Multiomics of Atlantic salmon testis

Purpose:Pubertal age is an important life-history trait that is underlined by a relatively simple genetic architecture in Atlantic salmon (Salmo salar). Nearly 40 percent of pubertal age variation in natural populations is explained by genetic variation cosegregating with the transcription cofactor gene vestigial-like 3 (vgll3). Using controlled-crossed salmon homozygous for either the late (L) or early (E) maturation conferring allele of vgll3, we investigated the molecular mechanisms mediating vgll3 association with pubertal age.Method:Salmon were produced by controlled crosses of gametes from alternative homozygous vgll3 genotypes from the Oulujoki stock obtained from the Finnish Natural Resources Institute (LUKE). Salmon were raised in common garden condition in a recirculating aquatic facility at the University of Helsinki with natural temperature and photo period. Individually tagged and genotyped males were sacrificed and dissected periodically during the second year of growth and testis samples were taken from phenotypically immature males. One testis from each fish was flash-frozen on liquid nitrogen and stored in -80 C until analysis for gene expression (RNA-seq). Another testis was frozen in a cryostorage buffer containing 10 percent DMSO with a 1 C/min cooling rate and stored in -80 C until analysis chromatin modifications (H3K27ac, H3K4me3, and VGLL3 ChIPmentation).Results:Our results showed that seasonal variation was a major driver of gene expression differences in the testis. In addition, multiple key puberty genes were upregulated in vgll3EE, compared to vgll3LL genotypes, indicating that vgll3 genotype mediates pubertal age differences by coordinated regulation of diverse cellular pathways including hormonal signalling, cell motility, TGFb-signalling, and cellular metabolism. Gene co-expression modules differentially expressed between vgll3 genotypes showed over-representation of corresponding cellular processes and functions, indicating that vgll3 genotype has large-scale influence on signalling pathway activity. Using ChIPmentation in paired samples from the same individuals, we identified enhancers (H3K27ac), promoters (H3K27ac & H3K4me3), and VGLL3 binding regions to test if vgll3 function directly mediates the differences in gene expression and cellular phenotypes observed. Vgll3 genotypes showed marked differences in the activity of VGLL3 regulatory elements that associated with unique cellular functions in each genotype, for example, signaling receptors and cell adhesion genes in vgll3EE and regulators of cell cycle progression in vgll3LL. Furthermore, the majority of DEGs between vgll3 genotypes were associated with VGLL3 binding regions, suggesting that differential expression may be directly mediated by functional differences in VGLL3 protein. Taken together, these results indicate that VGLL3 is widely associated with gene regulatory regions in immature testes and suggest that vgll3 genotype has wide-scale influence on cellular physiology and development through coordinated regulation of distinct genomic loci and cellular functions.Conclusions:Despite the relatively simple genetic architecture of pubertal age variation in Atlantic salmon, the mechanism acting through the transcription cofactor vgll3 integrates the regulation of multiple distinct signaling pathways and developmental programs. Overall, our results exemplify a hidden complexity of molecular mechanisms mediating the large, pleiotropic effect of single genes on alternative life histories.