Epigenetic Transgenerational Inheritance of Altered Sperm Histone Retention Sites

Abstract

A variety of environmental toxicants and factors have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Epigenetic alterations in the germline (sperm or egg) are required to transmit transgenerational phenotypes. The current study was designed to investigate the potential role of histones in sperm to help mediate the epigenetic transgenerational inheritance. The agricultural fungicide vinclozolin and the pesticide DDT (dichlorodiphenyltrichloroethane) were independently used to promote the epigenetic transgenerational inheritance of disease. Purified cauda epididymal sperm were collected from the transgenerational F3 generation control and exposure lineage male rats for histone analysis. A reproducible core of histone H3 retention sites was observed using an H3 chromatin immunoprecipitation (ChIP-Seq) analysis in control lineage sperm. Interestingly, the same core group of H3 retention sites plus additional differential histone retention sites (DHRs) were observed in the F3 generation exposure lineage sperm. Although new histone H3 retention sites were observed, negligible change in histone modification (methylation of H3K27me3) was observed between the control and exposure lineages. Observations demonstrate that in addition to alterations in sperm DNA methylation and ncRNA previously identified, the induction of differential histone retention sites (DHRs) also appear to be involved in environmentally induced epigenetic transgenerational inheritance.

Figure 1

Sperm core histone retention sites. (a) Chromosomal locations of the core histone H3 retention sites in sperm identified with red arrowhead on different chromosomes. Only sites that overlap in all samples are shown. (b) Comparison of sperm core histone retention sites between five different control lineage rats. The Venn diagram shows the overlap between sperm from five different rats for significant 100 bp windows of histone retention sites. The sites are defined using a read depth threshold of 150. (c) Overlap of core histone retention sites between the F3 generation DDT H3K27me3, DDT, vinclozolin and associated control core histones. (d) List of core histone site associated genes.

Figure 2

Sperm differential histone retention sites (DHRs). (a) DDT F3 generation sperm DHRs and (b) vinclozolin F3 generation lineage DHRs. The number of DHRs found using different p-value cutoff thresholds. The all window column shows all DHRs. The multiple window column shows the number of DHRs containing at least two significant windows. (c) DDT sperm DHR locations on the individual chromosomes. All DHRs at a p-value threshold of 1e-07 are shown. (d) Vinclozolin sperm DHR locations on the individual chromosomes. All DHRs at a p-value threshold of 1e-06 are shown. (e) Overlapping DMRs between the generation sperm DHRs F3 using p < 1e-06 for the vinclozolin results and p < 1e-07 for the DDT. (f) Overlapping DHRs between the two F3 generation DHRs using p < 1e-04 for the vinclozolin and p < 1e-04 for the DDT.

Figure 3

DHRs lengths. (a) The DDT F3 generation sperm DHR lengths (kilobases). All DHRs at a p-value threshold of 1e-07 are shown. (b) The vinclozolin F3 generation sperm DHRs lengths (kilobases). All DHRs at a p-value threshold of 1e-06 are shown. (c) The DDT H3K27me3 DHRs lengths (kilobases). All DHRs at a p-value threshold of 1e-06 are shown.

Figure 4

DDT sperm H3K27me3 DHRs. (a) The number of H3K27me3DHRs found using different p-value cutoff thresholds. The all window column shows all DHRs. The multiple window column shows the number of DHRs containing at least two significant windows. (b) Chromosomal DDT H3K27me3 DHRs locations on the individual chromosomes. All DHRs indicated with arrowheads at a p-value threshold of 1e-06 are shown. (c) Overlap of the core histones and the DDT H327me3 DHRs and the DDT or vinclozolin F3 generation sperm DHRs. (d) DHR associated gene categories and DHR associated genes for DDT DHRs (gray bar) and vinclozolin DHRs (black bar) with the number of genes versus the specific gene categories presented.