ATAC-Seq identifies regions of open chromatin in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus

- chromatin in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus.
- DNA-based biomarkers contributing to BRD resistance are potentially present in non-protein-coding regulatory regions of the genome, which can be determined using ATAC-Seq.
- The objectives of this study were to: (i) identify regions of open chromatin in DNA extracted from bronchial lymph nodes (BLN) of healthy dairy calves experimentally challenged with BRSV and compare them with those from non-challenged healthy control calves, (ii) elucidate the chromatin regions that were differentially or uniquely open in the BRSV challenged relative to control calves, and (iii) compare the genes found in regions proximal to the differentially open regions to the genes previously found to be differentially expressed in the BLN in response to BRSV and to previously identified BRD susceptibility loci..
- Seventy-six and 150 of the genes that were previously found to be differentially expressed using RNA-Seq, were located within 2 kb downstream of the differentially accessible regions, and of the regions uniquely open in BRSV challenged calves, respectively..
- Pathway analyses within ClusterProfiler indicated that these genes were involved in immune responses to infection and participated in the Th1 and Th2 pathways, pathogen recognition and the anti-viral response.
- Conclusions: The identified open chromatin regions are likely to be involved in the regulatory response of gene transcription induced by infection with BRSV.
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- In Ireland, the mortality rate for dairy calves between 0 and 6 months of age is 5.4% [2], while the pre-weaning dairy calf mortality rate in the US is 7.8% [3].
- BRD is a disease of the upper and lower respiratory tract which results in the formation of syncytial cells in the bronchiolar epithelium and lung parenchyma, and clinical signs which include an elevated rectal temperature, increased respiratory rate, nasal and ocular discharges, cough, dyspnea, decreased appetite and depressive-like behaviour [11, 12].
- Viral pathogens are generally responsible for the initiation of BRD and sec- ondary bacterial pathogens, many of which are normally commensal in the nasopharyngeal region of the upper respiratory tract, often proliferate and exacerbate the disease [13–15]..
- An understanding of the identity of the variation within the bovine genome which confers vari- ation in resistance to BRD is needed to incorporate gen- etic variants into breeding programmes designed to breed robust animals with increased resistance to BRD infection..
- Additionally, the transcriptional response to infec- tion with several pathogens involved in the bovine respira- tory disease complex (BRDC) in BLN [23], lung and multiple lymphoid tissues [24] has previously been de- scribed in US Angus x Hereford crossbred beef steers..
- However, there is a lack of knowledge regarding the non- protein-coding regions of the genome which are involved in the regulation of the transcriptional response to BRD..
- Among these QTL, the genetic variants which are lo- cated in the regulatory regions that are actively involved in the host response to BRD, are most likely to be.
- These active regulatory regions of the genome can be identified since the surrounding chromatin should be open and accessible by regulatory elements such as transcription factors..
- While we have previously identified key genes that are expressed during BRSV infection [22], there is a lack of information on the specific regions of the genome that regulate the re- sponse to BRSV infection.
- The identification of the re- gions of chromatin that are open in respiratory tissues during BRSV infection will indicate the genomic regions that are transcriptionally active during infection.
- The objectives of the study were to: (i) identify re- gions of open chromatin in the BLN of dairy calves ex- perimentally challenged with BRSV and also in control calves, (ii) elucidate the chromatin regions which were differentially or uniquely open in the BRSV challenged relative to the control calves, and (iii) compare the dif- ferentially open regions with the locations of genes pre- viously found to be differentially expressed in the BLN in response to BRSV and with the locations of previously identified BRD susceptibility loci .
- Approximately 96% of the reads were aligned to the UMD3.1 bovine reference genome assembly.
- Five per- cent of the reads mapped to the mitochondrial genome and 14% of the reads had a MAPQ score <.
- This indicates that these samples contained a large number of reads which could be aligned to multiple places in the reference genome with equal stringency.
- There were more regions of open chromatin detected in the BLN of the BRSV challenged calves than the control calves student’s T-test.
