Nucleosome Retention in Human Sperm Figure 1. Base composition predicts sites of nucleosome retention in human sperm. Nucleosome retention sites (red) across two representative genomic regions coincide with many transcription start sites and also with local peaks of high GC-content (black). Broader retention is seen at two transcription factors that regulate development, ALX3 (A) and FOXB1 (B), and this also correlates with broader regions of high GC- content. The plots were generated using the UCSC genome browser. GC-content correlates strongly with the number of sequenced reads from mononucleosome-enriched fractions of the sperm genome (C). In comparison, there is only a very weak correlation between GC-content and the number of sequenced reads from the input genomic control (D). GC-content is an excellent predictor of regions of nucleosome retention in sperm across the human genome (E). ROC curves are shown for predictions across the genome in 150 bp windows using either GC- or CpG-content. CpG islands are also excellent predictors of sites of nucleosome retention in sperm (x2 –test, p-value,2.2610216, see also Figure S6). doi:10.1371/journal.pgen.1002036.g001 sperm nucleosome retention sites across the entire genome (Figure 1E). In a receiver operating characteristic (ROC) analysis, the area under the curve (AUC) is equal to 0.89. This means that for a randomly chosen pair of windows, one with retained nucleosomes and one without, there is an 89% probability of GC- content correctly classifying the two regions. Using CpG-content as a predictor provides similar performance (Figure 1E), and nucleosomes are particularly retained in annotated CpG islands (Figure 1E). As genic regions tend to be GC-rich, we then split the genome into genic and non-genic portions (excluding 1 kb around transcription start sites from the non-genic regions) and evaluated the ability of base composition to predict nucleosome retention in both fractions of the genome. Prediction was equally good in both cases, with ROC AUC = 0.89 for both the genic and non-genic portions of the genome. Base composition accounts for the preferential retention of nucleosomes at transcription start sites Previously it was reported that nucleosome retention sites are enriched in gene promoters [3,5] (see also Figure S2). More than a third of nucleosome retention regions (9,068/25,121) are located within 50 bp of a known start site (Figure S2). In contrast, only 2.9% of retention sites (718/25,121) are located at the 39end of genes. Plotting the GC-content variation across all human genes reveals a peak at transcription start sites (Figure 2A), which closely mirrors both the nucleosome retention in sperm (Figure 2B) and the predicted in vitro nucleosome affinity variation (Figure 2D). In contrast, in a somatic cell (T-cell) nucleosome occupancy is not well predicted by base composition (Figure 2C), most likely because of the influence of transcription and additional DNA binding proteins [25,51]. Thus, whereas the typical nucleosome PLoS Genetics | www.plosgenetics.org 3 April 2011 | Volume 7 | Issue 4 | e1002036