In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycomb-mediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.

DNA methylation is an important epigenetic modification regulating various biological phenomena, including genomic imprinting and transposon silencing. It is known that methylation of the differentially methylated regions (DMRs) associated with paternally imprinted genes and of some repetitive elements occurs during male germ cell development in the mouse. We have performed a detailed methylation analysis of the paternally methylated DMRs (H19, Dlk1/Gtl2 and Rasgrf1), interspersed repeats [SineB1, intracisternal A particle (IAP) and Line1] and satellite repeats (major and minor) to determine the timing of this de novo methylation in male germ cells. Furthermore, we have examined the roles of the de novo methyltransferases (Dnmt3a and Dnmt3b) and related protein (Dnmt3L) in this process. We found that methylation of all DMRs and repeats occurred progressively in fetal prospermatogonia and was completed by the newborn stage. Analysis of newborn prospermatogonia from germline-specific Dnmt3a and Dnmt3b knockout mice revealed that Dnmt3a mainly methylates the H19 and Dlk1/Gtl2 DMRs and a short interspersed repeat SineB1. Both Dnmt3a and Dnmt3b were involved in the methylation of Rasgrf1 DMR and long interspersed repeats IAP and Line1. Only Dnmt3b was required for the methylation of the satellite repeats. These results indicate both common and differential target specificities of Dnmt3a and Dnmt3b in vivo. Finally, all these sequences showed moderate to severe hypomethylation in Dnmt3L-deficient prospermatogonia, indicating the critical function and broad specificity of this factor in de novo methylation.

In mammals, fusion of two morphologically distinct gametes, oocytes and spermatozoa, leads to the formation of totipotent embryos. Acquisition of totipotency is thought to be mediated by extensive epigenetic reprogramming of parental genomes, affecting DNA methylation and histone modifications, and possibly replication timing and transcriptional activity in parental specific manners [1][2][3][4] . It is currently unclear to what extent differential reprogramming of maternal and paternal genomes is due to differences in chromatin states inherited from the oocyte and spermatozoon [4][5][6][7][8][9][10][11] . Beyond DNA methylation 1,2,6,12 , it is unknown which types of parental chromatin states are maintained or reprogrammed in early embryos. If certain parental chromatin states did escape reprogramming in the early embryo, such information could constitute an "intrinsic intergenerational epigenetic program directing gene expression in the next generation 13 . If these chromatin states also escaped reprogramming during gametogenesis, the inheritance program would function transgenerationally 13 . An increasing number of studies point to inter-or transgenerational transmission of acquired phenotypic traits that are related to temporal exposure of (grand-)parents to alternative instructive environmental cues [14][15][16][17][18] . Mechanistically, such phenotypic changes may be related to (transient) alterations of an intrinsic inheritance program.A role of histones and associated posttranslational modifications in maternal and paternal transmission of intrinsic or acquired epigenetic information is largely unknown 13 . In many metazoans, male germ cells undergo during their final differentiation into sperm an extensive chromatin remodeling process during which 3 genomic DNA becomes newly packaged into a highly condensed configuration by sperm specific proteins. In mammals, removal of histones is generally not complete 10,11,[19][20][21][22][23][24] . Furthermore, remaining histones have been reported to stay associated with the paternal genome during de novo chromatin formation in the zygote following fertilization 9 .We and others recently showed that histones lasting in human sperm are to some extent enriched at regulatory sequences of genes 10,11 . We also demonstrated that H3K4me3-and H3K27me3-marked histones are retained at promoters of specific sets of genes in mouse spermatozoa 11. The extent of evolutionary conservation of nucleosome retention at gene regulatory sequences in spermatozoa and the mechanistic principles of such retention are, however, unknown.To address conservation and to dissect the molecular logic underlying nucleosome retention, we determined the genome-wide nucleosome occupancy in mouse spermatozoa that only contain 1% residual histones. We show here that combinatorial effects of sequence composition, histone variants and histone modifications uniquely determine the packaging of sperm DNA. Nucleosomes in sperm mainly localize to unmethylated CpG-rich sequences in a histone variant specif...

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.