panel B. Genes are sorted according to expression level and split in ten equally sized bins. The x-axis shows the average expression level of the genes in each bin. The y-axis shows the percentage of genes in a bin that have a start site within 50 bp from a region of nucleosome retention in sperm. From this figure it is also apparent that when controlling for expression level, the expression breadth of a gene correlates with the likelihood of nucleosome retention at the gene start (e.g. compare high expression level bins for genes expressed in one versus ten tissues). (EPS) Figure S4 Relationship between nucleosome retention at transcription start sites and the expression level or breadth of mRNAs detected as present in the mature sperm of at least seven out of thirteen healthy sperm donors. (A) The percentage of genes that retain nucleosomes at their start increases with gene expression level (522 genes per bin, x2 -test for trend in proportions = 158.9, p-value,2.2610216). (B) Expression breadth shows strong correlation with nucleosome retention at the gene start (x2 -test for trend in proportions = 512.8, p-val- ue,2.2610216). The number of genes per bin are; 166 (0 tissues), 632 (1 tissue), 293 (2 tissues), 304 (3 tissues), 273 (4 tissues), 233 (5 tissues), 283 (6 tissues), 318 (7 tissues), 419 (8 tissues), 763 (9 tissues), 5,222 (10 tissues). (C) When considering genes expressed in one tissue only, i.e. tissue-specific genes, the trend between nucleosome retention and expression level in the germline is greatly reduced (135 genes per bin, x2 -test for trend in proportions = 1.158, p-value = 0.28). (D) When considering genes expressed in all tissues, i.e. housekeeping genes, the trend between nucleosome retention and expression level in the germline is also greatly reduced (522 genes per bin, x2 -test for trend in proportions = 0.041, p-value = 0.84). All genes with testes expres- sion .0 and with present mRNA in at least seven out of thirteen healthy sperm donors are considered here (N = 5,222). Genes are considered to be expressed in a tissue when their expression level is . = 0.5 RPKM. Genes are ranked according to increasing expression level in testes and split in bins with the same number of genes. The x-axis shows the average expression level of the genes in each bin. The y-axis shows the percentage of genes in a References 1. Puwaravutipanich T, Panyim S (1975) The nuclear basic proteins of human testes and ejaculated spermatozoa. Exp Cell Res 90: 153–158. 2. Ward WS, Coffey DS (1991) DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 44: 569–574. 3. Arpanahi A, Brinkworth M, Iles D, Krawetz SA, Paradowska A, et al. (2009) Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences. Genome Res 19: 1338–1349. 4. Brykczynska U, Hisano M, Erkek S, Ramos L, Oakeley EJ, et al. (2010) Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat Struct Mol Biol 17: 679–687. 5. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, et al. (2009) Distinctive chromatin in human sperm packages genes for embryo development. Nature 460: 473–478. 6. Pogany GC, Corzett M, Weston S, Balhorn R (1981) DNA and protein content of mouse sperm. Implications regarding sperm chromatin structure. Exp Cell Res 136: 127–136. 7. de Yebra L, Ballesca JL, Vanrell JA, Bassas L, Oliva R (1993) Complete selective absence of protamine P2 in humans. J Biol Chem 268: 10553–10557. 8. Brewer L, Corzett M, Lau EY, Balhorn R (2003) Dynamics of protamine 1 binding to single DNA molecules. J Biol Chem 278: 42403–42408. 9. Ward WS (2009) Function of sperm chromatin structural elements in fertilization and development. Mol Hum Reprod 16: 30–36. 10. Cho C, Willis WD, Goulding EH, Jung-Ha H, Choi YC, et al. (2001) Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet 28: 82–86. 11. Cho C, Jung-Ha H, Willis WD, Goulding EH, Stein P, et al. (2003) Protamine 2 deficiency leads to sperm DNA damage and embryo death in mice. Biol Reprod 69: 211–217. Nucleosome Retention in Human Sperm bin that have a start site within 50 bp from a region of nucleosome retention in sperm. (EPS) Figure S5 Sperm H3K4me3, H3K27me3 and H2A.Z retention at the start sites of housekeeping, tissue-specific and developmental regulatory genes. (A–C) Retention of H3K4me3 at the start sites of the three gene classes is similar to nucleosome retention, shown in main Figure 3E–3G. (D–F) Retention of H3K27me3 appears to be enriched around the start sites of developmental regulators, as previously noted by Hammoud et al and Brykczynska et al. (G–I) Histone variant H2A.Z shows no enriched at gene start sites. This agrees with the observation that this histone variant is only enriched in pericentric heterochromatin. (EPS) Figure S6 Correlations between sperm nucleosome retention and GC- or CpG-content. Each point shows the correlation for all unique 150-bp windows in the human genome with the same number of G+C nucleotides (A) or CpG dinucleotides (B). GC- content correlates robustly with nucleosome retention over a wide range of window CpG-contents, whereas CpG content only correlates strongly with retention at high GC-contents. The analysis was carried out on windows with at least 5 reads from the histone and the input control dataset, as in Figure 1. Correlation values are Pearson correlation coefficients and error bars represent 95% confidence intervals. 95% of all windows fall within the data ranges shown here. (EPS) Acknowledgments We thank members of the Genetics Systems Laboratory for comments on the manuscript. Author Contributions Conceived and designed the experiments: TV BL. Performed the experiments: TV. Analyzed the data: TV BL. Wrote the paper: TV BL. 12. Haueter S, Kawasumi M, Asner I, Brykczynska U, Cinelli P, et al. (2010) Genetic vasectomy-overexpression of Prm1-EGFP fusion protein in elongating spermatids causes dominant male sterility in mice. Genesis 48: 151–160. 13. Gardiner-Garden M, Ballesteros M, Gordon M, Tam PP (1998) Histone- and protamine-DNA association: conservation of different patterns within the beta- globin domain in human sperm. Mol Cell Biol 18: 3350–3356. 14. Wykes SM, Krawetz SA (2003) The structural organization of sperm chromatin. J Biol Chem 278: 29471–29477. 15. Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CW (1987) Sequence-specific packaging of DNA in human sperm chromatin. Science 236: 962–964. 16. van der Heijden GW, Ramos L, Baart EB, van den Berg IM, Derijck AA, et al. (2008) Sperm-derived histones contribute to zygotic chromatin in humans. BMC Dev Biol 8: 34. 17. van der Heijden GW, Derijck AA, Ramos L, Giele M, van der Vlag J, et al. (2006) Transmission of modified nucleosomes from the mouse male germline to the zygote and subsequent remodeling of paternal chromatin. Dev Biol 298: 458–469. 18. Puschendorf M, Terranova R, Boutsma E, Mao X, Isono K, et al. (2008) PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos. Nat Genet 40: 411–420. 19. Carone BR, Fauquier L, Habib N, Shea JM, Hart CE, et al. Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell 143: 1084–1096. 20. Ng SF, Lin RC, Laybutt DR, Barres R, Owens JA, et al. (2010) Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring. Nature 467: 963–966. 21. Youngson NA, Whitelaw E (2008) Transgenerational epigenetic effects. Annu Rev Genomics Hum Genet 9: 233–257. PLoS Genetics | www.plosgenetics.org 10 April 2011 | Volume 7 | Issue 4 | e1002036