At the end of mammalian spermatogenesis, chromatin in differentiating germ cells is extensively remodeled, with the majority of nucleosomes being removed and ultimately exchanged by highly basic proteins named protamines. Residual nucleosomes are, to various degrees, retained at regulatory sequences in human and mouse sperm. Moreover, certain histone variants and modifications remain present in regulatory sequences of subsets of genes in spermatozoa, providing opportunities for paternal inheritance of chromatin states and epigenetic control of gene expression in the subsequent generation. Here we describe in detail a method that enables the generation of soluble chromatin samples from mouse and human spermatozoa within 1 d. These samples are amendable to chromatin immunoprecipitation and high-throughput sequencing of nucleosome-associated genomic DNA, which require several additional days. We also provide computational scripts that allow straightforward analysis of large genome-wide data sets by biologists with limited computational experience. This protocol will facilitate studies of mechanisms of chromatin remodeling and epigenetic reprogramming during spermatogenesis and of paternal epigenetic inheritance. Similarly, it will help in the study of the causes of human male infertility.
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We gratefully acknowledge J. Hofsteenge for critical advice during the development of this protocol. We thank T. Roloff (FMI functional genomics group), I. Nissen (Laboratory for Quantitative Genomics, Department of Biosystems Science and Engineering (D-BSSE), Basel) and the FMI animal facility for excellent assistance. S.E. is supported as a recipient of a Boehringer Ingelheim Fond fellowship. Research in the Stadler and Peters laboratories is supported by the Novartis Research Foundation and the Swiss Initiative in Systems Biology (Cell Plasticity - Systems Biology of Cell Differentation). In addition, the Peters laboratory receives funding from the Swiss National Science Foundation (31003A_125386 and National Research Programme NRP63—Stem Cells and Regenerative Medicine), the Japanese Swiss Science and Technology Cooperation Program, the FP7 Marie Curie Initial Training Network “Nucleosome4D” and the EMBO Young Investigator Program.
The authors declare no competing financial interests.
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Hisano, M., Erkek, S., Dessus-Babus, S. et al. Genome-wide chromatin analysis in mature mouse and human spermatozoa. Nat Protoc 8, 2449–2470 (2013) doi:10.1038/nprot.2013.145
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