- Global investigation of estrogen-responsive genes regulating lipid metabolism in the liver of laying hens. - In chickens, estrogen is critical for lipid metabolism in the liver. - Integrated analysis of the data of RNA-Seq and ChIP-Seq, identified 191 genes directly targeted by estrogen, including 185 protein-coding genes, 4 miRNAs, and 2 lncRNAs. - Conclusions: These results increase our understanding of the functional network of estrogen in chicken liver and also reveal aspects of the molecular mechanism of estrogen-related lipid metabolism.. - Liver is the central organ of lipid metabolism, especially in chicken, where more than 90% of the de novo synthe- sis of fatty acids takes place [1–3]. - Hence, the active lipid related genes in liver plays an im- portant role in adaption of the physiological changes from pre-laying to peak-laying stages in hens.. - Full list of author information is available at the end of the article. - All of the receptors may synergistically or antagonistically mediate multiple physiological and pathological processes by the interaction of all activation and inhibition pathways [14]. - Integrative analysis of the above RNA-seq data and ChIP-seq data was carried out to explore the genome-wide estrogen-responsive genes involved in lipid metabolism mediated via ERα in the chicken liver. - One of the estrogen-mediated lipid meta- bolic pathways regulated by miR-144 was proven. - Effects of estrogen on lipid metabolism in chicken liver To verify whether exogenous estrogen had a biological role in chicken, the mRNA expression levels of the two classic target genes of estrogen, APOB and APOV1, were detected in liver tissue. - An overview of the RNA-Seq data is presented in Table S1. - To identify the estrogen-responsive miRNA genes, miRNA-Seq of the livers from the 17β-estradiol treated and the control chickens was performed. - To study the functions of the differentially expressed estrogen-responsive genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. - To assess the possible participation of miRNAs in lipid metabolism, the potential target genes of the 10 estrogen- responsive miRNAs were predicted by means of miRDB, TargetScan, and PicTar. - A network of the differential expressed miRNAs and predicted target genes was constructed using Cytoscape (Fig. - Function annotation of the 109 potential target genes showed that the gene set was significantly enriched in lipid-related terms, including lipid biosynthetic process (p = 1.47E-05),. - We found that a low proportion (17.7%) of the binding sites were lo- cated <. - 10 kb upstream of the annotated TSS of genes (Fig. - 4a), while 41.4% of the binding sites were located downstream of the annotated TSS of genes (Fig. - All of the identified binding sites were used to perform an unbiased search for enriched motifs using MEME. - FIMO was then used to identify ERE se- quences in the identified binding sites. - Out of the 7000 unique ERα binding sites, 6492 putative EREs were identified (Fig. - Only of the binding sites occupy the putative EREs. - Approximately 47% of the ERα binding sites do not occupy any discernable EREs. - A total of 42.7% of the binding sites that occupied ERE were more than one pu- tative ERE (Fig. - All of the identified ERE sequences Table 1 The GO terms correlated to lipid metabolism. - b Enrichment of the FOXP1 binding motif in the 500 bp ChIP signal sequence. - c Distributions of the predicted EREs on the ER α -binding sites. - Notably, the trinucleotide spacer sequence NNN located in the middle of the ERE seems to be preferentially in the form of CNG. - Identification, functional analysis, and validation of the estrogen-responsive genes directly targeted by ER α via EREs. - To confirm the reliability of the analysis results, the rela- tionships among estrogen, gga-miR-144-3p, and the po- tential target genes PPARGC1B and DUSP16 were verified. - These findings indicate that estrogen suppresses the expression of PPARGC1B and DUSP16 by promoting the expression of miR-144-3p in the chicken liver.. - Estrogen can strongly affect lipid metabolism in the liver and adipose tissue of mammals [1, 3]. - We also found, using miRNA-Seq analysis, that miR-148a, which is linked to lipid metabolism [21, 22], was one of the most abundant miRNAs in the chicken liver. - We previously reported a transcriptome profile of the liver of pre-laying and peak-laying chickens [6]. - Thus, most of the DEGs were considered to be lipid-related. - The plasma estrogen level in female chickens reaches its peak before the onset of production of the first egg [7, 8], then drops gradually, but remains at a higher level than that in im- mature pullets [8]. - The TG content in the serum of chickens was significantly increased after estrogen treat- ment, and the oil red O staining of liver tissue showed more lipid droplets in the livers of estrogen-treated