Molecular responses of chicken embryos to maternal heat stress through DNA methylation and gene expression: a pilot study

Abstract

Climate change, with its repercussions on agriculture, is one of the most important adaptation challenges for livestock production. Poultry production is a major source of proteins for human consumption all over the world. With a growing human population, improving poultry's adaptation to environmental constraints becomes critical. Extensive evidence highlights the influence of environmental variations on epigenetic modifications. The aim of this paper is therefore to explore chickens' molecular response to maternal heat stress. We employed Reduced Representation Bisulfite Sequencing to generate genome-wide single-base resolution DNA methylation profiling and RNA sequencing to profile the transcriptome of the brains of embryos hatched from dams reared under either heat stress (32°C) or thermoneutrality (22°C). We detected 289 significant differentially methylated CpG sites (DMCs) and one differentially methylated region (DMR) between heat stressed and control groups. These DMCs were associated with 357 genes involved in processes such as cellular response to stimulus, developmental processes, and immune function. In addition, we identified 11 genes differentially expressed between the two groups of embryos, and identified ATP9A as a target gene of maternal heat stress on offspring. This study provides a body of fundamental knowledge on adaptive mechanisms concerning heat tolerance in chickens.

Keywords: DNA methylation; chicken; embryos; epigenetics; heat stress.

Figure 1.

Experimental design.

Figure 2.

Preprocessed data. (a) Number of CpGs kept after each step of the preprocessing workflow. The 22 individual samples are shown on the x-axis. (b) Average methylation level around TSS (Transcription Start Site) regions. TSS positions were retrieved from https://www.fragencode.org/data/1_ANOT_GRCg7b.GeneEnrichedAtlasFromENS107AndNCBI106.gtf.gz.

Figure 3.

Volcano plot of CpG methylation and DMCs between HS (Heat Stress) and CT (Control). The volcano plot shows the fold-change (x-axis) vs the significance (y-axis) of the 1 075 291 CpG sites analysed. A total of 138 CpGs were hypermethylated (Hyper, blue) in HS group compared to CT group and 151 CpGs were hypomethylated (Hypo, red) in HS group compared to CT group. The CpGs showing no differential methylation between groups are shown in black (NO). The volcano plot was generated using R software (version 4.2.2).

Figure 4.

Manhattan plot of differential methylation analysis between HS and CT groups. The y-axis represents the −log10(P) values for differential analysis of DNA methylation of 1 075 291 CpG sites between Heat Stressed (HS) and Control (CT) embryos. The x-axis represents the genomic distribution of CpG sites physically mapped along the chicken genome. The different colours represent the different chromosomes.

Figure 5.

Expression pattern of two lncRNA genes (LOC121110553, LOC121110554) across 47 tissues (https://gega.sigenae.org/). The 47 tissues and their respective four letter abbreviations are: adipose tissue (adip), blood (blod), bone marrow-derived macrophages (bmdm), brain (brai), bursa of Fabricius (burs), caecal tonsil (cctl), cecum (cecm), chorioallantoic membrane of an embryo (chor), colon (coln), cerebellum (crbl), cortex (crtx), dendritic cell (denC), duodenum (duod), embryon (ember), feather (feat), gizzard (gizz), Harderian gland (hard), heart (hert), hypothalamus (hypt), ileum (ileu), isthmus (isth), jejunum (jeju), kidney (kdny), liver (livr), lung (lung), lymphocyte B (lymB), lymphocyte T CD4 and CD8 (lymT), magnum (magn), monocyte, (mono), breast muscle (mscB), IEL-NK celles (nkil), optic lobe (optc), ovary (ovry), pancreas (pcrs), pineal gland (pine), pituitary (pitu), proventriculus (pvtc), retina (rtin), skin (skin), spleen (spln), testicule (test), thrombocyte (thro), thymus (thym), thyroid gland (thyr), trachea (trch), uterus (uter), and utricle (utri).

Figure 6.

Distribution of total CpGs and DMCs (hypermethylated and hypomethylated) across the different genomic regions.

Figure 7.

Gene Ontology functional analysis of the genes related to DMCs. The clustering heat map plot of the functional sets of gene ontology (GO) terms was obtained using ViSEAGO. GO functional analysis with count showing information content and a dendrogram on enriched GO terms based on BMA semantic similarity distance and Ward’s clustering criterion.

Figure 8.

Expression and methylation level per group (CT and HS) of the four DMCs for ATP9A.