节点文献
SIRT1通过在α/β珠蛋白基因簇远端调控区（MRE/LCR）特异位点的募集改变组蛋白修饰状态调节珠蛋白基因表达
【作者】 宋崴；
【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士
【摘要】 真核基因表达是一个复杂而精细的过程,是遗传调控和表观遗传调控综合作用的结果。作为基因表达调控的中心坏节,真核基因的转录调控可以分为三个层次:DNA水平、染色质水平和核水平。染色质水平作为调控真核基因表达的一种独特方式,日益受到人们重视。真核生物染色质的基本单位是核小体,它由146bp DNA和一个核心组蛋白八聚体组成。核心组蛋白的N-端可以发生乙酰化、甲基化、磷酸化和泛素化等多种共价修饰。组蛋白修饰可以独自发挥作用,也可以相互组合形成“组蛋白密码”,共同调节基因表达,而组蛋白乙酰化可能在调节染色体结构和功能上起核心作用。组蛋白乙酰转移酶和组蛋白去乙酰化酶具有相反的活性,它们的作用共同维持着核小体组蛋白上赖氨酸乙酰化水平的动态平衡。目前发现至少有三组蛋白具有组蛋白去乙酰化酶活性。Ⅲ类组蛋白去乙酰化酶不同于Ⅰ类和Ⅱ类组蛋白去乙酰化酶,它们是酵母Sir2(silent information regulator 2)的同系物,是NAD依赖的组蛋白去乙酰化酶,从细菌到人类都具有高度保守性。哺乳动物的SIRT1是Sirtuins家族中与Sir2同源性最高的一个成员,在许多生命过程,如细胞凋亡、细胞周期、DNA损伤修复、重组、长寿和基因沉默中都发挥了重要的作用。最近的研究表明,SIRT1可以与多种的转录因子,如p53,FOXO家族和MyoD相互作用,调节特异基因表达,从而参与凋亡、分化和发育等重要的生命过程。珠蛋白基因簇是研究染色质结构和基因表达关系的经典模型,它分为α-和β-两个基因簇。人α-珠蛋白基因簇只有两组功能基因(ζ,α2-α1),在个体发育过程中只有一个基因开关。人β-珠蛋白基因簇有三组功能基因(ε,~Gγ-~Aγ,δ-β),在个体发育过程中存在两个基因开关。此外,由一系列高敏位点(HSs)组成的主调控区(majorregulatory elements,MRE)和基因座控制区(locus control region,LCR)作为最主要的远端调控元件,分别调控α-和β-两类珠蛋白基因的表达。本实验中,我们首先用SIRT1干扰和SIRT1HY质粒稳定转染K562细胞,筛选后得到稳定的单克隆细胞株。然后分别在SIRT1干扰及过表达SIRT1HY突变体的K562细胞中,用MTT技术检测细胞增殖的变化;联苯胺染色结合半定量RT-PCR分析血红蛋白生成和珠蛋白基因表达的改变。为了进一步阐述SIRT1调节珠蛋白基因表达的分子机制,我们通过ChIP实验检测了SIRT1在α-和β-珠蛋白基因簇上的募集,并通过ChIP实验分别检测了α-珠蛋白基因簇MRE区(HS40、HS33、HS10),ζ-和α2-,α1-珠蛋白基因启动子区,以及β-珠蛋白基因簇LCR区(HS1-5),ε-、γ-和β-珠蛋白基因启动子区的H3乙酰化、H4乙酰化与H3-K4双甲基化修饰的变化。最后,检测了SIRT1对参与调控珠蛋白基因表达的红系相关转录因子表达的影响,并检测SIRT1对GATA-1乙酰化修饰的影响,通过ChIP实验检测SIRT1对GATA-1和NF-E2/P45在β-珠蛋白基因簇上募集情况的影响。结果显示:(1)在K562细胞中干扰SIRT1表达后抑制细胞增殖。(2)在K562细胞中干扰SIRT1表达促进血红蛋白的生成并伴随着珠蛋白基因mRNA的上调,并且在hemin诱导后仍保持这种上调趋势。(3)在K562细胞中过表达SIRT1HY抑制细胞增殖。(4)在K562细胞中过表达SIRT1HY促进血红蛋白的生成并伴随着珠蛋白基因mRNA的上调,并且在hemin诱导后仍保持这种上调趋势。(5)ChIP实验可检测到SIRT1在α-珠蛋白基因簇MRE区HS40和HS10的募集,以及在β-珠蛋白基因簇LCR区HS4和HS2的募集。(6)在K562细胞中抑制SIRT1表达和过表达SIRT1HY后,α珠蛋白基因簇MRE上的高敏位点HS40和HS10的组蛋白H3和H4乙酰化修饰水平明显增加,ζ-、α2-和α1-珠蛋白基因启动子区的H3和H4乙酰化修饰水平没有明显变化;而H3-K4双甲基化修饰水平在MRE区HS40、HS33和HS10,以及ζ-和α2-珠蛋白基因启动子区均有一定的上调,α1-珠蛋白基因启动子区没有明显变化。(7)在K562细胞中抑制SIRT1表达和过表达SIRT1HY后,β-珠蛋白基因簇LCR上的高敏位点HS5、HS4和HS2的组蛋白H3和H4乙酰化修饰水平明显增加,ε-、γ-和β-珠蛋白基因启动子区的H3和H4乙酰化修饰水平也有增加;而H3-K4双甲基化修饰水平在LCR和ε-、γ-和β-珠蛋白基因启动子区均有一定的上调。(8)在K562细胞中抑制SIRT1表达和过表达SIRT1HY,均未在mRNA水平和蛋白水平影响主要的红系相关转录因子的表达、乙酰化修饰和在珠蛋白基因簇上的募集。结果表明:(1)在K562细胞中,SIRT1可以调节珠蛋白基因的表达。(2)SIRT1调节珠蛋白基因的表达是去乙酰化酶活性依赖的。(3)在α-珠蛋白基因簇MRE区的HS40和HS10,以及β-珠蛋白基因簇LCR区的HS4和HS2上均可检测到SIRT1的募集。(4)SIRT1影响α-和β-珠蛋白基因簇的组蛋白修饰水平。(5)SIRT1可能不影响主要的红系相关转录因子的表达、乙酰化修饰以及在珠蛋白基因簇上的募集。综合以上结果,SIRT1可能通过募集到α/β珠蛋白基因簇远端调控区(MRE/LCR)的特定高敏位点,影响组蛋白修饰状态,并最终调节珠蛋白基因表达。
