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Parallel evolution of male germline epigenetic poising and somatic development in animals

Nature Genetics volume 48, pages 888–894 (2016)Cite this article

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A Corrigendum to this article was published on 28 September 2016

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Abstract

Changes in gene regulation frequently underlie changes in morphology during evolution, and differences in chromatin state have been linked with changes in anatomical structure and gene expression across evolutionary time. Here we assess the relationship between evolution of chromatin state in germ cells and evolution of gene regulatory programs governing somatic development. We examined the poised (H3K4me3/H3K27me3 bivalent) epigenetic state in male germ cells from five mammalian and one avian species. We find that core genes poised in germ cells from multiple amniote species are ancient regulators of morphogenesis that sit at the top of transcriptional hierarchies controlling somatic tissue development, whereas genes that gain poising in germ cells from individual species act downstream of core poised genes during development in a species-specific fashion. We propose that critical regulators of animal development gained an epigenetically privileged state in germ cells, manifested in amniotes by H3K4me3/H3K27me3 poising, early in metazoan evolution.

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Figure 1: Gene expression and chromatin state in the mammalian germ line.
Figure 2: The poised chromatin state in the mammalian germ line.
Figure 3: Core poised genes are conserved regulators of tissue patterning.
Figure 4: Gain of poising at genes with species-specific developmental roles.
Figure 5: Conservation of poising in the metazoan germ line.

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Change history

  • 18 July 2016

    In the version of this article initially published online, the following callouts to supplementary items or main figure panels were incorrect: in the legend to Figure 1, the callout to Supplementary Figure 4 should have referred to Supplementary Figure 2; on page 4 of the PDF, the callout to Figure 4a should have also called out Figure 4b; on page 6 of the PDF, the callout to Supplementary Table 4 should have referred to Supplementary Table 5 and the callout to Supplementary Figure 9b should have referred to Supplementary Figure 10b; and on page 9 of the PDF, the callout to Supplementary Table 2 should have referred to Supplementary Data. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank H. Skaletsky for statistical advice and analysis; R. Young for advice on ChIP-seq analysis and critical reading of the manuscript; and P. Reddien for critical reading of the manuscript. This project was funded by an HHMI award to D.C.P., by a Hope Funds for Cancer Research postdoctoral fellowship to B.J.L., and by a Burroughs-Wellcome Career Award to B.J.L.

Author information

Authors and Affiliations

  1. Whitehead Institute, Cambridge, Massachusetts, USA

    Bluma J Lesch & David C Page

  2. Infertility Center of St. Louis, St. Luke's Hospital, St. Louis, Missouri, USA

    Sherman J Silber

  3. Department of Biology, University of Texas at San Antonio, San Antonio, Texas, USA

    John R McCarrey

  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    David C Page

  5. Howard Hughes Medical Institute, Whitehead Institute, Cambridge, Massachusetts, USA

    David C Page

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  1. Bluma J Lesch
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Contributions

B.J.L. designed the project, conducted experiments, analyzed data, and wrote the manuscript. D.C.P. designed the project and wrote the manuscript. S.J.S. provided human testis samples and contributed to writing the manuscript. J.R.M. isolated germ cells for all samples and contributed to writing the manuscript.

Corresponding author

Correspondence to David C Page.

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Integrated supplementary information

Supplementary Figure 1 Assessment of sample purity and quality.

(a) Hematoxylin and eosin staining of formaldehyde-fixed, paraffin-embedded sections from a human testis biopsy collected concurrently with the sample used for cell sorting. Left, 10× magnification; right, 40× magnification of the boxed region at left. (b) Phase-contrast microscopy images of dissociated spermatogenic cells before StaPut and of sorted pachytene spermatocyte and round spermatid populations after StaPut. All images are shown at 40× magnification. A small population of contaminating red blood cells, which do not contain chromatin, was present in the human round spermatid sample shown (arrows). (c) Numbers of sorted cells (of a total of 100) from the fractions shown that are identifiable as belonging to the reported cell type population in each fraction.

Supplementary Figure 2 Correspondence between H3K4me3, H3K27me3, and expression levels.

(a–e) Heat maps showing mean expression level as a function of H3K4me3 and H3K27me3 quantile, as shown in Figure 1b. Each pachytene spermatocyte and round spermatid sample is shown separately for human (a), rhesus (b), mouse (c), bull (d), and opossum (e). The plots for human replicate 2 are identical to those shown in Figure 1b and are reproduced here for comparison.

Supplementary Figure 3 Correlations between biological replicates.

(a) Correlation of normalized, input-subtracted ChIP-seq signal or normalized RNA-seq counts by gene. Pearson’s correlation coefficient (r) is shown for each. P < 2.2 × 10−16 for all correlations. (b) Overlap between poised gene sets called for each human, rhesus, mouse, or opossum replicate.

Supplementary Figure 4 Principal-component and clustering analysis of H3K4me3, H3K27me3, and RNA-seq data.

(a) Principal-component analysis. Axes represent the first two principal components, labeled with the percentage of total variance explained by each. Left, H3K4me3 signal; middle, H3K27me3 signal; right, expression (FPKM). (b) Clustering of H3K4me3 data with and without inclusion of an outlier data set. Top, clustering of H3K4me3 data reproduced from Figure 1c; bottom, the same clustering analysis after removal of the pachytene sample from human replicate 1 (“human 1 p.s.”), the least well correlated of the biological replicates. Removal of the outlier does not alter the clustering result.

