Lxrs Dependent Chromatin Remodeling And Non-alcoholic Fatty Liver

BACKGROUND AND PURPOSE:Non-alcoholic fatty liver (NAFL) is a metabolic stress-related liverinjury and one of the metabolic diseases that has been a serious threat to thepeople's health. The pathogenesis of NAFL is not fully clear, but abnormallipid metabolisms leading to intrahepatic lipid (especially fatty acids andtriglycerides) deposition are considered to be the basis for the developmentof NAFL. Abnormal transcriptional regulations of the nuclear receptortarget genes will lead to lipid metabolism disorders of hepatic cells andthen will cause unusual intrahepatic lipid deposition, which is an importantmolecular mechanism for the development of NAFL. Liver X receptors(LXRs) are the key nuclear receptors to participate in varieties of hepaticlipid metabolisms such as cholesterol, bile acids and fatty acids. They havedouble effects that are to promote lowering cholesterol and to increasesynthesis of fatty acids and triglycerides. To study the regulationmechanism of LXRs on their target genes related to triglyceride synthesishas great significance for researching how LXRs cause lipid metabolism disorders, result in intrahepatic lipid deposition and promote thedevelopment of NAFL. Meanwhile we could find ways, which is to inhibitthe expression of target genes related to synthesis of triglycerides whiletarget genes that can reduce the level of cholesterols are activated, to solvethe double contradiction effects of LXRs.There ought to be local chromatin remodeling before or at the sametime of any signal molecules actioning on transcriptional regulatoryelements in genomic DNA, to promote the activation or inhibition of genetranscription. Thus chromatin remodeling is considered to be the final linkof cell signal transduction in transcriptional regulation. The chromatinremodeling includes two forms:histone post-translational modificationsand nucleosome remodelings, which are completed by chromatinremodeling factors that have enzyme activity. The transcriptional regulationof LXRs on their target genes about lipid metabolisms is also atranscription factor dependent-signal transduction process. So in theory,there should be chromatin remodeling events on LXR's target genes toparticipate in the regulation of these genes.No chromatin remodeling events related to LXRs have been reported.So this thesis is going to analyze the chromatin remodeling events on thepromoter and the transcriptional regulation areas of fatty acid synthase(FAS),which is a key target gene regulated by LXRs to promoteintrahepatic triglyceride synthesis and then to cause intrahepatic lipid deposition, in order to prove that the chromatin remodeling involves in thetranscriptional regulation of LXRs on FAS gene and to indicate thatchromatin remodeling is an important molecular mechanism of thedevelopment of NAFL. In the meantime, a theoretical basis could beprovided for seeking some activators or inhibitors of chromatin remodelingfactors that can be applied for solving the dual effects of LXRs in lipidmetabolism from this research.METHORDS:1. The time spectrum of FAS mRNA transcription expression in HepG2cells upon the LXRs agonist1) Identification of the fatty degeneration model of HepG2cells inducedby1uM LXRs agonist T0901317The concentration of triglycerides and the intracellular lipid depositionin HepG2cells were assesed by the measurement of intracellulartriglyceride and oil red O staining respectively48h after the induction of1uM LXRs agonist T0901317.2) Measurement of the time spectrum of FAS mRNA transcriptionexpression in HepG2cells upon LXRs agonist T0901317.The total RNAs in HepG2cells were collected at the time of2h,4h,6h,8h,12h,16h,24h and48h induced by LXRs agonist T0901317andthen reverse transcripted into cDNA respectively. Then the relative levelsof FAS mRNA expression at different time was detected by real-time PCR. 2. Chromatin remodelings on the promoter and transcriptional regulatoryregions of FAS gene upon the LXRs agonist1) Detection of the changes for histone modifications across the promoterand transcriptional regulatory regions of FAS gene upon the LXRs agonistHepG2cells were treated with LXRs agonist T0901317for0min,30min,60min,120min. Then the level of acetylation,phosphorylation andmethylation across the four regions of5'-2000bp upstream area oftranscription start site, LXRE, transcription start site and exon2at30min,60min,120min were compared to that at0min by methods of chromatinimmunoprecipitation and real-time PCR using ChIP-grade anti-histone H3and H4acetylation antibodies, anti-histone H3(S10) phosphorylationantibody and anti-H3(K4) trimethylation antibody.2) Detection of the changes for the whole level of histones across thepromoter and transcriptional regulatory regions of FAS gene upon theLXRs agonistHepG2cells were induced by LXRs agonist T0901317for0min,30min,60min,120min. Then the whole level of histone H3and H4acrossthe four regions of5'-2000bp upstream area of transcription start site,LXRE, transcription start site and exon2at30min,60min,120min werecompared to that at0min by methods of chromatin immunoprecipitationand real-time PCR using ChIP-grade anti-histone H3and H4antibodies(that didn't react with N end and reflected the whole levels of histones). 