DOI: 10.20350/digitalcsic/17069
title: Data supporting manuscript: "Transgenerational effects of heat shock on Drosophila melanogaster gene regulation and life history depend on adaptive environment"

description: [Description of methods used for collection/generation of data] A full description of the methodology is available in the materials and methods section of the associated manuscript ("Transgenerational effects of heat shock on Drosophila melanogaster gene regulation and life history depend on adaptive environment"), with extra detail provided in the supplementary material of the manuscript. We provide a very brief summary here. Experimental design: Drosophila melanogaster from two populations (Manzanares in Spain and Akaa in Finland) were brought to the lab, and lab populations were set up, using the offspring of 10 F0 females from each population. In the F2, the critical thermal limits (CTMax) of female flies from each population were tested using heat ramping experiments. Then in the F3 we looked at heat shock responses by comparing female flies subject to a heat stress (one hour at 37 C) with unheated controls. Immediately following heat shock, female ovaries were dissected for RNA-seq and ATAC-seq analysis (pooled samples of 30 individuals, triplicated for each treatment) to investigate the effects on the transcriptome and epigenome. The bodies of flies prepared for ATAC-seq were prepared for DNA WGS reseq in order to characterise the presence of transposable elements (TEs) in these populations. We quantified the effect of heat shock on life history traits by measuring the number of eggs produced by 24 heat shocked and 24 control females from each population, the number that developed into pupae and adults, the viability of the eggs, and the time taken to pupate and eclose. These traits were measured in 4 separate F4 cohorts based on eggs laid in a 48 hour period: 0-2 days, 2-4 days, 4-6 days and 12-14 days. We then maintained 96 lines (24 females per treatment, 2 populations) for a further 2 generations, and in the F6 generation we repeated our RNA-seq and ATAC-seq experiments to see if there were any detectable transgenerational effects of heat shock (from the F3) on the transcriptomes or epigenomes of their great-grand-offspring. We also repeated the measurement of life history in the F7 generation (three generations after the previous phenotypic measurements) to check for transgenerational effects on these traits. Bioinformatics and statistics: RNA-seq data was analysed using kallisto, ATAC-seq data was analysed using the nextflow nf-core/atac pipeline, and the two data sets were integrated using the R package intepareto. Reference TEs were characterised using Tlex3, while non-reference TEs were characterised using the consensus insertions discovered by two packages: Temp2 and PoPoolationTE2. TE results were integrated using custom command line scripts. Differential expression and differential accessibility were assessed using DESEq2, with set analysis used to compare results between populations and across generations. Linear regression was used to assess the relationship between expression and accessibility in the F3 and between expression in the F3 and the F6 among key sets of genes. Chi-square tests were used to test for associations between TEs and specific patterns of expression and accessibility, and functional enrichment of GO terms was tested for various sets of genes specifically those that were i) both differentially expressed and differentially accessible in the F3; ii) DEG in the F3 and F6, and iii) associated with TEs and directional change in expression or accessibility (in most cases, sets of genes were population specific). The effect of heat shock or ancestral heat shock on phenotypic traits was tested using a variety of statistical models: linear mixed effects models (lmer) for ctmax and time to eclosion and pupation, generalised linear models (glm) with binomial error distribution for viability data, and glm with poisson error distributions for egg, pupae and adult counts. In most cases cohort was included as a random effect, so most models were run using glmer.
description: Heat stress will increasingly affect populations, as climate change leads to higher temperatures and more extreme events such as heat waves. Adaptation is predicted to occur over long evolutionary timescales, but recent work suggests that interactions between the epigenome and transposable elements (TEs) could link environmental acclimation with rapid adaptation. However, combining all these factors in a tractable model system is difficult; consequently little is known about how these processes interact in natural genetic backgrounds to shape evolutionarily relevant phenotypes. To investigate these interactions, we carried out laboratory experiments to measure gene expression and chromatin accessibility responses to heat shock in female D. melanogaster from arid and temperate climates and their consequences for offspring phenotypes, and associated molecular responses with population variation in TEs. We then measured the same molecular and phenotypic traits three generations later to explore transgenerational inheritance. Expression and accessibility responses to heat shock varied between populations and were influenced by the presence of TEs, with more upregulated responses in the arid population. Effects of heat shock on transcription were still detectable after three generations, especially in the arid population, with many epigenetic genes transgenerationally expressed, although this was not driven by chromatin accessibility. Heat shock had negative phenotypic consequences on the initial offspring cohort, but later arid population cohorts developed quicker than controls, indicating hormesis, an effect still present in the great-great-grandoffspring. These results demonstrate the transgenerational inheritance of a potentially beneficial hormetic phenotype and associated patterns of gene expression in a natural insect population.
description: File List: This dataset consists of 11 main directories. Within each folder is a seperate ##_README.txt with more information about the files and subdirectories within. Below is a brief description of the 10 directories: 01_interchromate_kallisto - 24 subdirectories, one for each RNA-seq sample. Within each are the kallisto results, including the abundance.tsv file, which is used for DESeq2 analysis. 02_interchromate_nfcoreatac -1 subdirectory containing peaks files for postions of open chromatin. The .bam files used by interpareto were too large to be included in this data deposit. 03_interchromate_intepareto - 8 Rdata objects, containing matched and integrated RNA and ATAC results for all 8 pairwise comparisons considered in the study. 04_interchromate_TEresults - 3 subdirectories, one for Tlex3 results, one for Temp2 results, and one for PoPoolationTE2 results 05_interchromate_TEconsensus - 6 text files. Four .lst files, including reference (ref) and non-reference (den) insertions for P1 (population 1 = Akaa) and P2 (population 2 - Manzanares). Two .txt files, showing TE-gene associations for each population 06_interchromate_downstream - 3 Rdata objects and 7 summary .txt tables. Output from the InterChromaTE_downstream_analysis_final_250203.R script 07_interchromate_GOenrich - 10 summary. txt tables. Output from GO enrichment analyses in the InterChromaTE_downstream_analysis_final_250203.R script 08_interchromate_phenotypic - 7 text files containing ctmax measures from the F2, counts of eggs, pupae and adults from the offspring of F3 and F6 flies, and age-to-pupation and age-to-eclosion data for these same F3 and F6 cohorts. 09_interchromate_scripts - 13 bash scripts, 3 R scripts and an R function used in the analysis of the aforementioned data 10_interchromate_reference - 10 text files (.txt/.bed/.tsv/.fasta) that provide information about the iso1 TE references or the identity, position or orientation of genes and transcripts within the reference genome. 11_interchromate_meta - 5 text files (.txt/.csv) that were used as meta data in the analysis of this dataset.