The Role Of Epigenetic Mechanisms In Cold Acclimation Of Zebrafish Cells

As aquatic organisms,fish face a wide range of temperature changes during their life cycle.Cold stress has a huge impact on fish physiology and behavior,and even leads to death.Cold acclimation can enhance fish tolerance to low temperature.Therefore,it is of great significance to explore molecular regulation mechanisms of fish cold acclimation.Epigenetics plays an important role in the adaptation of plants and animals to environmental stress.However,there had been few studies on the role of epigenetics in the cold acclimation of fishes,especially the studies on histone modifications.The research on long non-coding RNA(lnc RNA)was still blank.Zebrafish as a common model organism,its optimum growth temperature is 28.5 °C,while wild-type zebrafish are subject to large fluctuations in daily(5.6 °C)and seasonal(15.0 ± 0.4 °C)temperature changes,can adapt to a wide range of temperature changes(6-38°C).Therefore,zebrafish were also commonly used to study the adaptation mechanism of fish to temperature changes.In the previous study,the DNA methylation level of zebrafish embryonic fibroblasts ZF4 detected by methylated DNA immunoprecipitation Sequencing(Me DIPSeq)was significantly altered during the cold acclimation process.Differential DNA methylation might be involved in the regulation of anti-oxidation,apoptosis,development,chromatin modification,immune system and other biological processes associated with cold acclimation.By comparing the transcriptomic data of ZF4 between control and cold acclimation group,we found that low temperature pressure led to a significant changes in the expression of some epigenetic enzymes,including the histone methyltransferase such as KMT2 and KMT7,the histone demethylases such as jmjd4,KDM4 A and KDM6 b,and the histone deacetylases such as HDAC8 and HDAC11,histone acetylase ep300.Furthermore,the chromatin co-immunoprecipitation(Ch IP)-q PCR method was used to find that the histone modifications in the promoter regions of p53,selenbp1 also changed under cold stress.These results indicated that DNA methylation and histone modification did participate in the process of cold acclimation in zebrafish cell.In this study,for exploring the role of lnc RNAs and histone modifications during cold acclimation,RNAseq and chromatin immunoprecipitation combined with high-throughput sequencing(Ch IP-seq)were used to detect the whole genome changes of lnc RNAs and histone modifications in normal cultured and cold acclimation ZF4 cells.The main results are as follows:1.To screen and identify lnc RNAs in response to low temperature stress in zebrafish,we extracted total RNA from ZF4 cells in normal cultured(28°C)and cold acclimation(18°C for 30 days)groups and performed the high-throughput sequencing.After aligning the sequencing data to the genome,the transcripts were reassembled.Among the reassembled transcripts,the known m RNAs and lnc RNAs were removed,and remaining transcripts with lengths of less than 200 nt,open reading frame greater than 300,coding potential were removed.8363 new lnc RNAs were identified according to the above criteria.Among these new lnc RNAs,62% were located in the intergenic region and 27% were located in the intron region.The comparison between novel lnc RNAs and known m RNAs revealed that more than 89% of the lnc RNAs had less than or equal to 2 exons and more than 86% of the m RNAs had more than 2 exons.More than 70% of lnc RNAs were less than 1 kb,and only about 33% of the m RNAs were less than 1 kb.The median FPKM values of lnc RNAs and m RNA were 0.9 and 2.6,respectively.In summary,the novel lnc RNAs have fewer exons,shorter transcripts,lower expression levels compared with known m RNAs.By comparing the expression profiles of lnc RNAs(including newly identified lnc RNAs and annotated lnc RNAs in NONCODE database)between control and cold acclimation group,it was found that 347 lnc RNAs were up-regulated and 342 lnc RNAs were down-regulated.Most of these differentially expressed lnc RNAs were positively related to the expression of adjacent genes(putative cis-target genes).Here GO and KEGG enrichment analyses of the differentially expressed cis-target genes showed the biological processes significantly enriched include ATP synthesis coupled electron transport,cell adhesion,cell migration,oxidation-reduction process,striated muscle tissue development and multicellular organism development and the KEGG pathways that were significantly enriched includes oxidative phosphorylation,focal adhesion,gap junction,and calcium signaling pathway.Furthermore,according to the free energy required for the interaction between lnc RNA and m RNA,the trans-target genes of the 20 lnc RNAs with the most obvious difference in expression levels were predicted and an inter-network map was constructed based on the interaction.Differentially expressed lnc RNAs and genes were verified by q PCR.The results of q PCR and RNA-seq showed good consistency,indicating that the RNA-seq data were reliable.In addition,this study also investigated the expression changes of four lnc RNAs and tp53 genes after coldacclimated cells returned to normal culture condition by q PCR.Transfered to normal culture conditions on day 7,the expression levels of tp53 and MSTRG.2788.1 showed no significant difference compared with normal cultured group.While the expression levels of NORDRET001625.2 and MSTRG2629.2 did not show significant differences on day 3.MSTRG.28882.1 still showed a low expression state on day 7.This suggested that lnc RNAs were regulated by