【Abstract】 Transcription is a complex process relies on the collective action of genetic and epigenetic regulations.Eukaryotic gene expression can be viewed within a conceptual framework in which regulatory mechanisms are integrated at three hierarchical levels,i.e. the sequence level,the chromatin level and the nuclear level.Recent studies have revealed that chromatin,the organized packing of DNA in the eukaryotic nucleus,plays a pivotal role in the regulation of gene expression.The basic repeating of chromatin,the nucleosome, includes two copies of the four core histones,H2A,H2B,H3 and H4,wrapped by 146 bp of DNA.Numerous studies in the past few years have witnessed that histone posttranslational modifications,including acetylation,methylation,phosphorylation and ubiquitylation,play an important role in eukaryotic gene regulation.It was already proposed that histone acetylation could have a central role in the regulation of chromatin structure and function.Two groups of enzymes,histone acetyltransferases(HATs) and histone deacetylases(HDACs),have been shown to maintain the delicate dynamic equilibrium in the acetylation level of lysine residues in nucleosomal histones.There are at least three classes of proteins with intrinsic HDAC activity.ClassⅢhistone deacetylases are distinct from classⅠandⅡHDACs as homologues of the yeast silent information regulator 2(Sir2).Sir2 is an NAD-dependent deacetylase that is broadly conserved from bacteria to humans.Mammalian SIRT1(Sir2α) is a member of the Sirtuins family which plays important roles in diverse processes,such as cell apoptosis,cell cycle,DNA damage repair,recombination,life span and gene scilencing.Recently,SIRT1 was shown to interact with various transcription factors such as p53,forkhead transcription factor (FOXO) family proteins,and MyoD,and regulating the expression of special genes to participate in stress tolerance,differentiation,and development.The globin gene clusters,consisting ofα-andβ-globin gene clusters,is an excellent model for elucidating the relationship between chromatin structure and gene regulation. The humanα-globin gene cluster includes an embryonicζgene and two fetal/adultαgenes lying close to the telomere of the short arm of chromosome 16(tel-ζ-α2-α1-cen).A single switch occurs as development proceeds.The human[β-globin gene cluster is located on the short arm of chromosome 11 and arranged spatially in the order of their expression during ontogeny,5’-ε-~Gγ-~Aγ-δ-β-3’.During development,two switches of globin gene expression occur.Furthermore,theα-globin MRE andβ-globin LCR,both consisting of a series of hypersensitive sites,activate high-levelα- andβ-globin gene expression,respectively.In our studies,we first transfected K562 cells with SIRT1 RNAi and SIRT1HY mutation constructs and selected stably transfected single clones for each of them.Next,in the SIRT1 down-regulated or the SIRT1HY over-expressed K562 cells,we detected the change of cellular proliferation with MTT assays.After that,benzidine staining and semi-quantitative RT-PCR were applied for detecting the changes of hemoglobin and globin genes expression respectively.To investigate the molecular mechanism of SIRT1 regulating globin