Dataset Information

Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

ABSTRACT:

Background

We have previously found substantial animal-to-animal and age-dependent variation in the response of Holstein fibroblast cultures challenged with LPS. To expand on this finding, fibroblast cultures were established from dairy (Holstein) and beef (Angus) cattle and challenged with LPS to examine breed-dependent differences in the innate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for expression differences was examined by methylated CpG island recovery assay sequencing (MIRA-Seq) analysis.

Results

The Holstein breed displayed a more robust response to LPS than the Angus breed based on RNA-Seq analysis of cultures challenged with LPS for 0, 2, and 8 h. Several immune-associated genes were expressed at greater levels (FDR < 0.05) in Holstein cultures including TLR4 at all time points and a number of pro-inflammatory genes such as IL8, CCL20, CCL5, and TNF following LPS exposure. Despite extensive breed differences in the transcriptome, MIRA-Seq unveiled relatively similar patterns of genome-wide DNA methylation between breeds, with an overall hypomethylation of gene promoters. However, by examining the genome in 3Kb windows, 49 regions of differential methylation were discovered between Holstein and Angus fibroblasts, and two of these regions fell within the promoter region (-2500 to +500 bp of the transcription start site) of the genes NTRK2 and ADAMTS5.

Conclusions

Fibroblasts isolated from Holstein cattle display a more robust response to LPS in comparison to cultures from Angus cattle. Different selection strategies and management practices exist between these two breeds that likely give rise to genetic and epigenetic factors contributing to the different immune response phenotypes.

SUBMITTER: Benjamin AL

PROVIDER: S-EPMC4806443 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Publications

Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

Benjamin Aimee L AL Green Benjamin B BB Crooker Brian A BA McKay Stephanie D SD Kerr David E DE

BMC genomics 20160324

<h4>Background</h4>We have previously found substantial animal-to-animal and age-dependent variation in the response of Holstein fibroblast cultures challenged with LPS. To expand on this finding, fibroblast cultures were established from dairy (Holstein) and beef (Angus) cattle and challenged with LPS to examine breed-dependent differences in the innate immune response. Global gene expression was measured by RNA-Seq, while an epigenetic basis for expression differences was examined by methylate ...[more]

PMID: 27009155

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Project description:BackgroundBy comparing fibroblasts collected from animals at 5-months or 16-months of age we have previously found that the cultures from older animals produce much more IL-8 in response to lipopolysaccharide (LPS) stimulation. We now expand this finding by examining whole transcriptome differences in the LPS response between cultures from the same animals at different ages, and also investigate the contribution of DNA methylation to the epigenetic basis for the age-dependent increases in responsiveness.ResultsAge-dependent differences in IL-8 production by fibroblasts in response to LPS exposure for 24 h were abolished by pretreatment of cultures with a DNA demethylation agent, 5-aza-2'deoxycytidine (AZA). RNA-Seq analysis of fibroblasts collected from the same individuals at either 5 or 16 months of age and exposed in parallel to LPS for 0, 2, and 8 h revealed a robust response to LPS that was much greater in the cultures from older animals. Pro-inflammatory genes including IL-8, IL-6, TNF-α, and CCL20 (among many other immune associated genes), were more highly expressed (FDR < 0.05) in the 16-month old cultures following LPS exposure. Methylated CpG island recovery assay sequencing (MIRA-Seq) revealed numerous methylation peaks spread across the genome, combined with an overall hypomethylation of gene promoter regions, and a remarkable similarity, except for 20 regions along the genome, between the fibroblasts collected at the two ages from the same animals.ConclusionsThe fibroblast pro-inflammatory response to LPS increases dramatically from 5 to 16 months of age within individual animals. A better understanding of the mechanisms underlying this process could illuminate the physiological processes by which the innate immune response develops and possibly individual variation in innate immune response arises. In addition, although relatively unchanged by age, our data presents a general overview of the bovine fibroblast methylome as a guide for future studies in cattle epigenetics utilizing this cell type.

