A Genome-wide Approach To Delineate Novel XBP-1 Targets Involved In Liver Metabolism

In eukaryotes,accumulation of unfolded or misfolded proteins in the endoplasmic reticulum(ER)lumen causes ER stress,activating the cellular unfolded protein response(UPR).Activation of the UPR restores ER homeostasis by increasing protein folding,enhancing degradation of misfolded proteins and inhibiting protein synthesis,thus adjusting the protein folding capacity of the ER to the folding demand.ER stress and disruption of protein homeostasis have recently been established as a significant mechanism contributing to liver diseases such as alcohol-induced liver disease.The involvement of ER stress in ethanol-induced organ damage has also been demonstrated in pancreas,and heart.Ethanol can exacerbate ER stress and promotes liver damage and dyslipidemia in fatty liver diseases.ER stress can induce acetaldehyde,oxidative stress,toxic lipid species and insulin resistance and all of these can impact liver metabolism.Previous study indicates that ER stress may directly be involved in hepatocellular carcinogenesis(HCC).ER stress pathways leading to disrupt liver metabolism will uncover potential therapeutic targets for treatment of human alcoholics with liver cancer.The X-box binding protein 1(XBP-1)is a transcription factor containing a bZIP domain.It was first identified by its ability to bind to the X-box,a conserved transcriptional element in the promoter of the human leukocyte antigen(HLA)DR alpha.The XBP-1 gene is located on chromosome 22 while a closely related pseudogene has been identified and localized to chromosome 5.The X-box binding protein 1(XBP-1)is a critical effector of the mammalian unfolded protein response(UPR)whose activity is regulated by IRE 1,an ER-resident kinase/ribonuclease that acts as an ER-stress sensor.Activation through oligomerization and autophosphorylation on release from grp78 results in the excision of a 26-base pair fragment from the mRNA of unspliced XBP1(XBP1u)by an unconventional splicing event that generates a spliced isoform of XBP1 with an altered reading frame XBP1s.XBP-1s is a trans-acting factor in the process of gene transcription.It not only regulates protein maturation and transport in the endoplasmic reticulum,but also regulates protein degradation.Therefore,-IRE1/XBP-1 plays an important role in regulating liver metabolism.XBP-1 is a key effector of mammalian unfolding protein response(UPR),but its mechanism is unclear.So we globally identified XBP-1 occupancy in mouse hepatic chromatin on a genome-wide level by chromatin immunoprecipitation coupled with high throughput DNA sequencing(ChIP-seq).We established fasted model and did the XBP-1 ChIP-seq experiment.Through bioinformatics analysis,the ChIP-seq datas are qualified.Gene ontology analysis reveals the functional significance of XBP-1 in liver metabolism.Interestingly,motif search reveals a sequence element,5'GCCACGT 3',consistent with a previously XBP-1-binding site.We show here that XBP-1 directly binds to the genomic sites for the genes in ER stress.These results suggest that we delineate novel XBP-1 targets involved in liver metabolism.In order to study the effect of XBP-1s on downstream target genes to regulate liver metabolism,we used KEGG pathway analysis.We can know that most of the signaling pathways are associated with the protein processing,secretion,and degradation in the endoplasmic reticulum which validated previous studies,and we also found novel pathway such as ubiquitination signaling pathways and endoplasmic reticulum-related degradation pathways.In order to verify that XBP-1 regulates these target genes transcription,we verify that these target genes were significantly down-regulated in IRE1 knockout mice.Interestingly we examined that some targets have significantly decreased in fasted model,which was consistent with the results of XBP-1 ChIP-seq peak result but increased in TM model.So we speculate that this physiological regulation is different from TM(non-physiological).This is one of the important discoveries of our subject.In summary,we have not only revealed the novel target gene of XBP-1 by XBP-1 ChIP-seq technique,but also found that ER stress regulation mechanism under physiological condition is different from TM-mediated mechanism.