Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men

Abstract

Background: The sperm chromatin of fertile men retains a small number of nucleosomes that are enriched at developmental gene promoters and imprinted gene loci. This unique chromatin packaging at certain gene promoters provides these genomic loci the ability to convey instructive epigenetic information to the zygote, potentially expanding the role and significance of the sperm epigenome in embryogenesis. We hypothesize that changes in chromatin packaging may be associated with poor reproductive outcome.

Methods: Seven patients with reproductive dysfunction were recruited: three had unexplained poor embryogenesis during IVF and four were diagnosed with male infertility and previously shown to have altered protamination. Genome-wide analysis of the location of histones and histone modifications was analyzed by isolation and purification of DNA bound to histones and protamines. The histone-bound fraction of DNA was analyzed using high-throughput sequencing, both initially and following chromatin immunoprecipitation. The protamine-bound fraction was hybridized to agilent arrays. DNA methylation was examined using bisulfite sequencing.

Results: Unlike fertile men, five of seven infertile men had non-programmatic (randomly distributed) histone retention genome-wide. Interestingly, in contrast to the total histone pool, the localization of H3 Lysine 4 methylation (H3K4me) or H3 Lysine 27 methylation (H3K27me) was highly similar in the gametes of infertile men compared with fertile men. However, there was a reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes. Finally, the methylation status of candidate developmental promoters and imprinted loci were altered in a subset of the infertile men.

Conclusions: This initial genome-wide analysis of epigenetic markings in the sperm of infertile men demonstrates differences in composition and epigenetic markings compared with fertile men, especially at certain imprinted and developmental loci. Although no single locus displays a complete change in chromatin packaging or DNA modification, the data suggest that moderate changes throughout the genome exist and may have a cumulative detrimental effect on fecundity.

Figure 1

Ac depiction of histone localization in each of the four donors in the HOXD cluster. Approximately, each donor has 5 million reads. The background reads in histone localization is minimal compared with patients (Fig. 2). Mapped sequencing reads were scored for enrichment (score) and for significance. Due to a low number of reads for each donor, statistical levels are much lower than the pooled (Fig. 2).

Figure 2

High-throughput sequencing of mononucleosomes reveals histone retention at the HOX loci, and relatively random histone association in most infertile patients. Mapped sequencing reads were scored for enrichment (score) and for significance. Regions significantly enriched for histone relative to the input control (sheared total sperm DNA) were identified using a 300-bp window metric. For display, we depict score and FDR window scores [–10 log(FDR)]. Note that an FDR of 20 is equal to <0.01 and FDR 40 <0.001. Each patient sample has ∼20 million reads.

Figure 3

H3K4me3 localization patterns are generally normal in the gametes of infertile men. This figure illustrates H3K4me3 retention aligned to the physical map of the HOXD locus. The y-axis depicts Storey q-value false discovery rate [–10log(FDR)]. Note that an FDR of 20 is equal to <0.01 and FDR 40 <0.001.

Figure 4

H3K27me3 localization patterns are generally normal in the gametes of infertile men. This figure illustrates H3K27me3 retention aligned to the physical map of the HOXD locus. The y-axis depicts Storey q-value false discovery rate [–10log (FDR)]. Note that an FDR of 20 is equal to <0.01 and FDR 40 <0.001.

Figure 5

A few maternally or paternally imprinted loci are improperly modified in the gametes of infertile men. (a) A depiction of two paternally imprinted genes (PEG10 and SGCE) that lack or have reduced levels of H3K4me3 in patients. (b) Representation of a maternally imprinted gene (ZNF264) that lacks K4me in fertile donors but acquires K4me in infertile men. (c, d) Represention of two properly marked maternally imprinted (H19) or paternally imprinted gene (PEG3). Asterisk represents the bisulfite amplicon.

Figure 6

DNA hypomethylation is largely maintained at developmental promoters of infertile men. Bisulfite sequencing of developmental promoters that lack or have reduced levels of K4me. CpGs are represented as open dots (if unmethylated) or filled dots (if methylated). Lines represent the number of alleles sequenced per patient.

Figure 7

DNA methylation patterns are altered at a subset of paternally imprinted loci in the gametes of infertile men. CpGs are represented as open dots (if unmethylated) or filled dots (if methylated).