Epigenetic transgenerational actions of vinclozolin on promoter regions of the sperm epigenome

Abstract

Previous observations have demonstrated that embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes transgenerational adult onset disease such as male infertility, kidney disease, prostate disease, immune abnormalities and tumor development. The current study investigates genome-wide promoter DNA methylation alterations in the sperm of F3 generation rats whose F0 generation mother was exposed to vinclozolin. A methylated DNA immunoprecipitation with methyl-cytosine antibody followed by a promoter tilling microarray (MeDIP-Chip) procedure was used to identify 52 different regions with statistically significant altered methylation in the sperm promoter epigenome. Mass spectrometry bisulfite analysis was used to map the CpG DNA methylation and 16 differential DNA methylation regions were confirmed, while the remainder could not be analyzed due to bisulfite technical limitations. Analysis of these validated regions identified a consensus DNA sequence (motif) that associated with 75% of the promoters. Interestingly, only 16.8% of a random set of 125 promoters contained this motif. One candidate promoter (Fam111a) was found to be due to a copy number variation (CNV) and not a methylation change, suggesting initial alterations in the germline epigenome may promote genetic abnormalities such as induced CNV in later generations. This study identifies differential DNA methylation sites in promoter regions three generations after the initial exposure and identifies common genome features present in these regions. In addition to primary epimutations, a potential indirect genetic abnormality was identified, and both are postulated to be involved in the epigenetic transgenerational inheritance observed. This study confirms that an environmental agent has the ability to induce epigenetic transgenerational changes in the sperm epigenome.

Figure 1. Chromosomal distribution of the transgenerational change in DNA methylation in promoter regions identified by MeDIP followed by comparative chip hybridizations.

The red arrow indicates regions with confirmed methylation change through mass spectrometry. Blue arrows indicate regions in which change was not able to be confirmed, either because of insufficient CpG site measurement through mass spectrometry or not changed. Grey arrows indicate regions where primers could not be designed to test them. Green arrow indicates a transgenerational copy number variation (CNV) event in Fam111a.

Figure 2. Comparison of the methylation signal in regions where transgenerational methylation change is confirmed between vinclozolin and control.

Analysis of methylation through MeDIP followed by comparative hybridization is graph on right with genome location and log signal intensity presented with shaded area being the differential methylation region. Heavy red line indicates control and blue line indicates vinclozolin and arrow indicates transcriptional start site and direction. The graph on left is the bisulfite mass spectrometry analysis of CpG sites within the bar in shaded area of right graph indicated with percent methylation data presented for each gene (a-n). Horizontal axis shows chromosomal localizations. Inset legend presented in Figure 4.

Figure 3. Refer to Figure 2 Legend.

Figure 4. Refer to Figure 2 Legend.

Figure 5. Comparison of the transgenerational methylation change observed between vinclozolin and control in (a) KCNE2 and (b) RGD1561412/Olr735.

Analysis of methylation through MeDIP followed by comparative hybridization is graph on top with genome location and log signal intensity presented with shaded area being the differential methylation region. Heavy red line indicates control and blue line indicates vinclozolin and arrow indicates transcriptional start site and direction. The graph on bottom left is the bisulfite mass spectrometry analysis of CpG sites within bar in shaded area of the top graph indicated with percent methylation data presented. Horizontal axis shows chromosomal localizations. In addition, comparison between methylation for these genes in individual animal samples of DNA is shown using pyrosequencing, bottom right graph with percent methylation presented for individual animals (n = 6) mean± SEM for specific CpG in the differential methylation region. Horizontal axis shows chromosomal localizations.

Figure 6. Identification of a DNA sequence motif EDM1 (Environmental Induced Differential Methylation Consensus Sequence 1).

(a) Logo representation of the motif EDM1. This motif was obtained with the GLAM2 tool of MEME suite from the set of 16 regions that had been confirmed with mass spectrometry to present transgenerational changes in methylation. Glam2 score value for this motif is 194.184 (b) Results obtained when scanning EDM1 with GLAM2SCAN for prevalence of matches against four sets of sequences: (i) the 16 promoters containing the regions positively confirmed to be changed, (ii) the 48 promoters containing the regions confirmed to show change in the array, (iii) a set of 125 random promoters, and (iv) a set of 75 imprinted promoter regions from mouse and rat databases. Results shown are from matches in GLAM2SCAN scoring equal or higher than the cut-off value of 20.

Figure 7. Analysis of similarities between EDM1 and eukaryotic transcription factor binding sites with the STAMP tool.

The five top changes and respective significance values are shown with E value, sequence alignment and logo for each.

Figure 8. Identification of differential DNA methylation site and associated copy number variation.

(a) Comparison of the transgenerational methylation change observed between vinclozolin F3 generation sperm (VNG) and control F3 generation sperm (CTR) in Fam111a. Analysis of methylation through MeDIP followed by comparative hybridizations of methylated DNA immuno-precipitations and mass spectrometry analyses are shown. (b) Comparative genomic hybridization signals between F3 generation VNG and CTR sperm are shown for Fam111a. (c) Comparative genomic hybridization signals between VNG and CTR are shown for RGD1561412/Olr735.