The Study On Apo CIII Enhancer Regulating The Chromatin Modification Of Human Apolipoprotein AI/CIII/AIV/AV Gene Cluster

Eukaryotic genome is packaged into chromatin,which plays vital role in regulating gene expression,as well as DNA replication and DNA repair.Regulating at chromatin level includes at least two aspects,ATP dependent chromatin remodeling and histone covalent modification.They are also important epigenetic signals due to cellular memory mechanisms.Different histone modifications result in condensed or decondensed chromatin structure,which is relative to gene activation or gene repression.Among the histone modifications,acetylated histone H3(H3 Ac) and methylated histone H3 lysine 4(H3K4 di-Me) represent the transcriptional active chromatin.According to the range of histone modification,chromatin can be opened locally at cis-elements,or opened extensively across enhancer,promoter and even the intergenic regions between them.The extensively opened chromatin may provide loose chromatin structure for active transcription.The formation of such an extensively loose chromatin is relative to enhancer-promoter interaction.By RNA-Trap and chromatin conformation capture assays, a loop structure of the communicated enhancer and promoter was confirmed.There are three models for the formation of the loop structure overcoming long distance---looping model,linking model and tracking model.Recent studies suggest the correlation between intergenic transcription and extensive chromatin opening.Intergenic transcription may contribute to the tracking process from an enhancer to a promoter.Apolipoprotein Aâ… /Câ…¢/Aâ…£/Aâ…¤genes play important roles in lipids metabolism. Oberrant expression of these genes results in atherosclerosis,metabolism syndrome or other cardiovascular disease.The apoCâ…¢enhancer is a common enhancer for the liver and the intestine specific expression of apo Aâ… ,apo Câ…¢and apo Aâ…£,but not apo Aâ…¤.Many efforts involved in studing the cis-elements,trans-factors or signal transduction pathways for apolipoprotein gene expression.However,there is no report on how the apoAâ… gene cluster is regulated at the chromatin level.Since the importance of chromatin in eukaryotic gene's expression,and the importantce of epigenetics in the development of diseases resulting from circumstance,in this study,we want to disclose how the apoCâ…¢enhancer affects the chromatin of the apoAâ… gene cluster so as to regulate apo gene expression.To study the potential role for apo Câ…¢enhancer regulating apo Aâ…¤promoter,we constructed the apo Câ…¢enhancer-apo Aâ…¤promoter luciferase report vectors,and observed the expression of luciferase after transient transfection.As expected,apoCâ…¢enhancer increased the expression level of apo Aâ…¤in vitro.However,in our previous transgenic mice experiments,apoCâ…¢enhancer didn't regulate apo Aâ…¤gene in vivo.Considering chromatin regulation may contribute to the difference between the in vitro and in vivo results,we firstly studied the relationship between the transcriptional level and the histone modification level in HepG2,Caco-2 and HeLa cells.In ChIP assays, antibodies against acetylated H3 and di-methylated H3K4,which represent opened chromatin structure,were used.To study the enhancer's role in chromatin modification in vivo,we then compared the different histone modification level of the apo Aâ… gene cluster in livers between normal transgenic mice and enhancer deleted transgenic mice.The results from HepG2,Caco-2 cells and the liver of transgenic mice showed that H3 Ac and H3K4 di-Me had similar modification pattern,i.e.,extensively opened chromatin in the region of apoAâ… -apoCâ…¢-apoAâ…£,except for the low acetylation level at apo Aâ…¤promoter and a site in the apo Aâ…¤-apoAâ…£intergenic region.It seemed that the extensive histone acetylation modifications prefered to apoAâ… -apoCâ…¢region in HepG2 cells and in the liver,while they prefered to apoAâ…¤-apoAâ…£region in Caco-2 cells.However,only local histone acetylation presented on the apo Aâ…¤promoter,and there were low acetylation between apo Aâ…¤-apoAâ…£in HepG2 cells.So the extenisively opened chromatin didn't continue to apo Aâ…¤region.In livers of apo Câ…¢enhancer deleted transgenic mice,the histone modification level of apo Aâ… -apoCâ…¢-apoAâ…£region were decreased extensively.Since the potential correlation among intergenic transcription,chromatin opening and enhancer-promoter communication,we measured the intergenic transcripts on apo Aâ… gene cluster by RT-PCR,and compared the intergenic transcriptional level between the normal and the enhancer deleted transgenic mice.At the same time,we predicted the potential regulatory elements in the apoAâ…¤-apoAâ…£region by bioinformatics.The possible mechanisms for apoCâ…¢enhancer function were discussed.The results showed that there were tissue specific intergenic transcripts throughout the apo AI gene cluster,even at the low histone modification sites,whereas the transcripts downstream the apo Aâ…¤gene also expressed in the apoAâ… gene cluster non-expression cells.Apo Câ…¢deletion resulted in the transcriptional decrease at sites of apo Aâ… -apoCâ…¢and apo Aâ…£-apoCâ…¢intergenic region. A potential nuclear matrix attachment region(MAR),characterized with palindrome AT-rich,just downstream the apoAâ…£gene,was predicted by bioinformatics.The hisotne H3 acetylation and H3K4 methylation were particularly low near the potential MAR.According to the results mentioned above,we conclude that,1) Potential communication exsits between apo Câ…¢enhancer and apo Aâ…¤promoter.2) In apo Aâ… gene cluster,acetylated histone H3 relative to transcriptional activity,whereas di-methylated H3K4 relative to the transcriptional possibility.3) Extensive histone H3 acetylation across the apo Aâ… /Câ…¢/Aâ…£gene are in accordance with transcriptional level,while the chromatin of apo Aâ…¤promoter is insulated from the extensive acetylation domain.4) Tissue specific intergenic transcripts throughout the apoAâ… gene cluster may participate in gene regulation. 5) apoCâ…¢enhancer regulates not only the histone modification of apo Aâ… -apoCâ…¢-apoAâ…£region,but also the intergenic transcription of this region.Whether the potential MAR obstacles the spreading of extensive chromatin opening mediated by apoCâ…¢enhancer,and whether intergenic transcripts from the repeat sequences involved in the particular low histone modification by the RNAi mechanism,are interesting questions need to be studied in the near future.