Project description:BACKGROUND: Linkage disequilibrium (LD) maps can provide a wealth of information on specific marker-phenotype relationships, especially in areas of the genome where positional candidate genes with similar functions are located. A recently published high resolution radiation hybrid map of bovine chromosome 14 (BTA14) together with the bovine physical map have enabled the creation of more accurate LD maps for BTA14 in both dairy and beef cattle. RESULTS: Over 500 Single Nucleotide Polymorphism (SNP) markers from both Angus and Holstein animals had their phased haplotypes estimated using GENOPROB and their pairwise r2 values compared. For both breeds, results showed that average LD extends at moderate levels up to 100 kilo base pairs (kbp) and falls to background levels after 500 kbp. Haplotype block structure analysis using HAPLOVIEW under the four gamete rule identified 122 haplotype blocks for both Angus and Holstein. In addition, SNP tagging analysis identified 410 SNPs and 420 SNPs in Holstein and Angus, respectively, for future whole genome association studies on BTA14. Correlation analysis for marker pairs common to these two breeds confirmed that there are no substantial correlations between r-values at distances over 10 kbp. Comparison of extended haplotype homozygosity (EHH), which calculates the LD decay away from a core haplotype, shows that in Holstein there is long range LD decay away from the DGAT1 region consistent with the selection for milk fat % in this population. Comparison of EHH values for Angus in the same region shows very little long range LD. CONCLUSION: Overall, the results presented here can be applied in future single or haplotype association analysis for both populations, aiding in confirming or excluding potential polymorphisms as causative mutations, especially around Quantitative Trait Loci regions. In addition, knowledge of specific LD information among markers will aid the research community in selecting appropriate markers for whole genome association studies.

Project description:BackgroundOne of the goals of livestock genomics research is to identify the genetic differences responsible for variation in phenotypic traits, particularly those of economic importance. Characterizing the genetic variation in livestock species is an important step towards linking genes or genomic regions with phenotypes. The completion of the bovine genome sequence and recent advances in DNA sequencing technology allow for in-depth characterization of the genetic variations present in cattle. Here we describe the whole-genome resequencing of two Bos taurus bulls from distinct breeds for the purpose of identifying and annotating novel forms of genetic variation in cattle.ResultsThe genomes of a Black Angus bull and a Holstein bull were sequenced to 22-fold and 19-fold coverage, respectively, using the ABI SOLiD system. Comparisons of the sequences with the Btau4.0 reference assembly yielded 7 million single nucleotide polymorphisms (SNPs), 24% of which were identified in both animals. Of the total SNPs found in Holstein, Black Angus, and in both animals, 81%, 81%, and 75% respectively are novel. In-depth annotations of the data identified more than 16 thousand distinct non-synonymous SNPs (85% novel) between the two datasets. Alignments between the SNP-altered proteins and orthologues from numerous species indicate that many of the SNPs alter well-conserved amino acids. Several SNPs predicted to create or remove stop codons were also found. A comparison between the sequencing SNPs and genotyping results from the BovineHD high-density genotyping chip indicates a detection rate of 91% for homozygous SNPs and 81% for heterozygous SNPs. The false positive rate is estimated to be about 2% for both the Black Angus and Holstein SNP sets, based on follow-up genotyping of 422 and 427 SNPs, respectively. Comparisons of read depth between the two bulls along the reference assembly identified 790 putative copy-number variations (CNVs). Ten randomly selected CNVs, five genic and five non-genic, were successfully validated using quantitative real-time PCR. The CNVs are enriched for immune system genes and include genes that may contribute to lactation capacity. The majority of the CNVs (69%) were detected as regions with higher abundance in the Holstein bull.ConclusionsSubstantial genetic differences exist between the Black Angus and Holstein animals sequenced in this work and the Hereford reference sequence, and some of this variation is predicted to affect evolutionarily conserved amino acids or gene copy number. The deeply annotated SNPs and CNVs identified in this resequencing study can serve as useful genetic tools, and as candidates in searches for phenotype-altering DNA differences.

Dataset Information

Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

Background

Results

Conclusions

Publications

Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs without major differences in the methylome.

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