Genome-wide DNA methylome variation in two genetically distinct chicken lines using MethylC-seq

Abstract

Background: DNA cytosine methylation is an important epigenetic modification that has significant effects on a variety of biological processes in animals. Avian species hold a crucial position in evolutionary history. In this study, we used whole-genome bisulfite sequencing (MethylC-seq) to generate single base methylation profiles of lungs in two genetically distinct and highly inbred chicken lines (Fayoumi and Leghorn) that differ in genetic resistance to multiple pathogens, and we explored the potential regulatory role of DNA methylation associated with immune response differences between the two chicken lines.

Methods: The MethylC-seq was used to generate single base DNA methylation profiles of Fayoumi and Leghorn birds. In addition, transcriptome profiling using RNA-seq from the same chickens and tissues were obtained to interrogate how DNA methylation regulates gene transcription on a genome-wide scale.

Results: The general DNA methylation pattern across different regions of genes was conserved compared to other species except for hyper-methylation of repeat elements, which was not observed in chicken. The methylation level of miRNA and pseudogene promoters was high, which indicates that silencing of these genes may be partially due to promoter hyper-methylation. Interestingly, the promoter regions of more recently evolved genes tended to be more highly methylated, whereas the gene body regions of evolutionarily conserved genes were more highly methylated than those of more recently evolved genes. Immune-related GO (Gene Ontology) terms were significantly enriched from genes within the differentially methylated regions (DMR) between Fayoumi and Leghorn, which implicates DNA methylation as one of the regulatory mechanisms modulating immune response differences between these lines.

Conclusions: This study establishes a single-base resolution DNA methylation profile of chicken lung and suggests a regulatory role of DNA methylation in controlling gene expression and maintaining genome transcription stability. Furthermore, profiling the DNA methylomes of two genetic lines that differ in disease resistance provides a unique opportunity to investigate the potential role of DNA methylation in host disease resistance. Our study provides a foundation for future studies on epigenetic modulation of host immune response to pathogens in chickens.

Fig. 1

Global profile of the chicken DNA methylome. a The percentage of methylcytosines identified in chicken lungs. b Distribution of DNA methylation level in the CG context. The y axis means the fraction of all mCs that display each methylation level (x axis), where the methylation level is the mC/C ratio at each cytosine. c Blue dots indicate methylcytosine density in Leghorn lungs in 10-kb windows throughout the chromosome 1. The positive and negative value of y axis is the methylation density of the sense and antisense strand respectively. d-f Logo plots of the sequences proximal to sites of CG, CHG and CHH DNA methylation in each sequence context

Fig. 2

Distribution of methylated cytosines in different genome regions. a Proportion of methylated CpG islands in different genomic regions. b Relative methylation level in gene regions (Different areas were divided by dotted lines)

Fig. 3

Promoter relative methylation level of different gene categories in the chicken genome. Box plots showed the methylation level of each gene category. Each category was compared with coding protein. miRNA (P <2.2E-16), misc_RNA (P = 0.8381), rRNA (P = 0.1472), snoRNA (P = 1.99E-14), snRNA (P = 0.08) and tRNA (P = 9.56E-4)

Fig. 4

a Promoter relative methylation level of different temporal groups. The species used for each temporal group were: TG1 (African malaria mosquito, fruitfly, nematode, Schistosoma and yellow fever mosquito), TG2 (medaka, pufferfish, trout and zebrafish), TG3 (clawed frog and tropical frog), TG4 (all chicken genes not found in the above species). b Gene body relative methylation level of different temporal groups

Fig. 5

DNA methylation distribution in the repeat sequences and pseudogenes. a Absolute methylation level (blue line) and relative methylation level (red line) in repeat elements regions. b Promoter relative methylation level of pseudogenes and corresponding genuine genes

Fig. 6

Relationship between DNA methylation and expression levels of genes in the chicken. a-b Average methylation degree across gene promoters and gene bodies. Genes were classified into five quintiles according to mRNA expression level: 1st quintile was the lowest and 5th was the highest. The promoter was defined as the region spanning from 1.5 kb upstream to 0.5 kb downstream of the transcript start site. c-d Box plots showed average methylation degree of promoters and gene bodies in each gene expression quintile

Fig. 7

Genome wide gene expression and DNA methylation variation degree between Fayoumi and Leghorn. The transcription and DNA methylation p-value of each gene between two lines were calculated by χ ² test