Nucleosome Retention in Human Sperm Figure 5. Nucleosome retention in sperm is linked to the formation of DNA methylation-free regions in the early embryo. Most (74%, 11,264/15,237) CpG islands that remain unmethylated in ES cells overlap nucleosome retention sites in sperm. In contrast, only 29% (1774/6,127) of the CpG islands that are methylated in ES cells overlap sperm nucleosome retention sites (A). CpG islands that are unmethylated in ES cells are enriched for H3K4me3 in mature sperm (B) compared to CpG islands that are methylated in ES cells (C). H3K27me3 shows moderate enrichment in sperm around CpG islands that are unmethylated in ES cells (D) compared to around CpG islands that are DNA methylated in ES cells (E). doi:10.1371/journal.pgen.1002036.g005 Figure 6. A model for nucleosome retention in human sperm. During the histone to protamine transition nucleosomes are retained at GC-rich sites which have high intrinsic affinity for nucleosomes. This results in nucleosome retention at the start sites of many genes, especially at the start sites of housekeeping genes and master regulators, as well as at distal regulatory elements. Regions that retain nucleosomes in sperm are also frequently established as free from DNA- methylation (‘Me’) in the early embryo, further suggesting a connection between the transmission of paternal nucleosomes and the establish- ment of gene regulation in the early embryo. doi:10.1371/journal.pgen.1002036.g006 [3,4,5] (see also Figure S5). Consistent with this, H3K27me3 marked regions are very similar in mature sperm and in the early embryo, suggesting that this epigenetic state might be stably maintained across generations from one germline to the next [4,5]. Similarly, we suggest that the retention of nucleosomes and activation marks (Figure S5) at housekeeping genes might be important for marking transcription start sites on the paternal genome, facilitating gene activation in the embryo. The retention of nucleosomes at distal regulatory regions defined by DNase I hypersensitive sites is also consistent with a model in which the transmission of paternal nucleosomes and their modifications influences gene expression in the early embryo. Further, we suggest that the need to retain nucleosomes in sperm may explain why widely and highly expressed genes have high nucleosome occupancy encoded at their transcription start sites in the human genome, in contrast to the nucleosome-free regions encoded in the yeast genome [29,58]. Nucleosome retention in sperm is linked to the establishment of DNA methylation-free regions in the early embryo Also consistent with a connection between nucleosome retention and gene expression in the embryos is the observation that nucleosome retention sites are established as free from DNA methylation in the early embryo. During early embryogenesis the genome-wide removal of methylation marks is followed by a wave of non-specific methylase activity [45]. Many CpG islands are protected from de novo methylation, and these islands are accurately predicted by their nucleosome retention in sperm PLoS Genetics | www.plosgenetics.org 7 April 2011 | Volume 7 | Issue 4 | e1002036