Genetic regulation of sperm DNA methylation in cattle through meQTL mapping

Abstract

Background: DNA methylation (DNAm) plays an important functional role and is influenced by genetic variants known as methylation QTLs (meQTLs). The majority of meQTL studies have been conducted in human blood. Despite its unique landscape, the genetic regulation of sperm DNAm remains largely unexplored. In this study, we leveraged DNAm measured in sperm from 405 Holstein bulls using reduced representation bisulfite sequencing (RRBS) and performed sequence-level genome-wide association studies for 166,985 variable CpGs (s.d. >5%). We reported heritability estimates and have mapped both cis-meQTLs and trans-meQTLs.

Results: Heritability estimates ranged from 0 to 1 and averaged 0.26 across all selected CpGs, with 76% of estimates above 0.1. The meQTL mapping revealed that 32.9% of the CpGs had a cis-meQTL, 3.6% had a trans-meQTL and 1.0% had both cis- and trans-meQTLs. The cis-CpGs were located on average 261 kb (absolute mean) from their cis-meQTL top SNPs (defined by the most significant association). MeQTLs were enriched in featured genomic annotations, including regions surrounding transcription start sites and ATAC-seq peaks. We also identified spurious trans-associations by analyzing data across multiple genome assemblies, including the construction of a partial pangenome. Additionally, eight trans-meQTL hotspots, defined as variants associated with at least 30 trans-CpGs, were identified and overlapped with genes involved in epigenetic regulation. Using peripheral blood mononuclear cell DNAm from 54 out of the 405 bulls, we did not observe a similar effect of the trans-meQTL hotspots to that one observed in sperm.

Conclusions: For the first time, meQTLs have been detected and characterized in bovine sperm, contributing to a better understanding of the transmission of paternally inherited DNAm marks. These findings provide useful information for further research aimed at integrating epigenetic information into the prediction of performance traits.

Keywords: Cattle; DNA methylation; GWAS; MeQTL mapping; Semen.

Fig. 1

DNA methylation measured by RRBS. (a) Scatter plot of CpG methylation levels, showing the mean methylation ratio (x-axis) vs. the standard deviation (s.d.) (y-axis) for all analyzed CpGs (N = 1,212,361). Black dots, above the red line (s.d. >0.05), were retained for the study (N = 166,985). The upper and right-sided histograms represent the overall distribution of methylation levels and standard deviations, with retained CpGs highlighted in dark grey. (b) Distribution of the retained CpGs (N = 166,985) per Mb across the genome, with alternating colors distinguishing chromosomes

Fig. 2

Distribution of heritability estimates () for 166,985 CpGs with variable DNA methylation. Dashed lines represent the first quartile, the median and the third quartile

Fig. 3

Mapping of cis- and trans-methylation QTLs. (a) Distribution of meQTLs types for the 166,985 CpGs. (b) Distribution of the distance between the CpG and the top cis-SNP. (c) Significant trans associations between SNPs located on BTA9 and CpGs located on BTA3. “N” indicates the number of associated trans-CpG per SNP. Purple line = BTA9:64,209,331 − 64,210,048 bp. Blue line = BTA3:117,543,159 − 117,544,310 bp (containing up to 19 trans-CpGs). (d) Pangenome bubble within the 35 to 70 Mb region of BTA9 extracted from a pangenome graph constructed with ARS-UCD1.2 and 8 Holstein genome assemblies. Purple segment = BTA9:64,209,331 − 64,210,048 bp. Pink segment = BTA9:64,209,243 − 64,209,316 bp. Blue segment = region with a 99.74% identity with BTA3:117,543,159 − 117,544,310 bp. The reported genomic coordinates corresponded to positions within the ARS-UCD1.2 reference genome. (e) Number of trans-CpGs (y-axis) associated to each SNP (chromosome coordinates on x-axis). Red line = 30 CpGs

Fig. 4

Enrichment in genomic annotations of independent cis-CpGs, trans-CpGs and their corresponding top cis- and trans-meQTL SNPs (selected by lowest P-value) for common annotation databases (“cpgIslandExt” Table [36] from “CGI” to “open_sea”; Ensembl release 112 [35] from “TSS200” to “INTERGENIC”; and RepeatMasker [36] from “DNA” to “SINE”) or novel catalogs (CAGE annotation track [37] from “TSS+−200_CAGE_testis” to “INTERGENIC_CAGE” and ATAC-seq bovine catalog [38] from “ATAC_seq_peaks”, from which only “ubiquitous” or “testis” peaks were retained). The error bars represent the 95% confidence interval. *P < 0.05, **P < 0.01, ***P < 0.001