Data for: A complex mechanism translating variation of a simple genetic architecture into alternative life histories

Pubertal age is an important life-history trait that is underlined by a relatively simple genetic architecture in Atlantic salmon (Salmo salar). Nearly 40% 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. 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 conditions in a recirculating aquatic facility at the University of Helsinki with natural temperature and photoperiod. 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 at -80 °C until analysis for gene expression (RNA-seq). Another testis was frozen in a cryostorage buffer containing 10% DMSO with a 1 °C/min cooling rate and stored at -80 °C until analysis chromatin modifications (H3K27ac, H3K4me3, and VGLL3 ChIPmentation). Our results showed that seasonal variation was a major driver of gene expression differences in the testes. In addition, multiple key puberty genes were upregulated in vgll3EE, compared to vgll3LL genotypes, indicating that the 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 an over-representation of corresponding cellular processes and functions, indicating that the vgll3 genotype has a 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 are 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 a wide-scale influence on cellular physiology and development through coordinated regulation of distinct genomic loci and cellular functions. 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.

Methods
Overall design:

Immature testis samples from 20 fish (11 vgll3EE and 9 vgll3LL) were analysed for gene expression. Chromatin profiles were analysed for 19 fish (9 vgll3EE and 10 vgll3LL), of which 17 were shared between RNA-seq samples. Samples represented a continuum of sampling dates from early April up to December of the second year of the fish.

RNA-seq methods:

Total RNA was extracted from flash-frozen testes using the Macherey-Nagel Nucleospin 96 RNA kit and quantified using a Qubit instrument. 100 ng of total RNA was used for RNA-seq library construction using an Illumina stranded mRNA kit and manufacturer instructions. Libraries were sequenced at the University of Helsinki Institute of Biotechnology sequencing service using a NextSeq500 instrument and 75 bp paired-end reads.

ChIPmentation methods:

Cryostored testis tissue was removed from -80 °C storage and rapidly thawed in a 37 °C water bath. Thawed tissue was rinsed with ice-cold D-PBS and transferred to a clean tube. Tissue was homogenised in 250 μl ice-cold D-PBS using an OMNI Bead Ruptor Elite instrument with 7 ml tubes and 2.4 mm ceramic beads, and a program with one burst of 5s with speed 2.4. The cell suspension was filtered through a Flowmi Cell Strainer and inspected under a microscope with Trypan blue staining. Cell suspension was diluted with room temperature D-PBS to a final volume of 450 μl and cross-linked with Diagenode ChIP crosslinking gold in 1X concentration for 30 min, followed by fixation with 1% formaldehyde for 2 min. Formaldehyde was quenched in 0.125 M glycine for 5 min and cells were collected with centrifugation at 400 g for 10 min. Cells were washed two times with 500 ul ice-cold PBS and centrifuged at 400 g for 10 min in between washes. 

Cells were subject to ChIPmentation with Thermo MAGnify ChIP-kit and Illumina Tn5 reagents as follows. Cells were collected using centrifugation, resuspended in 50 μl lysis buffer supplemented with protease inhibitors, and lysed on ice for 5 min. Chromatin was sheared in 50  μl volumes using a Bioruptor device with settings high power and 3x eight cycles of 30 s on, 30 s off. Debris was pelleted by centrifugation and sheared chromatin was diluted to four equal aliquots of 100 μl using dilution buffer supplemented with protease inhibitors. One aliquot of sheared chromatin was reserved as input control. The remaining three aliquots were immunoprecipitated in 4 °C o/n using 1  μg of Abcam ab4729, 2  μg of Abcam ab8580, and 10 μg of a custom procured anti-VGLL3 antibody on ThermoFisher Dynabeads Protein A/G. Beads were subsequently washed following MAGnify kit protocol, with an additional final wash using 150 μl of ice-cold 10 mM Tris (pH 8). Bead-bound chromatin was then treated in 20 μl volume of tagmentation reaction containing Illumina Tn5 transposase for 5 min at 37 °C. Input controls were treated with tagmentation reaction for 5 min at 55 °C. Tagmentation was terminated by adding 7.5 volumes of RIPA buffer and incubation on ice for 5 min. Chromatin was subsequently washed twice with 150 μl of ice-cold RIPA and TE buffer. Crosslinks were reversed using a proteinase-K treatment and ChIPment DNA was captured using Macherey-Nagel NucleoMag magnetic beads. ChIPmentation libraries were measured using a Qubit instrument and a control PCR was run with Nextera sequencing oligos to assess library amplification on agarose gel. Finally, libraries were indexed, pooled, and sequenced at the University of Helsinki Institute of Biotechnology sequencing and FIMM sequencing services using NextSeq500 (75 bp paired-end) and Novaseq6000 (150 bp paired-end) instruments, respectively.