- The Bedtools Jac- card score was used to measure of the similarity of ROCs between two samples based on the ratio of the number of base pairs present in the intersection to the number present in the unique union of ROCs predicted for each sample.
- Samples 4 and 5 from the control calves had lower Jaccard scores than the rest of the sam- ples.
- Following removal of the Jaccard scores for these calves, the mean Jaccard score for the control calves in- creased to 0.54.
- Fifty percent (28,635) of the ROCs were within 2 kb upstream of protein-coding (non-.
- Of the protein- coding genes expressed in the BLN had a ROC either within the gene or within 2 kb up- stream of the gene.
- Twenty-two percent (1450) of the protein coding genes not expressed in the BLN had a ROC either within the gene or within 2 kb upstream of the gene.
- Of the protein-coding genes expressed in the BLN had a ROC within the gene body..
- This plot was generated in Diffbind and illustrates the similarity of the BRSV challenged ( n = 12) and control ( n = 4) calves ’ bronchial lymph node samples based on regions of accessible chromatin (ATAC-Seq ROCs).
- Bronchial lymph node tissue samples from BRSV challenged calves (Calf IDs 7 to 18) are coloured in pink and from control calves (Calf IDs 1, 2, 3 and 6) are coloured in purple.
- There were and 1034 genes located in or within 2 kb downstream of the ROCs pre- dicted to be differentially accessible by the DeSeq2, EdgeR and both analyses, respectively (Fig.
- There were 169, 110 and 76 genes located in or within 2 kb downstream of the differentially accessible ROCs predicted in the DeSeq2, EdgeR and both analyses, respectively, and that were also found to be differentially expressed in the BLN RNA-Seq analysis [22] (Fig.
- There were 2966 and 400 genes located in or within 2 kb downstream of the ROCs which were unique to BRSV challenged calves and unique to control calves, respectively (Fig.
- There were 150 and 24 genes located in or within 2 kb down- stream of the ROCs which were unique to BRSV chal- lenged calves and unique to control calves, respectively, and were also found to be differentially expressed in the BLN RNA-Seq analysis [22] (Fig.
- ROCs unique to BRSV challenged calves.
- There were 91 enriched KEGG pathways among the closest downstream genes to the ROCs revealed by the Diffbind occupancy analysis to be uniquely open in the BRSV challenged calves (Additional file 10).
- There were 187 enriched GO BP terms, 20 enriched GO MF and 41 enriched GO CC terms among the closest downstream genes to the ROCs shown by the Diffbind occupancy analysis to be uniquely open in the BRSV challenged calves (Additional file 10)..
- Differentially expressed genes (BRSV challenged vs..
- “cell-substrate adherens”, “cell-substrate” and “focal ad- hesion”, among the closest downstream genes to the ROCs shown to be uniquely open in the control calves by the Diffbind occupancy analysis..
- Genes within 2 kb downstream of a ROC uniquely found in the control calves which were also differentially expressed (both up- and down-regulated) in the bron- chial lymph node, and their associated fold changes, P- values and FDR-values from our BLN RNA-Seq study [22], were input into IPA.
- There were 237 differentially accessible ROCs identified by either DeSeq2 or EdgeR within 40 of the BRD suscep- tibility loci identified by Neibergs et al.
- 3 Flow chart illustrating the results of the Diffbind analysis.
- Furthermore, there were 8 uniquely accessible ROCs de- tected in the BRSV-challenged calves by the Diffbind oc- cupancy analysis located within 5 BRD QTLs identified in Israeli Holstein male calves by Lipkin et al.
- [25], 11 uniquely accessible ROCs identified in BRSV-challenged.
- Enriched KEGG pathways among the closest downstream genes to the ROCs found to be differentially accessible in bronchial lymph node tissue samples between BRSV challenged and control calves by both DeSeq2 and EdgeR.
- This plot was produced in ClusterProfiler based on the results of the “ EnrichDAVID ” function.
- ROCs in the BLN tissue of healthy (control) and BRSV- challenged Holstein-Friesian calves.
