Figure 4. GC-content predicts sperm nucleosome retention at individual genes. GC-content (A,C,E) and sperm nucleosome retention (B,D,F) around the transcription start site of housekeeping genes (A–B), tissue-specific genes (C–D), and transcription factors that regulate development (E–F). Each row of the heat map is an individual gene. Genes are clustered according to their GC-content and the same gene ordering is used in the nucleosome retention plots. In both cases values are calculated in 150 bp windows. doi:10.1371/journal.pgen.1002036.g004 Nucleosome Retention in Human Sperm nucleosome retention is accounted for by local base composition variation along the human genome. Nucleosome retention in sperm is linked to the establishment of DNA methylation-free regions in the early embryo One of the most striking epigenetic events in the early embryo is the de novo genome-wide re-establishment of DNA methylation at CpG sites [55]. Recently it was shown that many, but not all, CpG islands are protected from this de novo wave of methylation [41]. Given that nucleosomes are also enriched at many CpG islands in sperm (Figure 1E) we investigated whether these two phenomena might be linked. Strikingly, we observed a strong association between nucleosome retention at CpG islands in sperm and the establishment of unmethylated regions in the early embryo (Figure 5A). Considering all CpG islands in the genome, sperm nucleosome retention predicts the establishment of an unmethy- lated region with a precision of 86%, and correctly identifies 74% of all unmethylated regions. Unmethylated CpG islands in the early embryo are also strongly associated with H3K4me3 in mature sperm (Figure 5B), and to a lesser degree with H3K27me3 (Figure 5D). Thus, the retention of nucleosomes in sperm, and the modification H3K4me3, are directly or indirectly linked to the establishment of DNA methylation-free regions in the early embryo. Discussion Nucleosome retention in sperm, rather than occupancy in the soma, may be the major functional consequence of base composition variation in the human genome In mature sperm only a minority of the genome remains bound by nucleosomes [5]. We have shown here that nucleosome retention sites defined genome-wide in sperm by MNase digestion are strikingly predicted by the fine-scale GC-content variation along the human genome. In both genic and non-genic regions of the genome, base composition is likely to be the primary determinant of nucleosome retention (Figure 6). High GC-content is associated with an increased binding affinity for nucleosomes in vitro [31,32], which suggests that intrinsic binding preferences may account for much of the retention biases observed in sperm. It also suggests that the major consequence of intrinsic binding affinity variation along the human genome may be chromatin organization in transcription- ally quiescent sperm, rather than that in transcriptionally active somatic cells. Consistent with this, inhibiting RNA polymerase in yeast results in nucleosome occupancy that more closely matches the predicted in vitro binding preferences [24]. We note, however, that in vitro binding preferences, as quantified in a sequence-based model, are a slightly poorer genome-wide predictor of nucleosome retention in sperm than GC-content alone (data not shown). This suggests that other factors such as competition with protamines or transition proteins, CpG-binding proteins [56] or the process of DNA demethylation earlier in germline development [57] might also be important. Nucleosome retention may relate to the need for robust gene activation and silencing in the early embryo The transmission of paternal nucleosomes [16] and their modifications [17] to the zygote could influence gene activity in the embryo [19,20,21]. For example, the inheritance of H3K4me3 and H3K27me3 at developmental loci might be important for establishing a robust silent or ‘poised’ state in the early embryo PLoS Genetics | www.plosgenetics.org 6 April 2011 | Volume 7 | Issue 4 | e1002036