Supplementary Figure 5 Effect of changing ChIP and expression thresholds on the number of poised genes called.

(a) Top left, numbers of five-mammal poised genes called as expression and ChIP thresholds vary; H3K4me3 and H3K27me3 thresholds are set as equal in all conditions. Top right, numbers of five-mammal poised genes called as H3K4me3 and H3K27me3 thresholds vary relative to each other; the expression threshold is held constant at FPKM ≤ 5. Dashed lines show the gene space included by the criteria used in this study. Bottom, example values for numbers of five-mammal poised genes called using different combinations of H3K4me3, H3K27me3, and expression thresholds. (b) Numbers of genes meeting each threshold independently (expression, H3K4me3, or H3K27me3) in each sample. (c) Numbers of poised genes for each overlap condition among five mammalian species.

Source data

Supplementary Figure 6 Effect of changing ChIP and expression thresholds on the number of poised genes called in only one of five mammalian species.

(a) Left, total numbers of species-specific poised genes called as H3K4me3 and H3K27me3 ChIP thresholds vary; the expression threshold is held constant at FPKM ≤ 5. The red dashed line on the plot shows the gene space included by the criteria in this study. Right, sample values for numbers of total species-specific poised genes called using different combinations of H3K4me3, H3K27me3, and expression thresholds. (b) Numbers of species-specific poised genes called for individual species as H3K4me3 and H3K27me3 ChIP thresholds vary, with the expression threshold held constant at FPKM ≤ 5.

Supplementary Figure 7 ChIP signal at sequential ChIP–qPCR targets.

(a) Input-subtracted ChIP signal tracks for the promoters targeted in mouse sequential ChIP–qPCR experiments (Fig. 2c). (b) Input-subtracted ChIP signal tracks for the promoters targeted in opossum sequential ChIP–qPCR experiments (Fig. 2d).

Supplementary Figure 8 Characteristics of core poised genes.

(a) Distribution of core poised genes on human chromosomes. Top, absolute numbers of poised genes on each human chromosome; middle, numbers of poised genes corrected for chromosome length; bottom, numbers of poised genes corrected for chromosome gene density, expressed as the percentage of all genes on the chromosome that are poised. (b) Conservation of promoter regions (1 kb upstream of the transcription start site) for human core poised genes, human genes with conserved retention of H3K27me3 only, human-specific poised genes, and all other genes. Horizontal bars represent the median. ***P < 0.001 by two-sided Welch t test. phastCons score is derived from multiple alignments of 99 vertebrate genomes to the human genome. (c) Class distribution of all transcription factors encoded in the human genome (compare to Fig. 3b).

Source data

Supplementary Figure 9 Additional examples of genes poised specifically in only one of five mammalian species.

(a) Human-specific poised genes. HIPK2 has gained a human-specific enhancer that functions in early limb development8. ITGB2 exhibits human-specific gain of active chromatin at its promoter in developing brain9. LMF1 is expressed in human but not mouse or bull placenta32. (b) Mouse-specific poised gene. Smug1 is expressed in mouse but not human or bovine placenta32. For HIPK2, ITGB2, and LMF1, only the region surrounding the TSS is shown to fit all tracks on the same page.

Supplementary Figure 10 Poised genes in Drosophila germ cells and in human and mouse spermatozoa.

(a) Polycomb enrichment at orthologs of five-mammal poised genes in the Drosophila melanogaster germ line. ChIP–microarray data are from ref. 50. P value was calculated by two-sided Welch t test. (b) Fraction of poised genes marked by both H3K4me3 and H3K27me3 in mature human and mouse spermatozoa. Data are from ref. 19 (human) and ref. 20 (mouse). ***P < 1 × 10−15 (Fisher’s exact test).

Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Note and Supplementary Table 10. (PDF 3986 kb)

Supplementary Table 1

Summary of Illumina libraries and alignments. (XLSX 47 kb)

Supplementary Table 2

Poised genes by species. (XLSX 318 kb)

Supplementary Table 3

Two-way poised gene overlaps. (XLSX 46 kb)

Supplementary Table 4

Core poised genes and assigned protein function categories. (XLSX 63 kb)

Supplementary Table 5

GO category enrichments. (XLSX 387 kb)

Supplementary Table 6

Genes poised in only one of five mammalian species. (XLSX 115 kb)

Supplementary Table 7

Motifs gained in promoters of genes poised in only one of five mammalian species. (XLSX 57 kb)

Supplementary Table 8

Poised gene set overlaps among five mammalian species. (XLSX 49 kb)

Supplementary Table 9

Poised genes in chicken male germ cells and gene set overlaps among six amniote species. (XLSX 218 kb)

Supplementary Data

Tab-delimited text files containing input data. (ZIP 8609 kb)

Supplementary Code

R scripts used for analyses in the manuscript. (ZIP 20 kb)

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Lesch, B., Silber, S., McCarrey, J. et al. Parallel evolution of male germline epigenetic poising and somatic development in animals. Nat Genet 48, 888–894 (2016). https://doi.org/10.1038/ng.3591

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