RNA-seq And ChIP-seq Integrative Analvsis Reveals The Molecular Mechanism Of Estrogen Regulating Liver Lipid Metabolism In Chickens

Background:A large number of studies have pointed out that the most significant physiological change in chickens from the early stage of laying to the peak stage of laying is the sharp increase of estrogen,and the change of estrogen level is the key factor affecting the liver metabolic activity of poultry during the laying period.The main mechanism of action for estrogen to regulate gene expression or change gene transcriptional activity is via the binding of estrogen receptor(ER)and estrogen response element(ERE)on the target gene.Estrogen can also participate in a variety of metabolic processes by regulating non-coding RNA(ncRNA).However,the mechanism of lipid metabolism in poultry is different from that in mammals,and there is still a lack of systematic research on the molecular regulation mechanism of lipid metabolism in chicken.Therefore,we previously obtained the differentially expressed mRNA database in the liver of chicken in peak-laying stage and the differentially expressed mRNA database in the liver of estrogen-treated chicken by RNA-seq.Study purpose:This study aimed to reveal the regulatory mechanism of estrogen on liver lipid metabolism in chickens.On the basis of the previous mRNA databases,the role of non-coding RNA in the regulation of lipid metabolism in chicken liver by estrogen will be further studied,the target genes directly binding to estrogen receptor will be screened,and the regulatory network of estrogen on genes related to lipid metabolism will be revealed at the whole genome level.Methods:Integrated analysis of the database of differentially expressed mRNAs were applied,functional pathway enrichment analysis and protein-protein interaction(PPI)analysis were performed on the selected candidate genes.High-throughput transcriptome sequencing of chicken liver with estrogen treatment was carried out to screen out estrogen responsive miRNAs and lncRNAs.The miRNAs-target mRNAs co-expression network and the lncRNAs-target mRNAs co-expression network were predicted and constructed,the functional annotation was applied on potential target genes.Chromatin immunoprecipitation sequencing(CHIP-seq)technology was used to screen the DNA fragments interacting with ER? in chicken liver in the whole genome level.The key mRNAs and miRNAs were filtered by integration analysis for further study.The genomic synteny,gene structure,evolutionary event and functional domains of the ACSL gene family members were analyzed using bioinformatics methods,followed by the verification of the regulatory effects and regulatory mechanism of estrogen on ACSLs.Dual-luciferase reporter system was used to determine the targeting relationship between miR-144-3p and PPARGC1B or DUSP16.The effect of miR-144-3p on PPARGC1B and DUSP16 was verified by over-expression and inhibition experiments in vitro.Results:1.Through integrative analysis,123 estrogen responding candidate genes were found,of which 99 genes were significantly up-regulated and 24 genes were significantly down-regulated in the peak-laying stage or the estrogen treatment group.These genes are significantly enriched in metabolic process,biological regulation process and other lipid metabolism pathways.Close and abundant protein interactions were found among 13 genes,including DHCR24,DHCR7,AACS,CYP51A1,MSMO1,FDFT1,STARD4,NSDHL,LSS,MVD,HMGCR,ACAA1 and ACSL1.2.A total of 575 known miRNAs were detected in chicken liver by RNA-seq.11 differentially expressed miRNAs were detected in the estrogen-treatment group,including 5 up-regulated miRNAs and 6 down-regulated miRNAs.There were 2562 potential target genes predicted for the differentially expressed miRNAs.By integrating with the previous database,a total of 122 estrogen responsive miRNA-target mRNA pairs were found,and the co-expression network was obtained.The 109 potential estrogen responsive target genes in the co-expression network were significantly enriched in metabolism process and lipid metabolism pathways.3.A total of 5690 lncRNAs were detected in chicken liver by RNA-seq,including 17 differentially expressed lncRNAs,of which 7 were up-regulated and 10 were down-regulated in estrogen-treatment group.A total of 4152 cis target genes and 199 trans target genes were predicted.These potential target genes were significantly enriched in biological regulation process and lipid metabolism pathways.The co-expression network of lncRNAs and target genes suggested the potential roles of differentially expressed lncRNAs in lipid metabolism.4.A total of 7000 unique ERa binding sites were identified in chicken liver,including 4307 protein coding genes,147 miRNAs and 2 lncRNAs.33.97%ERa binding sites were located within 50kb upstream of transcription start site(TSS).The motif analysis showed that the trinucleotide spacer sequence NNN located in the middle of the ERE seems to be preferentially in the form of CNG.By integrating with the previous databases,12 mRNAs and 4 miRNAs were found directly interacting with ER?.ACSL1 and miRNA-144-3p were selected for further study.5.ACSL2 gene was evolutionarily lost in birds and mammals.Chicken ACSL gene family had highly conserved ATP/AMP classical motif and FACS classical motif.of phylogenetic tree showed that ACSL1,ACSL5,ACSL6,and ACSL3,ACSL4 were clustered into two main branches in the phylogenetic tree,respectively.ACSL1 was highly expressed in abdominal fat,heart,kidney and pancreas,followed by liver and glandular stomach.The expression of ACSL1 decreased significantly from pre-laying stage to peak-laying stage.Estrogen inhibited the expression of ACSL1,ACSL3 and ACSL4,promoted the expression of ACSL6,and had no effect on the expression of ACSL5.Estrogen down-regulated the expression of ACSL1 via ER?,therefore promoted the lipid synthesis in the liver of peak-laying chicken.6.Estrogen significantly up-regulated the expression of miR-144-3p,and down-regulated the expression of PPARGC1B and DUSP16.The expression of miR-144-3p increased significantly,while the expressions of PPARGC1B and DUSP16 decreased significantly from pre-laying stage to peak-laying stage.PPARGC1B and DUSP16 were the the positive target genes of miR-144-3p.miR-144-3p inhibited the expression of PPARGC1B and DUSP16 by binding with 467-473nt seed region on PPARGC1B 3'UTR and 1923-1929nt seed region on DUSP16 3'UTR.Estrogen directly up-regulated the expression of miR-144-3p through ER?,accordingly inhibited the expressions of PPARGC1B and DUSP16,therefore regulated the lipid metabolism in chicken liver.Conclusion:This study elucidated two mechanisms of estrogen regulating liver lipid metabolism in chickens.Direct regulation pathway:estrogen directly inhibits the expression of ACSL1 through the mediation of ER?,therefore promotes the lipid synthesis in liver.Indirect regulation pathway:estrogen upregulates the expression of miR-144-3p through ER?,therefore inhibits the expression of its target genes and promotes the lipid synthesis in liver.In this study,we obtained a list of mRNAs,miRNAs and lncRNAs that play important roles in the estrogen regulation of chicken lipid metabolism pathway through multiple omics integrative analysis and mutual verification,this study provided the basis and laid the foundation for future researches.