摘要
真核生物与原核生物基因组结构的最大差别是真核生物的基因组紧密压缩和包装在染色质结构中。染色质的高级结构是真核生物基因转录进行的障碍,为了使基因转录正常进行,核心组蛋白的结构必须发生改变。近年来人们发现了能够通过改变染色质构型来影响(活化或抑制)基因转录的蛋白质复合物,统称为核小体重塑复合物(nucleosome remodeling complexes)。Gcn5(general control non-derepressible 5)是SAGA这种在酵母中主要的转录相关HAT复合物的接触反应亚基,主要的作用靶位为组蛋白H3,通过乙酰化修饰发挥重要作用。我们选取hsp70家族ssa4基因,通过Real-time RT-PCR,染色质免疫沉淀,免疫共沉淀,RT-PCR等实验技术进行分析证明组蛋白乙酰转移酶Gcn5可以结合到ssa4基因的启动子区,通过调控启动子区组蛋白H3的乙酰化水平,对基因的转录调控发挥作用。并且我们发现,HDAC也参与了ssa4基因的转录调控,在TSA处理的野生型菌株和组蛋白去乙酰化酶缺失突变菌株中检测ssa4基因的表达情况,证明酵母中组蛋白去乙酰化酶Rpd3对ssa4基因的表达具有明显的调控作用。总之,可逆的组蛋白乙酰化修饰对ssa4基因的转录调控具有十分重要的作用。组蛋白乙酰转移酶Gcn5直接参与了ssa4基因的转录调控,它与组蛋白去乙酰化酶的拮抗作用决定了ssa4基因的转录状态。本论文对热休克基因ssa4转录调控的研究,为进一步深入探讨其转录机理提供了有价值的实验证据。
The eukaryotic genome is packaged into chromatin, which acts as a constant barrier to transcription and other cellular processes that require access to DNA. Therefore the structure of chromatin has to be modified before active transcription can proceed. Gcn5 (general control non-derepressible 5) is the catalytic subunit of SAGA (Spt-Ada-Gcn5-acetyltransferase), a major transcription-related HAT complex in yeast. Histone H3 is the primary target for both of the complex, and the complex display significant functional redundancy. We chose ssa4 as the target gene for biochemical and molecular studies. Our results indicated that histone acetylation was required for the ssa4 transcription activation. Histone acetyltransferases (HATs) Gcn5 affecteds ssa4 gene transcription and its HAT activity was required for the function of regulating transcription. The transcriptional regulatory function of Gcn5 was direct because it could be recruited to ssa4 gene. The HATs modulated the transcription of ssa4 genes through affecting the acetylation status of histone H3. Our data also showed that the mRNA level of ssa4 gene was increased in the cells treated with TSA or the cells with the deletion of histone deacetylases (HDACs), indicating that HDACs were involved in the transcription of ssa4 gene. Data presented in this report provide further insight into and direct evidence of the functions of Gcn5 in hsp ssa4 gene transcriptional in yeast.
引文
[1]Paranjape S M,Kamakaka R T,Kadonaga J T. Role of chromatin structure in the regulation of transcription by RNA polymeraseⅡ[J]. Annu Rev Biochem,1994,63(): 265-297.
[2]Davie J R. Covalent modifications of histones: expression from chromatin templates [J]. Curr Opin Genet Dev, 1998, 8(): 173-178.
[3]Allfrey V, Faulkner R M, Mirsky A E. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis[J]. Proc Natl Acad Sci U S A, 1966,51(): 786-794.
[4]Roth SY, Denu JM, Allis CD. Histone Acetyltransferases[J]. Annu Rev Biochem,2001, 70():81-120.
[5]Khorasanizadeh S. The nucleosome: from genomic organization to genomic regulation[J]. Cell, 2004, 116(): 259-272.
[6]Berger SL. Gene activation by histone and factor acetyltransferases[J]. Curr Opin Cell Biol, 1999, 11():336-341.
[7] Allfrey VG, Faulkner RM, Misky AE. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci. USA. 1964, 51:786-793.
[8] Berger SL. Gene activation by histone and factor acetyltransferases. Curr Opin Cell Biol. 1999, 11:336-341.
[9] Cress WD, Seto E. Histone Deacetylases, transcriptional control and cancer. J Cell Physiol. 2000, 184:1-16.
[10] Wu C. Chromatin remodeling and the control of gene expression. J Biol Chem.1997, 272:28171-4.
[11] Peterson CL. HDAC's at work: everyone doing their part. Mol Cell. 2002, 9:921-2.
[12] Turner BM. Histone acetylation and an epigenetic code. Bioessays. 2000, 22:836-45.
[13] Peterson CL, Logie C. Recruitment of chromatin remodeling machines. J Cell Biochem. 2000, 78:179-85.
[14] Hebbes TR, Thorne AW, Crane-Robinson C. A direct link between core histone acetylation and transcriptionally active chromatin. EMBO J. 1988, 7:1395-1402.
