P-523 Single molecule chromatin footprinting reveals only low occurrence of nucleosomes at multiple loci throughout chromatin of human and mouse spermatozoa

Abstract

Abstract Study question What fraction of spermatozoa from mouse and human semen contain nucleosomes at specific loci? What chromatin features affect nucleosome retention in spermatozoa? Summary answer On average, 1-2% mouse spermatozoa and 5-9% human spermatozoa contain nucleosomes at sampled genomic loci, with moderately higher frequencies at unmethylated regions of DNA. What is known already Nucleosomes constitute the basic packaging and gene regulatory unit of DNA in eukaryotic cells. During final stages of spermatogenesis in mammals, however, most nucleosomes are replaced by protamines. Previous global chromatin immunoprecipitation-sequencing studies reported the presence of nucleosomes at gene regulatory and other genomic regions. Such nucleosomes were proposed to convey intergenerational inheritance of epigenetic information. Additional studies revealed that infertile men with decreased success of assisted reproductive therapy have protamine deficiency or displayed more widely distributed nucleosomes in their sperm genome. Direct links between altered levels of nuclear proteins and fertilizing potential of sperm have, however, not been established. Study design, size, duration We conducted a case study on nucleosome retention levels in sperm from four selected fertile human samples. This study was complemented in mice, in which we characterized the dynamic changes in nucleosome frequencies at genomic loci during differentiation of haploid spermatids into spermatozoa, and their responses in mutant spermatozoa lacking DNA methylation at certain loci. Participants/materials, setting, methods We used the single-molecule sequencing technique called Nucleosome Occupancy and Methylome Sequencing to quantify nucleosome occupancy rates at selected loci in spermatozoa from ejaculated semen of four fertile semen donors. We also profiled round spermatids, early and late elongating spermatids and spermatozoa of C57BL/6J wild-type mice as well as spermatozoa of mice conditionally deficient for the methyltransferases Dnmt3a and Dnmt3b, lacking DNA methylation at certain genomic regions. Main results and the role of chance To our knowledge, this is the first study to quantify nucleosome occupancy in mouse male gametes and human spermatozoa at single-molecule resolution. In mice, we observed that chromatin remodelling during spermatid elongation resulted in a progressive loss of nucleosomes and their positioning, a transient gain in global chromatin accessibility which was succeeded by increasing chromatin inaccessibility and nuclear compaction. In both human and mouse sperm, we profiled nucleosome occupancy levels at different genomic loci and measured only low percentages, ranging from ∼1-2% in mouse and 5-9% in human. In both organisms we observed a near two-fold higher nucleosome occupancy at loci being unmethylated at their DNA over loci being highly methylated. Mice partially deficient in genomic DNA methylation manifested a similar fold increase in nucleosome occupancy in absence of DNA methylation. These data reveal a moderate modulatory role for DNA methylation established in spermatogonial stages in regulating nucleosome eviction during spermiogenesis. The overall low nucleosome occupancy frequencies in mammalian spermatozoa suggest a stochastic or random rather than a programmed role of nucleosomes in regulating paternal gene expression during ensuing embryonic development. Limitations, reasons for caution Amplicon-based sequencing provides deep sequencing coverage for selected genomic loci. We lack, however, information on the distribution of nucleosomes among loci within the genome of single spermatozoa. In human, we studied a limited number of human donors. Wider implications of the findings This study resolves a long-standing question on the frequency and local distribution of nucleosomes in murine and human spermatozoa. The variability in nucleosome occupancy between spermatozoa within and between samples of human individuals inspires further investigations into the relationship between chromatin states and quality of individual spermatozoa for embryonic development. Trial registration number SNCTP000003654