Studies in mice and men suggest that changes in environmental conditions such as nutrition, exposure to environmental pollutants or parental care during early postnatal life can affect the development, physiology and fitness of offspring. The epigenetic mechanisms underlying the transmission of such traits over one or multiple generations are largely unknown but DNA methylation, RNA and nucleosomes have been proposed as mediators of such inheritance. At the end of male germ cell development chromatin is extensively remodeled, resulting in a highly compact nuclear architecture in spermatozoa that is compatible with fertilization. During this process, most but not all nucleosomes are removed and replaced by protamines. The host laboratory recently demonstrated retained nucleosomes localize at CpG islands of the genome that are present within regulatory regions of genes, suggesting that these regions may confer epigenetic inheritance between generations. Preliminary data implicate different histone H3 variants and modifications in the nucleosome eviction and retention process.Here I aim at dissecting the mechanism of nucleosome retention in mouse sperm. Using a quantitative biochemical approach, I will test the possibility that nucleosome retention at CpG islands is an indirect consequence of high intrinsic affinities of protamines for GC-poor DNA. I will determine the composition of evicted versus retained nucleosomes in terms of histone variants and post-translational modifications, by performing immunoprecipitation coupled to mass spectrometry. Importantly, I will investigate whether nucleosome retention is controlled by a “retention factor” using a mass spectrometry approach combined with cellular and genetic assays. The proposed research will provide mechanistic insights into the mechanisms of nucleosome retention during spermatogenesis, thereby laying the foundations for further functional studies in nucleosome-based paternal epigenetic inheritance.