gene expression,we detected SIRT1 recruitment onα/βglobin gene cluster distal regulatory element and globin genes promoter.Additional ChIP analyses were performed to detect the change of active histone modifications,including H3 acetylation, H4 acetylation and H3-K4 dimethylation states,across the human globin gene clusters in the SIRT1 down-regulated and the SIRT1HY over-expressed K562 cells.Finally,we checked the expression of major erythroid related transcription factors by RT-PCR and western blot,the acetylation of GATA-1 by IP(immunoprecipitation),and the recruitment of GATA-1 and NF-E2/P45 onβ-globin gene cluster by ChIP(Chromatin immunoprecipitation) in both kinds of transfected cells to find whether the expression and post-translational modification of those factors have been affected by SIRT1 disturbance.Our experiments showed that:(ⅰ) SIRT1 down-regulation in K562 cells inhibits cellular proliferation.(ⅱ) SIRT1 down-regulation in K562 cells promotes hemoglobin and globin genes expression before and after being induced by hemin.(ⅲ) SIRT1HY over-expression in K562 cells inhibits cellular proliferation.(ⅳ) SIRT1HY over-expression in K562 cells promotes hemoglobin and globin genes expression before and after being induced by hemin.(ⅴ) SIRT1 can be recruited to HS40,HS10 ofα-globin MRE and HS4,HS2 ofβ-globin LCR.(ⅵ) Histone H3 and H4 acetylation increased obviously in HS40 and HS10 ofα-globin MRE,with no change been observed forζ-,α2-,andα1-globin promoter;while histone H3-K4 dimethylation increased modestly acrossα-globin cluster except for α1-globin promoter in SIRT1 down-regulated and SIRT1HY over-expressed K562 cells. (ⅶ) Histone H3 and H4 acetylation increased obviously in HS5,HS4 and HS2 ofβ-globin LCR,with no change in HS3 and HS1 ofβ-globin LCR and modest increase observed inε-,γ-,andβ-globin promoter;while histone H3-K4 dimethylation increased modestly across wholeβ-globin cluster in SIRT1 down-regulated and SIRT1HY over-expressed K562 cells. (ⅷ) The expression,acetylation and recruitment toβ-globin gene cluster of major erythroid related transcription factors were unaffected in SIRT1 down-regulated and SIRT1HY over-expressed K562 cells.From the results described above,several conclusions can be drawn:(ⅰ) SIRT1 could regulate globin genes expression in K562 cells.(ⅱ) SIRT1 regulates globin genes expression in a deacetylase-dependent manner.(ⅲ) SIRT1 recruitment on hypersensitive site HS40,HS10 ofα-globin MRE and HS4,HS2 ofβ-globin LCR.(ⅳ) SIRT1 influences histone modifications status ofα-/β-globin cluster.(ⅴ) SIRT1 may not affect major transcription factors expression,acetylation and recruitment on globin gene cluster. Therefore,SIRT1 may regulate globin genes expression through recruiting to particular HSs of MRE/LCR and modulating histone modifications status.