various mechanisms,and the time required for their expression levels return to normal also showed different.In conclusion,the low-temperature pressure results in significant changes in the expression profile of lnc RNAs in zebrafish cells.The results of this study lay the foundation for further exploration of the role of lnc RNAs in fish adaptation to low temperature stress.2.To explore the role of histone modification response to low temperature stress in zebrafish.In this part of the study,ZF4 cells in normal cultured(28°C)and cold acclimation(18°C for 30 days)were also used as experimental materials.Ch IP-seq experiments were performed using antibodies against histone modifications such as H3K4me3,H3K9me3,H3K27 ac,and H3K27me3.Through bioinformatics analysis,these modifications showed differently degrees changes under low temperature stress.For example,the overall levels of H3K4me3 and H3K9me3 decreased,while the levels of H3K27 ac and H3K27me3 increased.After cold acclimation,the proportions of H3K4me3,H3K27 ac and H3K27me3 peaks in the gene promoter region all reduced.While H3K27me3 reduced most obviously.More than 50% of the lost peaks,but only less than 10% of the gained peaks of H3K27me3 were located in the promoter region.GSEA results showed that H3K4me3,H3K9me3 and H3K27 ac were positively correlated with gene expression,while H3K27me3 was negatively correlated with gene expression.It indicated that these histone modifications indeed participate in the regulation of gene expression during cold acclimation.By comparing the distribution of the differentially expressed gene and differentially enriched histone modifications on each chromosome,we found that the expression levels of genes on the long arm of chromosome 4 were very low,and most of these genes expression levels decreased after low temperature treatment.Ch IP-seq data showed that H3K4me3 and H3K27 ac were considerable deleted in this region,and H3K9me3 and H3K27me3 were enriched.After cold acclimation,the H3K4me3,H3K9me3 and H3K27 ac enrichment levels decreased overall,while H3K27me3 increased.This suggests that the down-regulation of the gene expression in this region were regulated by histone modifications.To verify the accuracy of Ch IP-seq experiments,the enrichment of H3K4me3,H3K9me3,H3K27me3 and H3K27 ac on the promoter and genebody of epha4 a,kdm4aa,tnfb,and ccnd2 a,cxcl18b,ppp1r3 db,casp3a,casp7 were verified by Ch IP-q PCR.The verification results showed that the Ch IP-q PCR was consistent with the Ch IP-seq results.The coding genes that appeared differentially enriched histone modifications were performed the GO and KEGG enrichment analysis.The results showed that there were only minor overlaps in the biological processes or pathways between these four histone modifications,suggesting that different histone modifications might be regulated by different transcription factors.The GO enrichment results showed that most of the genes losing H3K4me3 enrichment related to ion transport and the genes gaining H3K4me3 participate in biological processes including nucleosome assembly,chromatin silencing,and defense response to Gram-positive bacterium;the genes gaining H3K27 ac involved in biological processes include negative regulation of cell migration,positive regulation of cell proliferation,MHC mediated antigen processing,etc;most of the genes gaining H3K27 ac enrichment were related to ribosome biogenesis;most genes regulated by H3K27me3 were related to development and some of them also involved in angiogenesis;genes controlled by H3K9me3 were also mostly related to development,and involved in the regulation of iron ion homeostasis,protein ubiquitination and so on.The genes regulated by these four histone modifications involved in the KEGG pathways include MAPK signaling pathway,FOXO signaling pathway,p53 signaling pathway,etc.The above pathways were also observed in many studies about zebrafish response to cold stress.It's interesting to find that elevation of H3K27 ac might enhance the activation of p53 signaling pathway.But the H3K27 ac in the promoter regions of several genes(casp3a,casp7,etc.)involved in apoptosis pathways decreased and most of these genes expression decreased at the same time.This may be a way for the zebrafish to adapt to the low temperature environment by inhibiting cell proliferation,promoting DNA repair and at the same time inhibiting apoptosis.According to the histone modification markers,this study also predicted the typicalenhancer and super-enhancer associated with cold acclimation.The results showed that the super-enhancer had a stronger effect on genes expression.The biological functions regulated by super-enhancer were explored by GO and KEGG enrichment analysis of super-enhancer target genes.It was found that biological processes that are significantly enriched include transcription,DNA-templated,somitogenesis,angiogenesis,lymphangiogenesis,etc.Significantly enriched pathways included MAPK signaling pathway,focal adhesion,VEGF signaling pathway,and Wnt signaling pathway.The levels of H3K27 ac enrichment in the enhancer element of angiogenesis-associated genes were increased,whereas the H3K27me3 enrichment levels were decreased.It was suggested that zebrafish might activate VEGF signaling pathway by regulating these two histone modifications at promoter or enhancer and promoted angiogenesis to enhance oxygen diffusion and meet oxygen demand under cold pressure.This part of the study revealed the potential role of histone modifications and improved the regulatory mechanisms of epigenetics in zebrafish cold acclimation.