[15] Grunstein M. Histone acetylation in chromatin structure and transcription. Nature. 1997, 389:349-352.
[16] Fletcher TM, Hansen JC. The nucleosomal array: structure /function relationships. Crit. Rev. Eukaryote Gene Expr. 1996, 6:149-188.
[17] Brownell JE, Zhou J, Ranalli T, Kobayashi R, Edmondson DG, Roth SY, Allis CD. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell. 1996, 84: 843-851.
[18] Smith ER, Allis CD, Lucchesi JC. Linking global histone acetylation to the transcription enhancement of X-chromosomal genes in Drosophila males. J. Biol Chem. 2001, 276:31483- 31466.
[19] Lusser A, Kolle D, Loidl P. Histone acetylation: lessons from the plant kingdom. Trends Plant Sci. 2001, 6:59-65.
[20] Allis CD, Chicoine LG, Richman R, Schulman IG. Deposition-related histone acetylation in micromuclei of conjugating Tetrahymena. Proc Natl Acad Sci. USA. 1985, 82:8048-8052. Johnson CA, Turner BM. histone deacetylases: complex transducers of nuclear signals [J]. Semin Cell Dev Biol, 1999, 10: 179-188.
[21] Howe L, Brown CE, Lechner T, et al. Histone acetyltransferase complexes and their link to transcription [J]. Cri Rev Eukaryot Gene Expr, 1999, 9: 231-243.
[22] Khochbin S, Verdel A, Lemercier C, et al. Functional significance of histone deacetylase diversity [J]. Curr Opin Genet Dev, 2001, 11: 162-166.
[23] Marks PA, Miller T, Richon VM. Histone deacetylases [J]. Current Opinion in Pharmacology, 2003, 3: 344-351.
[24] Santos-Rosa H, Caldas C. Chromatin modifier enzymes, the histone code and cancer [J]. Eur J Cancer, 2005, 41: 2381-2402.
[25] Christina M, Grozinger CM, Stuart LS. Deacetylase enzymes: biological functions and the use of small-molecule inhibitors [J]. Chem Biol, 2002, 9: 3-16.
[26] Zhang Y, Ng HH, Erdjument-Bromage H, et al. Analysis of the NURD subunits reveals a histone deacetylase core complex and a connection with DNA methylation [J]. Genes Dev, 1999, 13: 1924-1935.
[27] Li J, Wang J, Nawaz Z, et al. Both corepressor proteins SMRT and N-CoR exist in large protein complexs containing HDAC3 [J]. EMBO J, 2000, 19: 4342-4350.
[28] Miska EA, Karlsson C, Langley E, et al. HDAC4 deacetylase associates with and represses the MEF2 transcription factor [J]. EMBO J, 1999, 18: 5099-5107.
[29] Guenther MG, Lane WS, Fischle W, et al. A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness [J]. Genes Dev, 2000, 14: 1048-1057.
[30] Huang EY, Zhang J, Miska EA, et al. Nuclear receptor corepressors partner with classⅡ histone deacetylase in a Sin3-independent repression pathway [J]. Genes Dev, 2000, 14: 45-54.
[31] Kao HY, Downes M, Ordentlich P, et al. Isolation of a novel histone deacetylase reveals that classⅠand classⅡ deacetylases promote SMRT-mediated repression [J].Genes Dev, 2000, 14: 55-66.
[32] Wen YD, Perissi V, Staszewski LM, et al. The histone deacetylase-3 complex contains nuclear receptor corepressors [J]. Proc Natl Acad Sci USA, 2000, 97: 7202-7207.
[33] Dressel U, Bailey PJ, Wang SC, et al. A dynamic role for HDAC-7 in MEF2 mediated muscle differentiation [J]. J Biol Chem, 2001, 276: 17007-17013.
[34] Wang AH, Bertos NR, Vezmar M, et al. HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor [J]. Mol Cell Biol, 1999, 19: 7816-7827.
[35] Rito6sa F. A new puffing pattern induced by temperature shock and DNP in Drosophila [J]. Experientia, 1962, 18: 571-573.
[36] Tisseres A, Mitchell AK. Some new proteins induced by temperature shock in Drosophila [J]. J Mo1 Biol, 1974, 84: 389-398.
[37] Horwitz J. a-Crystaline can function as a molecular chaperone Proc [J]. Nail Acad Sci USA, 1992, 89: 10449-10453.
[38] Yuki Sugiyama. Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation [J]. J Biol Chem, 2000, 275: 1095-1104.
[39] 朱玉贤, 李毅. 现代分子生物学(第2版) [M]. 北京:高等教育出版社, 2002, 7:281-283.
[40] Yeh CH,Chang PL,Yeh KW,et al. Expression of a gene encoding a 16.9-kDa heat-shock protein Oshspl6.9, in Escherichia coil enhances thermotolerance [J]. Proc Natl Acad Sci, 1997, 94: 10967-10972.