- Changes in chroma- tin states in response to disease status provide an insight into the regulation of the host’s transcriptional response to infection [33] and the corresponding epigenetic modi- fications directly induced by the pathogen [34]..
- This plot was produced in ClusterProfiler based on the results of the.
- The majority of the ROCs found in the BLN of these dairy calves were located in protein coding genes which had previously been found to be expressed in the BLN tissue of these calves [22].
- However, there are no published data describing the transcriptional re- sponse of individual cell types to BRSV, while there are studies describing changes in gene expression in bulk tissues [22–24], such as lung and lymph node tissues, in- cluding our RNA-Seq study [22] which elucidated the al- terations in global gene expression induced in the same BLN tissue, from the same calves, and by the same BRSV challenge, as described in the present study..
- Enriched pathways among the closest downstream genes to the ROCs predicted to be differentially accessible by both DeSeq2 and EdgeR, which were also predicted to be differentially expressed in the RNA-Seq analysis ( P <.
- There were more ROCs in the BLN from BRSV challenged calves than the control calves.
- This suggests that significant chromatin remodelling occurs in the BLN in response to BRSV challenge to enable the transcriptional activation of anti-viral and inflammation response genes, as the BLN is the site of antigen presen- tation and activation of Th1 and Th2 immune effector cells [39]..
- The enriched pathways and GO terms associated with the closest downstream genes to the differentially access- ible ROCs and the ROCs that were uniquely accessible in the BRSV challenged calves are primarily involved in the immune response, particularly the anti-viral re- sponse.
- This is important as it shows that the differen- tially and uniquely accessible regions of chromatin in the BLN of the BRSV challenged calves function as ex- pected, in the immune-based transcriptional response to the virus.
- “chemokine signalling pathway”, “Toll-like receptor sig- nalling pathway”, “TNF signalling pathway”, “NF-kappa B signalling pathway” and “Leukocyte transendothelial migration”, further emphasize the increased transcrip- tion of genes involved in the innate and anti-viral im- mune responses.
- “interferon-gamma production”, “anti- gen receptor-mediated signalling pathway”, “immune response-activating signal transduction” and “T-helper cell differentiation”, demonstrate the inflammatory T- cell response in the BLN to BRSV challenge [39].
- There were enriched GO terms associated with the closest downstream genes of ROCs uniquely present in the BRSV-challenged calves that were related to neuro- transmitter synapses, including dopaminergic synapse, serotonergic synapse, GABAergic synapse, glutamatergic synapse and cholinergic synapse.
- Integrating the ATAC-seq detected ROCs with the dif- ferentially expressed genes from the BLN RNA-Seq study [22] revealed the dependence between differen- tially expressed genes and differentially accessible or uniquely accessible ROCs in the BRSV challenged rela- tive to control calves.
- Differentially expressed genes in- volved in the innate immune response, the complement system, antigen presentation, the Th1 and Th2 signalling pathways, interferon signalling and pathogen recognition all had a differentially accessible ROC located either within, or within 2 kb upstream, of the gene.
- The identified ROCs which are differentially or uniquely open in the BRSV challenged calves are in- volved in the transcriptional and regulatory response (in- cluding transcription factor binding, DNA methylation, mRNA splicing and mRNA stability) to infection with BRSV.
- However, as the differen- tially and uniquely accessible ROCs in the BLN in re- sponse to BRSV challenge are involved in the regulation of immune responses, they may be interrogated in future large scale or functional studies for the identification of variants associated with resistance to BRSV infection..
- There were several differentially accessible and uniquely accessible ROCs in the BLN of BRSV-challenged calves that were located in BRD susceptibility loci previously identified by GWAS .
- As 90% of variants identified by GWAS studies are lo- cated in non-protein-coding regions of the genome [42], elucidating the variants associated with BRD resistance which are located in differentially or uniquely open ROCs in the BRSV challenged calves could identify their roles in the regulation of gene expression in immune and anti- viral responses and could lead to improved disease resist- ance through selection on these variants.
