Author Summary In most cells, DNA is packaged by protein complexes called nucleosomes. In sperm, however, nucleosomes are only retained at a small fraction of the genome, particularly at the start sites of genes. In this work, we show that the sites at which nucleosomes are retained in sperm are specified by variation in the base composition of the human genome. At a fine scale, the human genome varies extensively in the content of GC versus AT base pairs, and we find that in both genic and non-genic regions this predicts very well where nucleosomes are retained in mature sperm. These regions include transcription start sites, especially for genes that are expressed in all cells and for genes that regulate development. We also report that regions that retain nucleosomes in sperm are likely to be protected from DNA methylation in the early embryo, suggesting a further connection between the presence of nucleosomes on the paternal genome and the establish- ment of gene regulation in the embryo. Based on these results, we propose that an important selective pressure on base composition evolution in mammalian genomes may be the requirement to organize chromatin in sperm in a way that facilitates gene regulation in the early embryo. poly(dA:dT) motifs destabilize the formation of nucleosomes [30,31]. Indeed it has long been speculated that fine-scale base composition variation in mammalian genomes may relate to chromatin structure [33]. Many transcription start sites and regulatory regions are GC-rich and are predicted to have high intrinsic nucleosome affinity [34]. However, in vivo analysis of a number of mouse GC-rich promoters reached the opposite conclusion: GC-rich promoters were depleted of nucleosomes in vivo [35]. Thus, although GC-rich sequences have high intrinsic binding specificity for nucleosomes, in somatic cells other processes such as transcription may have a more important influence on nucleosome occupancy over functionally important regions of the genome. GC-content peaks are found at the promoters of many human genes, where they are termed CpG islands because of the elevated frequency of CpG bases [36]. High CpG-content promoters are associated with both widely expressed housekeeping genes [36,37] and with developmental regulators such as transcription factors [38,39]. One major epigenetic feature of CpG islands is that they tend to be largely devoid of DNA methylation [40]. CpG sites in mammalian genomes are highly methylated, but many CpG islands are established as unmethylated regions in the early embryo (although they may later gain methylation in some cases upon differentiation [41,42]). Indeed genome-wide mapping has shown that most (but not all) CpG islands are unmethylated in human embryonic stem (ES) cells [43]. This methylation-free state, combined with the presence of transcription activation marks such as tri-methylation of histone H3 lysine 4 (H3K4me3) may maintain CpG islands accessible or ‘poised’ for transcription initiation [41]. Many CpG islands are also known to be unmethylated and associated with H3K4 methylation in sperm [4,5,42]. Across the genome in general, however, DNA methyl- ation has been reported as enriched on nucleosome bound DNA [44]. In the early embryo, genome-wide erasure of DNA methylation is followed by the de novo establishment of methylation patterns [45]. A subset of CpG sites must therefore be protected from this non-specific methylase activity. This protection may be linked to the binding of transcriptional activators [41,46,47] or the presence Nucleosome Retention in Human Sperm of H3K4me3-containing nucleosomes [48]. Importantly, DNA hypo-methylation is the reason why CpG islands maintain their high CpG content: methylated CpG sites have an elevated mutation rate due to the spontaneous deamination of methylcyt- osine to thymine, which leads to the genome-wide depletion of CpG dinucleotides outside of unmethylated regions [42,49]. Given the lack of transcription, we reasoned that the major influence of GC-content on chromatin organization might occur in the male germline rather than in somatic cells. Here we test this idea, and show that nucleosome retention in human sperm is indeed strikingly related to fine-scale base composition variation. Across both genic and non-genic regions of the genome, nucleosome retention sites are extremely well predicted by GC- composition. The retention of nucleosomes at GC-rich sequences with high intrinsic nucleosome affinity accounts for the previously reported enrichment of nucleosomes both at transcription start sites and at genes that regulate development. It also means that nucleosomes are retained at the start sites of most universally expressed genes, which may be important for their activation in the early embryo. Further, we report a striking association at CpG islands between nucleosome retention in sperm, and the establishment of unmethylated regions in the early embryo. This suggests that paternal nucleosome retention may assist in the establishment of these regions, possibly through the retention of H3K4me3-marked histones. Our findings suggest that chromatin organization in the male germline, rather than that in somatic cells, is the major functional consequence of fine-scale base composition variation in the human genome. We suggest that the selective pressure on this may be the requirement to propagate paternal epigenetic information to the embryo. Results Nucleosomes are retained in mature sperm at GC-rich loci Sites of nucleosome retention in mature human sperm were identified genome-wide by Hammoud and co-workers using micrococcal nuclease (MNase) digestion followed by deep sequencing. Comparing mononucleosome fragments to a sonicat- ed input control, 25,121 genomic regions were identified with statistically significant enrichment for sperm nucleosomes [5]. Mapping these regions onto the genome shows that they overlap peaks of high GC-content (Figure 1A, 1B). In genic regions, these peaks frequently occur at transcription start sites (Figure 1A) and also more broadly across some genes, particularly developmental regulators (Figure 1A, 1B). Considering the whole genome, there is indeed a striking correlation between GC-content and the number of sequenced mononucleosome fragments isolated from sperm (Figure 1C; Pearson correlation = 0.68; p-value,2.2610216). This is not accounted for by the known GC-bias of Solexa sequencing [50] (Figure 1D, Pearson correlation = 0.12; p-value,2.2610216). Further, GC-content also correlates with nucleosome enrichment as quantified by microarray hybridization in a second study using two different extraction protocols (micrococcal nuclease digestion and salt extraction followed by restriction digestion) [3] (Figure S1). Base composition is an excellent predictor of nucleosome retention sites across the human genome To formally assess the extent to which base composition predicts nucleosome retention in sperm, we divided the genome into non- overlapping 150-bp windows, and ranked these windows by their GC-content. Comparing this ranking to retention sites demon- strates that base composition alone is an excellent predictor of PLoS Genetics | www.plosgenetics.org 2 April 2011 | Volume 7 | Issue 4 | e1002036