[41] Shopland LS, Hirayoshi K, Fernandes M, et al. HSF access to heat shock elements in vivo depends critically on promoter architecture difined by GAGA factor, TFⅡD, and RNA polymeraseⅡbinding sites [J]. Genes & Dev, 1995, 9: 2756-2769.
[42] Leibovitch BA, Lu Q, Benjamin LR, et al. GAGA factor and the TFⅡD complex collaborate in generating an open chromatin structure at the Drosophila melanogaster hsp26 promoter [J]. Mol Cell Biol, 2002, 17: 6148-6157.
[43] Tsukiyama T, Becker P, Wu C. ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor [J]. Nature, 1994, 367: 525-532.
[44] Tsukiyama T, Wu C. Purification and properties of an ATP-dependent nucleosome remodeling factor [J]. Cell, 1995, 83: 1011-1020.
[45] Sandaltzopoulos R, Becker PB. Heat shock factor increases the reinitiation rate from potentiated chromatin templates [J]. Mol Cell Biol, 1998, 18: 361-367.
[46] Garcia-Bermejo L, Vilaboa NE, Perez C, et al. Modulation of heat-shock protein 70 (hsp70) gene expression by sodiumbutyrate in U-937 promonocytic cell: relationshipswith differentiation and apoptosis [J]. Exp Cell Res, 1997, 236 (1): 268-274.
[47] Deckert J, Struhl K. Histone acetyltion at promoters is differentially affected by specific activators and repressors [J]. Mol Cell Biol, 2001, 21 (8): 2726-2735.
[48] Nightingale KP, Wellinger RE, Sogo JM, et al. Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin [J]. EMBO J, 1998, 17: 2865-2876.
[49] Li Q, Herrler M, Landsberger N, et al. Xenopus NF-Y pre-sets chromatin to potentiate p300 and acetylation-responsive transcription from the Xenopus hsp70 promoter in vivo [J]. EMBO J, 1998, 17: 6300-6315.
[50] Ovakim DH, Heikkila JJ. Effect of histone deacetylase inhibitors on heat shock protein gene expression during Xenopus development [J]. Genetics, 2003, 36: 88-96.
[51] Smith ST, Petruk S, Sedkov Y, et al. Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex [J]. Nature Cell Boil, 2004, 6: 162-167.
[52] Burdon RH. Heat shock proteins in relation to medicine [J]. Mol Aspects Med, 1993, 14(2):83-165.
[53] 刘德立. 热激蛋白Hsp70的新功能区--钙调蛋白结合区[J]. 生命的化学, 1993, 13(1): 24-25.
[54] 张君岚, 黄善生, 凌亦凌. 不同家族热休克蛋白的生理功能及病理意义[J]. 中国病理生理杂志, 1998, 14(5): 549-553.
[55] Lee JE,Kim YJ,Kim JY,et al. The 70kDa heat shock protein suppresses matrix metalloproteinases in astrocytes [J]. Neuroreport, 2004, 15(3): 499-502.
[56] Gabai VL,Meriin AB,Mosser DD,et a1. Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance [J]. Biol Chem, 1997, 272(29): 18033-180377.
[57] 郑磊, 颜晓慧. 热应激时热休克蛋白70及其细胞保护作用[J]. 国外医学--卫生学分册, 1999, (26)4: 209-212.
[58] Burke D, Dawson D, Stearns T. Methods in Yeast Genetics, A Cold Spring Harbor Laboratory Course Manual, USA [M]. Cold Spring Harbor Laboratory Press, 2000, Edition: 103-105.
[59] Hirata Y, Andoh T, Asahara T, et al. Yeast glycogen synthase kinase-3 activates Msn2p-dependent transcription of stress responsive genes [J]. Mol Biol Cell, 2003, 14: 302-312.
[60] Totter EW, Camilla M-F Kao, Berenfeld L, et al. Misfolded proteins are competent to mediate a subset of the responses to heat shock in Saccharomyces cerevisiae [J]. J Biol Chem, 2002, 277: 44817-44825.
[61] Kadosh D, Struhl K. Repression by Ume6 involves recruitment of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to target promoters [J]. Cell, 1997, 89: 365-371.
[62] Kurdistani SK, Robyr D, Tavazoie S, et al. Genome-wide binding map of the histone deacetylase Rpd3 in yeast [J]. Nat Genet, 2002, 31: 248-254.
[63] Robyr D, Suka Y, Xenarios I, et al. Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases [J]. Cell, 2002, 109: 437-446
[64] Chen T, Sun H, Lu J, et al. Histone acetylation is involved in hsp70 gene transcription regulation in Drosophila melanogaster [J]. Arch Biochem Biophys, 2002, 408: 171-176.
[65] Smith ST, Petruk S, Sedkov Y, et al. Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex [J]. Nature Cell Boil, 2004, 6: 162-167.
[66] Thomson S, Hollis A, Hazzalin C, et al. Distinct stimulus-specific histone modifications at hsp70 chromatin targeted by the transcription factor heat shock factor-1[J]. Molecular Cell, 2004, 15: 585-594.