DOI: 10.5061/dryad.vhhmgqp1g
title: Data for: A complex mechanism translating variation of a simple genetic architecture into alternative life histories

description: 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.
description: Overall design: Immature testis samples from
 20 fish (11 <em>vgll3EE</em> and 9
 <em>vgll3LL</em>) were analysed for gene expression. Chromatin
 profiles were analysed for 19 fish (9 <em>vgll3EE</em> and 10
 <em>vgll3LL</em>), 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.
description: # Data for: A complex mechanism translating variation of a simple genetic
 architecture into alternative life histories 
 [https://doi.org/10.5061/dryad.vhhmgqp1g](https://doi.org/10.5061/dryad.vhhmgqp1g) This dataset contains custom R code to analyse RNA-seq and ChIPmentation data produced from Atlantic salmon testes. RNA-seq count tables, MACS3 output files, other output files, and the salmon genome version used in the analysis are included in the dataset as stand-alone files or RData objects. ## Description of the data and file structure Most primary RNA-seq data is contained in the "featureCountsEnsembl.RData" object that contains summarised RNA-seq counts over gene models (from Rsubread featureCounts). Counts can also be accessed as a stand-alone file "geneCountsEnsembl.txt". Analysis of RNA-seq data is carried out with code in the RNAseqAnalysisRsubreadCountsInteractionContinuous.Rmd file and subsequently with code in the RNAseqAnalysisWGCNAwithRUVcounts.Rmd file. ChIPmentation peak data are provided as .xls or bedgraph output files from MACS3. Transcription factor motif overrepresentation results are provided as .txt files from HOMER. ChIPmentation data are analysed with code in the ChIPmentationAllSamples.MACS3.Rmd, ChIPmentationGenotypes.MACS3.Rmd and ChIPmentationWGCNA.Rmd files. Comparison with gene expression requires that the RNA-seq analysis scripts are run first and the results saved as RData objects (provided in the dataset). ## Sharing/Access information Links to other publicly accessible locations of the data: * SRA accession PRJNA1042649 ## Usage notes The scripts in this dataset were run on R version 4.2.0 and require the following libraries: * DESeq2 * vsn * AnnotationHub * WGCNA * ggplot2 * magrittr * GenomicFeatures * ChIPpeakAnno * reshape2 * clusterProfiler * viridis * stringr * biomaRt * UpSetR * pheatmap * RColorBrewer * ggrepel * igraph * network * sna * ggraph * visNetwork * threejs * networkD3 * ndtv The workflow of the analyses follows the steps: 1. RNAseqAnalysisRsubreadCountsInteractionContinuous.Rmd 2. RNAseqAnalysisWGCNAwithRUVcounts.Rmd 3. ChIPmentationAllSamples.MACS3.Rmd 4. ChIPmentationGenotypes.MACS3.Rmd 5. ChIPmentationWGCNA.Rmd ## Files This submission contains the following files (by format): ### .txt files Files containing read counts for RNA-seq data. * geneCountsEnsembl.txt Files containing results from differential expression analysis. Filename describes tested effect (genotype "LLvsEE", sampling date "days", interaction between genotype and sampling date "int"). * sig_LLvsEE_Annot_ensgenes_NCBIannot.txt * sig_days_Annot_ensgenes_NCBIannot.txt * sig_int_Annot_ensgenes_NCBIannot.txt Files containing results from gene coexpression network analysis. Gene annotations for module genes. * magenta_ensgenes_NCBIannot.txt * cyan_ensgenes_NCBIannot.txt * coral1_ensgenes_NCBIannot.txt * lavenderblush3_ensgenes_NCBIannot.txt * lightyellow_ensgenes_NCBIannot.txt * darkgreen_ensgenes_NCBIannot.txt * blue_ensgenes_NCBIannot.txt * salmon4_ensgenes_NCBIannot.txt * midnightblue_ensgenes_NCBIannot.txt Files containing edges and nodes for specific coexpression modules. * CytoscapeInput-nodes-magenta.txt * CytoscapeInput-edges-magenta.txt * CytoscapeInput-nodes-blue.txt * CytoscapeInput-edges-blue.txt * VGLL3_blue_neighbours.txt Files containing results from transcription factor binding motif analysis in HOMER: * motifs_VGLL3StrongPromotersEnhancersEEknownResults.txt * motifs_VGLL3StrongPromotersEnhancersLLknownResults.txt Files containing ChIPmentation peaks called by MACS3 including genomic locations, read pileups, fold enrichments, and p-values. Filenames describe the antigen (H3K27ac, H3K4me3, VGLL3) and the *vgll3* genotype (EE, LL). * H3K27ac_peaks.txt * H3K4me3_peaks.txt * VGLL3broad_peaks.txt * H3K27acEE_peaks.txt * H3K4me3EE_peaks.txt * VGLL3EE_peaks.txt * VGLL3LLdown_peaks.txt * H3K27acLLdown_peaks.txt * H3K4me3LLdown_peaks.txt Files containing results of ChIPmenation and RNA-seq analysis. Each row is an expressed gene assigned to a VGLL3 promoter or enhancer. Gene sets are either unique to *vgll3* genotypes ("EEunique", "LLunique") or shared between genotypes ("shared"). * VGLL3PromotersEnhancersEEunique_ensgenes_NCBIannot.txt * VGLL3PromotersEnhancersLLunique_ensgenes_NCBIannot.txt * VGLL3PromotersEnhancersShared_ensgenes1_NCBIannot.txt * VGLL3PromotersEnhancersShared_ensgenes2_NCBIannot.txt ### RData files These files contain results and data of RNA-seq analysis in R. * featureCountsEnsembl.RData * RNAseqAnalysisRsubreadCountsInteractionContinuous.RData ### RMarkdown files These files contain scripts used in the analysis. * RNAseqAnalysisRsubreadCountsInteractionContinuous.Rmd * RNAseqAnalysisWGCNAwithRUVcounts.Rmd * ChIPmentationAllSamples.MACS3.Rmd * ChIPmentationGenotypes.MACS3.Rmd * ChIPmentationWGCNA.Rmd ### Bedgraph files These files (.bdg) contain ChIPmentation read pileups for visualization in a genome browser or R. Filename specifies individual sample (four digits), tissue (gonad "Gn"), and antigen (H3K27ac, H3K4me3, VGLL3). Information on the genotype of each individual sample and the sampling date can be found in the RNAseqAnalysisRsubreadCountsInteractionContinuous.RData object under "design". ### Other formats Genome files, genome annotations, and black-list regions were used in the analysis. * Salmo_salar-GCA_905237065.2-unmasked.sizes.genome * Salmo_salar-GCA_905237065.2-2021_07-genes.gtf * Aligned.primary.filtered.sortedByCoord.reheaded.RGadded.dupRem.blackList.bed