Introduction
The European seabass (Dicentrarchus labrax ) is a marine fish of great importance for Mediterranean aquaculture. Disease outbreaks, especially viral infections, represent severe threats that can interfere with the progress and sustainability of intensive aquaculture systems. K nowledge of the complex molecular mechanisms and signaling pathways underlying host response to pathogen is of crucial importance to improve understand ing of genome function and regulation. Changes in chromatin structure, in terms of chromatin accessibility for transcription machinery, and histone modifications (HMs) are considered to play a key role in transcriptional regulation.
As part of the AQUA-FAANG (www.aqua-faang.eu ) project, the present work aims to profile in vivo and in vitro response to stimulation with viral (Poly I:C) mimics. RNA-seq, ATAC-seq and ChIP -seq methodologies were applied in order to define a comprehensive genome-wide map of chromatin states and gene expression upon stimulation with viral mimics.
Material and methods
In vitro challenge was conducted on head kidney isolated leucocytes stimulated with PBS or Poly I:C (6 replicates/condition). Cell cultures were collected 12 hours post-infection (hpi ) and employed for RNA-seq, ATAC-seq and ChIP -seq library preparation. For in vivo challenge, adult individuals were stimulated by injection with PBS or Poly:IC (6 replicates/condition). Animals were sacrificed 24 hpi and head kidney sampled for RNA-seq, ATAC-seq and ChIP-seq library preparation. The histone marks investigated: H3K4me3 (promoter regions) , H3K27ac (active enhancer and promoter regions) and H3K27me3 (associated with Polycomb repression) . All sequencing data analyses were conducted by using dedicated NF-core pipelines (https://nf-co.re ). Differentially expressed genes (DEGs) analysis was conducted with EdgeR and functional enrichment analysis on the DEGs were performed using the package clusterProfiler . ATAC-seq data were employed in order to investigate for differential DNA accessibility between PBS and Poly:IC stimulated samples by using DiffBind package. ATAC- seq and ChIP-seq peaks from the in vivo and the in vitro experiments were employed to predict genome-wide chromatin states using the software ChromHMM.
Results
Differential expression analysis identified a total of 516 and 1467 DEGs from in vitro and in vivo datasets, respectively. The two challenges showed peculiar response not only in terms of number of DEGs, but also in terms of enriched biological pathways . The top GO terms in over-expressed genes in in vitro stimulated leucocytes were mainly associated with immune system activation (i.e. GO:006955-immune response, GO:0098542 – defense response to other organism) , in this context IFN- stimulated genes and IFN regulatory factors are highly significant. In vivo challenged head kidney showed a great enrichment on biological processes involved in DNA replication and RNA processing while immune related pathways are associated to down-regulated genes .
Analysis of ATAC-seq peaks revealed w eak or no differential DNA accessibility between treatments for in vivo and in vitro challenges, respectively. Accessible chromatin regions were found to cover up to the 20% of the genome with variations mainly due to cell source rather than treatment indicating that those regions are transcriptionally enabled prior to stimulation.
HMs analysis through ChromHMM software allowed to define the first chromatin state map of non-stimulated and Poly I:C stimulated cells. Comparison between groups highlighted a 4-fold increase of active state regions in Poly I:C stimulated samples.
Conclusion
The present study provides the first genome-wide functional annotation map of the European seabass response to immune stimulation. This represent a significant increase on our fundamental understanding of the genomic basis for immune function and disease resistance in an important aquaculture species.
Acknowledgements
Funding: Horizon 2020 Grant n. 817923 (AQUA-FAANG). Animals used in the experiment: Valle Cà Zuliani Società Agricola srl (Italy).