- Their identification would allow their in- clusion in the genotyping assays routinely used by the international beef and dairy cattle industries for the gener- ation of healthier, more robust cattle with a greater poten- tial to resist BRSV infection and subsequent BRD..
- Differen- tially and uniquely accessible regions of chromatin found in calves experimentally challenged with BRSV are lo- cated close to genes involved in the immune response, particularly the anti-viral response.
- These ROCs may harbour genetic variants that create biological variation in the transcriptional and regulatory response to infection by BRD.
- Further inter- rogation of the variants in these regions for their associ- ation with risk of BRD will identify markers that will be broadly useful across breeds for use in breeding.
- [32] “Omni- ATAC” protocol with a modification of the transposase enzyme quantity from 2.5 μl to 5 μl.
- FC Illumina, Inc., San Diego, California) were used in the transpos- ition reaction.
- to determine the optimal number of additional PCR cy- cles to be performed on each of the libraries.
- The num- ber of additional PCR cycles was determined by plotting the linear reaction versus cycle and determining the cycle number which corresponded to one third of the maximum florescence intensity [32].
- The quality of the ATAC-Seq libraries was examined using an Agilent 2100 Bioanalyser (Agilent Technologies Ireland Ltd..
- “Action of the Toes.
- Consensus ROCs for BRSV challenged calves and for control calves were gen- erated with the requirement that peaks overlapped in at least 66% of the samples within each of the BRSV chal- lenged or control sample groups.
- The ROCs which were unique to the BRSV challenged calves and to the control calves were identified.
- Bedtools (version 2.27.1) closest was used to determine the closest genes downstream of the identified ROCs..
- Three gene lists were created with each containing the closest genes within 2 kb downstream of the ATAC-Seq ROCs which were: 1) differentially accessible between the BRSV challenged and control calves in both Diff- bind’s EdgeR and DeSeq2 analyses, 2) uniquely present in the BRSV challenged calves, and 3) uniquely present in the control calves.
- The three gene lists were merged in R (version 3.6.1) with the genes found to be differentially expressed in the RNA-Seq dataset (P <.
- 2) [22], to obtain three new lists comprising only the genes which were differentially expressed in the BLN.
- algorithm, which predicts increases or decreases in func- tions based on directional changes in the differentially expressed genes was used to predict differences in the over-represented cellular and molecular functions.
- The closest genes downstream of the ROCs that were either differentially accessible or unique to the BRSV-challenged calves were identified using Bedtools (version 2.27.1) closest..
- These plots were generated in Diffbind and illustrate the similarity of the BRSV challenged ( n = 12) and control ( n = 6 and/or n = 4) calves ’ bronchial lymph node samples based on ATAC-Seq ROC.
- Regions (peaks) unique to BRSV challenged calves identified through Diffbind ’ s occupancy analysis..
- 0.05) among the closest downstream genes to the regions found to be differentially accessible between BRSV challenged and control calves by both DeSeq2 and EdgeR..
- downstream genes to the regions uniquely open in the BRSV challenged calves..
- SEM: Standard error of the mean.
- This project was funded by the Irish Department of Agriculture and the Department of Agriculture, Environment and Rural Affairs, Northern Ireland, as part of the US-Ireland R&D partnership call (RMIS_0033 Project 16/RD/US- ROI/11).
- All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and with the approval of the Agri-Food and Biosciences Institute Northern Ireland Ethical Review Committee..
- Failure of respiratory defenses in the pathogenesis of bacterial pneumonia of cattle.
- Bacterial pathogens of the bovine respiratory disease complex.
- Immunological response to single pathogen challenge with agents of the bovine respiratory disease complex: an RNA-sequence analysis of the bronchial lymph node transcriptome.
- Tissue tropism in host transcriptional response to members of the bovine respiratory disease complex.
- Functional annotation of the cattle genome through systematic discovery and characterization of chromatin states and butyrate-induced variations.
- T cell-mediated host immune defenses in the lung.
- Basic mechanisms controlling lymph transport in the mesenteric lymphatic net

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