chickens than in livers of the control group. - Some estrogen-responsive genes are regulated during the laying period because of the increased plasma estro- gen level, and they therefore participate in liver lipid metabolism. - regulation of lipid metabolism by estrogen signaling in the chicken liver.. - Therefore, the chicken liver is one of the best models to study lipid. - In this study, a total of 7000 ERα binding sites and 113 of the 962 estrogen-responsive genes dir- ectly targeted by estrogen via ERα binding to the special ERE sequence were identified in the chicken genome by ChIP-Seq. - Unexpectedly, most of the identified binding sites were located away from the annotated TSS and only a small proportion. - 5%) of the binding sites were located <. - Furthermore, more than 60% of the binding sites were located in the gene body and downstream of genes. - However, considering that many genes occupy alternative TSS, the binding sites that were located at a gene body may also be lo- cated at the promoter of one of the gene’s transcript iso- forms. - suppression of the transcription of genes by ERα, but. - 5 Confirmation of the identified genes directly targeted by estrogen. - a, d FPKM/TPM data of the genes detected by RNA-Seq. - b, e Effects of 17 β -estradiol on the mRNA expression of the genes detected by qRT-PCR. - Although numerous interacting molecules at the genome scale were identified, high resolution of the complexes at one specific genomic locus remains lacking. - 6 MiR-144-3p targets and suppresses the expression of PPARGC1B and DUSP16 in the chicken liver. - a, b The potential miR-144-3p binding sites in the 3 ′ -UTR of PPARGC1B (a) and DUSP16 (b). - The exact location and the sequence of the binding sites are indicated. - h, i Expression patterns of miR-144-3p and DUSP16 and PPARGC1B mRNAs in response to 17 β -estradiol in the chicken liver according to qRT-PCR analysis. - j Comparison between the expression curve of miR-144-3p and the egg production curve of the Lushi green-shelled-egg chickens. - k, l Expression levels of miR-144-3p and DUSP16 and PPARGC1B mRNAs in the chicken liver at different ages of chickens. - One of the estrogen-responsive miRNAs, miR-148a, can target more than 10 genes, such as MSK1 [38] and P27 [39]. - These results increase our understanding of the non-coding RNA mediated estrogen regulation net- work in the liver of chickens.. - Thus, identification of noncoding genes tar- geted by estrogen is helpful for understanding the mech- anism of the estrogen signaling pathway.. - In conclusion, our integrated analysis of RNA-Seq data and ChIP-Seq data identified estrogen receptor func- tional sites in the chicken genome and all of the genes, including protein-coding genes, miRNAs and lncRNAs, regulated via estrogen in the liver of chicken (Fig. - These results increase our understanding of the functional net- work regulated by estrogen in liver of chicken and pro- vide insight into estrogen mediated lipid metabolism.. - A portion of the fresh liver tissue was embedded with optimum cutting temperature compound for oil red O staining. - The rest of the liver was snap-frozen in liquid nitrogen and stored at − 80 °C in a freezer until use.. - In this study, the FDR was used to de- termine the threshold of the p value in multiple tests and analyses.. - The expression of the identified miRNAs was normalized by calculation of TPM. - MAST was employed for motif alignment to exclude duplicate motifs, whereas Tomtom was used for annotation of the discovered motifs.. - All of the constructed vectors were confirmed by PCR and sequencing (BGI, Shenzhen, China). - The expression levels were measured in terms of the cycle threshold (Ct) and then normalized to the ex- pression of β -actin using the 2. - Expression of the miRNA was detected by stem-loop qRT-PCR. - TW, JK, WZ and WD participated in management of the experimental animals and the sample collection. - The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.. - doi.org/10.1021/bi00330a007.. - In vivo lipogenesis in the domestic chicken. - https://doi.org . - Lipid biosynthesis and transport in the domestic chick (Gallus domesticus). - https://doi.org X . - Binding to two oocyte-specific members of the low density lipoprotein receptor gene family. - doi.org/10.1074/jbc . - Systematic analysis of the regulatory functions of microRNAs in chicken hepatic lipid metabolism. - https://doi.org/10.1038/srep31766.. - https://doi.org/10.1186/s . - https://doi.org/10.1507/endocrj . - https://doi.org/10.1051/rnd:19860311.. - doi.org . - https://doi.org/10.1210/. - https://doi.org/10.1016/S . - https://doi.. - https://doi.org/10.1038/ng1901.. - Insulin inhibits the estrogen-dependent expression of the chicken very low density apolipoprotein II gene in Leghorn male hepatoma cells. - 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