Figure 2. The characteristic GC-content signature of human genes account for sperm nucleosome retention at transcrip- tion start sites. Human genes show a characteristic base composition Nucleosome Retention in Human Sperm signature with high GC-content at their start sites (A), which correctly predicts high nucleosomes in sperm (B). In contrast, in a somatic tissue (resting T-cells), nucleosomes are positioned around a strong nucleo- some free region at the start site, most likely due to transcription related processes (C). The high GC-content of transcription start sites means that they have high intrinsic nucleosome binding preferences (D), which correlates well with nucleosome retention in sperm, but not occupancy in somatic cells. The average plots were generated for the 4 kb region centered at the start site of all human protein-coding genes for the GC-content (A), the normalized nucleosome retention score (B), the predicted binding preferences (nucleosome model score from Kaplan et al) (D) and the shifted somatic nucleosome read count (C) measured in 150 bp windows. doi:10.1371/journal.pgen.1002036.g002 occupancy across all genes in mature sperm is very well predicted by base composition, in somatic cells this is not the case. Nucleosomes are retained at the start sites of most housekeeping genes Although mature sperm are transcriptionally inactive [52], it is possible that nucleosome retention relates to transcription earlier during male germline development. We compared retention at gene start sites to the transcription of genes in the male germline as quantified by deep sequencing [53]. Both highly-expressed and widely-expressed genes preferentially retain nucleosomes at their start sites (Figure S3). However, the association with expression level is largely accounted for by the association with the expression breadth of a gene (Figure S3). This is also confirmed when only considering mRNA detected in mature sperm (Figure S4). Indeed we find that 61% of ubiquitously expressed ‘housekeeping’ genes retain nucleo- somes at their start sites (Figure S3B), which contrasts with only 21% of tissue-specific genes. This may relate to the need to robustly express housekeeping genes in the early embryo (see Discussion). As for the general relationship between retention sites and transcription initiation sites, this preferential nucleosome retention is accounted for by local base composition variation: housekeeping genes (Figure 3A) have higher GC content at their start sites than tissue- specific genes (housekeeping genes typically have CpG-island promoters, Figure 3B), higher nucleosome affinity (Figure 3M, 3N),and higher nucleosome retention in sperm (Figure 3E, 3F). This is not the case for somatic cells (Figure 3I, 3J), where in general base composition is a poor predictor of nucleosome occupancy at the start sites of housekeeping genes. Considering the variation in base composition and nucleosome retention in sperm within and across all individual housekeeping genes confirms these conclusions (Figure 4). Base composition accounts for the retention of nucleosomes at genes for transcription factors that regulate development It was previously shown that sperm nucleosomes are also pre- ferentially found in the promoters of genes that regulate development, particularly those encoding transcription factors such as HOX proteins [3,5]. Indeed, 59% of genes (318/539) annotated with the Gene Ontology terms ‘DNA-dependent regulation of cellular transcription’ and ‘development’ retain nucleosomes at their start sites. Develop- mental transcription factors, like housekeeping genes, are also typically transcribed from CpG-island promoters. However the start sites of these developmental regulators lie within broader GC-rich regions and predicted nucleosome affinity peaks, in contrast to the sharper peaks observed at housekeeping gene starts (Figure 3C, 3O, Figure 4E). This correctly predicts the broader sperm nucleosome peak at develop- mental regulators (Figure 3G, Figure 4F), but not their nucleosome occupancy in somatic cells, where their start sites are generally depleted of nucleosomes (Figure 3K). PLoS Genetics | www.plosgenetics.org 4 April 2011 | Volume 7 | Issue 4 | e1002036