3. Chromatin remodelings on the promoter and transcriptional regulatoryregions of FAS gene dependent on LXR-alpha1) Detection of the influence of LXR-alpha gene silencing on chromatinremodelings across the promoter and transcriptional regulatory regions ofFAS geneFirstly, three LXR-alpha shRNA plasmids were transfected into HepG2cells for24h and the cells which were trasnsfected with the negativeplasmid or no plasmids were regarded as controls. The best plasmid forsilencing LXR-alpha gene was chosen by comparing the mRNA andprotein levels of LXR-alpha through RT-PCR and Western blot. Then24hafter HepG2cells were transfected with the best plasmid and the negativeplasmid, the total RNAs in cells were collected at the time of2h,4h,6h,8h,12h,16h,24h and48h induced by LXRs agonist T0901317and thenreverse transcripted into cDNA respectively. The relative levels of FASmRNA at different time in the two groups were compared by real-time PCR.Finally, HepG2cells were transfected with the best plasmid and thenegative plasmid for24h following by induction of LXRs agonistT0901317for30min. The levels of histone H3and H4acetylation, H3(S10)phosphorylation, H3(K4) trimethylation as well as total histone H3and H4across the promoter and transcriptional regulatory regions of FAS genewere compared between the silent plasmid group and negative plasmidgroup of HepG2cells. 2) Detection of the influence of LXR-alpha gene overexpression onchromatin remodelings across the promoter and transcriptional regulatoryregions of FAS geneFirstly, the LXR-alpha cDNA recombinant plasmid were transfectedinto HepG2cells for24h and the cells which were trasnsfected with blankplasmid or no plasmids were regarded as controls. The overexpressioneffect of LXR-alpha gene was assessed by comparing the mRNA andprotein levels of LXR-alpha through RT-PCR and Western blot. Then thetotal RNAs in cells were collected and then reverse transcripted into cDNArespectively at the time of12h,24h,36h and48h after HepG2cells weretransfected with the LXR-alpha cDNA recombinant plasmid or blankplasmid. The relative levels of FAS mRNA in cells at different time wereanalyzed by real-time PCR. Finally, the levels of histone H3and H4acetylation, H3(S10) phosphorylation, H3(K4) trimethylation as well astotal histone H3and H4across the promoter and transcriptional regulatoryregions of FAS gene were detected in different groups of HepG2cellswhich were transfected with the LXR-alpha cDNA recombinant plasmidfor12h,24h or36h.4. Inhibitory effect of histone acetyltransferase, garcinol on thetranscriptional expression of FAS gene induced by LXRsFirstly, the appropriate concentration of garcinol was selected bydetecting the survival rate of HepG2cells under different concentrations of garcinol through MTT. Then the levels of histone H3and H4acetylationacross the promoter and transcriptional regulatory regions of FAS genewere assessed30min after induction of the optimal concentration ofgarcinol together with T0901317. Finally the FAS mRNA and proteinexpression levels in cells treated with both the optimal concentration ofgarcinol together with T0901317were comtpared to these in cells treatedwith T0901317alone by methods of real-time PCR and Western blot.RESULTS:1. The time spectrum of FAS mRNA transcription expression in HepG2cells upon the LXRs agonist1) The triglyceride synthesis and the intracellular lipid accumulation inHepG2cells increased obviously upon1uM LXRs agonist T0901317.2) The FAS mRNA levels in4h,6h,8h,12h,16h,24h and48h groups ofcells increased in a time-dependent manner compared to that in theuntreated group (0h group) under1uM LXRs agonist T0901317. And thehighest level was at16h while the all the increases were statisticallysignificant (P<0.05, P<0.01).2. Chromatin remodelings on the promoter and transcriptional regulatoryregions of FAS gene upon the LXRs agonist1) There were varing degrees of changes in modifications of histoneacetylation, phosphorylation and trimethylation across5500bp range of thepromoter and the transcriptional regulatory regions of FAS gene at the early stage of its transcription activated by LXRs (within2h's induction byT0901317). The changes occurred in LXR response element (LXRE) at30min were the most dominant, which were the increased levels of histoneH3and H4acetylation as well as H3(K4) methylation and decreased levelsof H3(S10) phosphorylation. And there were also some different changesfor acetylation, methylation and phosphorylation on transcription start site,5'-2000bp upstream area and exon2simultaneously.2) Changes of the whole levels of histone H3and H4took place across5500bp range of the promoter and the transcriptional regulatory regions ofFAS gene at the early stage of its transcription activated by LXRs (within2h's induction by T0901317). The levels of histone H3and H4in all theregions were affected by T0901317distinctly. The levels of the twohistones became higher at30min, and the change of histone H3was moreobvious. The level of histone H3mainly increased while that of histone H4mainly decreased,60-120min after induction.3． Chromatin remodelings on the promoter and transcriptional regulatoryregions of FAS gene dependent on LXR-alpha1) LXR-alpha shRNA plasmid1had the optimal silencing effect amongthe three constructed LXR-alpha shRNA plasmids. The FAS mRNA levelsafter LXR-alpha gene had been silenced were not only lower than thatbefore LXR-alpha silencing, but also fluctuated around the0h basis level.Compared with the non-silent group, the decreasd level of FAS mRNA in silent group had statistically significance from6h (P<0.05), and thedifferences at12h and16h were the most dominant (P<0.01). LXR-alphagene silencing could cause different degrees of changes in the levels ofhistone H3and H4acetylation, H3(K4) methylation as well as the totalhistone H3and H4across the promoter and transcriptional regulatoryregions of FAS gene,in which those on LXRE location were the mostremarkable. No significant changes in the level of histone H3(S10)phosphorylation could seen after the silence of LXR-alpha gene.2) The LXR-alpha cDNA recombinant plasmids could elevate the mRNAand protein levels of LXR-alpha gene in HepG2cells. The FAS mRNAlevels increased in a time-dependent manner from24h after HepG2cellshad been transfected with LXR-alpha cDNA recombinant plasmid. And thelevels at36h and48h had statistical significance (P<0.05) compared to thatat0h. During the FAS gene transcription induced by overexpression ofLXR-alpha, there were changes in histone histone H3and H4acetylation,H3(K4) methylation as well as the total histone H3and H4across thepromoter and transcriptional regulatory regions of FAS gene, which had thesame trend with the changes cauesd by LXRs agonist T0901317. The levelof histone H3(S10) phosphorylation had slight change, which wascompletely different from that induced by LXRs agonist T0901317.4. Inhibitory effect of the inhibitor of histone acetyltransferase--garcinolon the transcriptional expression of FAS gene induced by LXRs The survival rates of HepG2cells were79.45%,49.05%,9.5％and8.1%respectively under the concentrations of5uM,10uM,20uM and50uM garcinol, in which those of more than10uM had significantdifference compared to blank group (P<0.05, P<0.001) and theconcentration of5uM had little effect on cells. The levels of histone H3and H4acetylation on the four sites of the promoter and transcriptionalregulatory regions of FAS gene were lower induced by5uM garcinoltogether with T0901317than those induced by T0901317alone while thedifferences were statistically significant (P<0.05). Changes occurred onLXRE and the transcription start site were the most predominant (P<0.01),which were even lower than the basic level of the blank group. Theinhibitory effect of garcinol on FAS gene mRNA expression activated byLXRs started from12h, peaked at16h and continued until48h. All thedifferences were statistically significant (P<0.05). The inhibition of proteinexpression began at24h and the strongest inhibitory effect occurred at48hwhile the differences had statistical significance (P <0.05).CONCLUSION:1. FAS mRNA expression levels started from12h in a time-dependentmanner, peaked at16h and continued until48h activated by LXRs agonistT0901317.2. There were different degrees of changes in two chromatin remodelingevents—histone modifications and nucleosome remodelings across the 5500bp range of transcriptional regulatory areas upstream and downstreamtranscription start site at the early stage of FAS transcription induced byLXRs agonist T09013173. Histone acetylation and methylation as well as nucleosome remodelingrelated to FAS transcription were proved be dependent on LXR-alpha bymethods of the gene silence and gene overexpression.4. The inhibitor of histone acetyltransferase--gacinol, could inhibit FASgene transcription and expression induced by LXRs via changing histoneacetylation levels on the transcription regulatory regions of FAS gene.