| 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.
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