Central bars represent medians, the boxes encompass 50% of the data points, and the error bars indicate 90% of the data points. We detected no statistical difference in H3K27ac enrichment at autosome enhancer-like ERVs vs. sex chromosome enhancer-like ERVs: P = 0.307, Mann-Whitney U test. e, Bar chart shows enhancer-like ERVs distribution across genomic entities (intergenic, intronic, etc.) in autosomes versus the sex chromosomes: P = 3.6 × 10-5, Chi-square test with Yates’s correction. f, The consensus sequence of RLTR10B, listed in the Dfam database, contains two A-MYB binding motifs (GGCAGTT).

Extended Data Fig. 5 The generation of CRISPRa embryonic stem cell lines, and the evaluation of CRISPR-deletion mice.

a, qRT-PCR analyses of CRISPRa embryonic stem (ES) cells show expression level changes of the dCas9-VPR transgene 24 h after doxycycline (Dox) induction. Expression levels were normalized to the endogenous housekeeping gene Hprt. Upon addition of Dox, all ES cell clones evinced overt dCas9-VPR mRNA expression. Because clone #6 exhibited the highest upregulation of dCas9-VPR transcript, we restricted further experiments to clone #6. b, Representative image of CRISPRa ES cell colonies at day 4 after transduction with the sgRNA lentiviral construct. We validated the degree of sgRNA expression through observations of the red fluorescent reporter protein DsRed. Scale bar, 200 µm. c, Testis sections from wild-type (WT; left) and Zfy2 enhancer-deletion mice (right) at postnatal day 28 (P28). The sections were stained with hematoxylin and eosin. Scale bars, 100 μm. In our observations of Zfy2 enhancer-deletion samples, we noted no gross changes to testis morphology; however, we observed multinucleated cells (arrowheads).

Extended Data Fig. 6 The synteny of mouse meiosis-specific enhancer-like ERVs in rats and other placental mammals.

a, Pie charts indicate the genomic distribution of enhancer-like ERVs in the following genomes: mouse (mm10) and rat (rn6). Between the two species, genomic feature enrichment statistically differs: *** P < 0.001, Chi-square test with Yates’s correction. b, Representative track views show evolutionary conservation in regions adjacent to enhancer-like ERVs across several placental mammals. Red highlights indicate enhancer-like ERV loci; such loci exhibit low levels of conservation across placental mammals, including rats, a species closely related to mice.

Extended Data Fig. 7 MER57E3 is enriched in KRAB-ZF-encoding genes that have rapidly evolved in primates or humans.

a, Representative track views show H3K27ac ChIP-seq enrichment for whole, adult human testis tissue and RNA-seq signal in human KIT+ and PS. Red highlights indicate enhancer-like MER57E3s that overlap high levels of H3K27ac deposition. b, Pie charts indicate the genomic distribution of enhancer-like MER57E3 loci in the human genome (hg38). Most enhancer-like MER57E3s are located within the first intronic regions of KRAB-ZF-encoding genes.

Extended Data Fig. 8 A-MYB is highly expressed in both mouse and human spermatocytes.

a, Testis sections from mice at 12 weeks of age immunostained with antibodies raised against A-MYB (red) and γH2AX (green), and counterstained with DAPI (gray). The Roman numerals indicate stages of the seminiferous epithelium cycle. Scale bars, 20 µm. b, Representative testis sections from humans at 29-to-65 years of age immunohistochemically stained with an antibody raised against A-MYB (brown), counterstained with hematoxylin. Images of human testis sections were sourced and adapted from the Human Protein Atlas (www.proteinatlas.org/ENSG00000185697-MYBL1/tissue/testis). Scale bars, 20 µm.

Supplementary information

Reporting Summary

Supplementary Data 1

List of differentially expressed TE copies between two spermatogenic stages.

Supplementary Data 2

List of locations of meiosis-specific enhancer-like ERVs and their H3K27ac enrichments.

Supplementary Data 3

List of genes adjacent to meiosis-specific enhancer-like ERVs.

Supplementary Data 4

List of upregulated genes in Dox+, A-MYB+ and Dox+;A-MYB+ conditions.

Supplementary Data 5

List of locations of human enhancer-like ERVs in testis.

Supplementary Data 6

List of sequence information used in this study.

Supplementary Data 7

List of next-generation sequencing data from other sources.

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Sakashita, A., Maezawa, S., Takahashi, K. et al. Endogenous retroviruses drive species-specific germline transcriptomes in mammals. Nat Struct Mol Biol 27, 967–977 (2020). https://doi.org/10.1038/s41594-020-0487-4

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