自体乙酰化诱饵蛋白酵母双杂合筛选体系的建立
摘要
以DNA为模板的细胞核活动如：转录、复制、重组和修复都发生于染色体中。 而染色体结构与核心组蛋白H2A、H2B、H3和H4密切相关。染色体的基本单位─核小体由两个分子的H2A、H2B、H3、H4与其外围缠绕的约150bpDNA片段组成。核小体的形成还需要组蛋白－组蛋白之间，组蛋白－DNA之间的结合反应，这些反应主要发生于核心组蛋白中心结构域，即：组蛋白折叠区域。所有核心组蛋白N’末端为“活动尾”，这些“活动尾”突出于核小体之外而无固定构像。同时敲除酵母细胞组蛋白H3和H4基因或H2A和H2B基因将导致细胞死于G2/M期。
许多翻译后的修饰可以发生在组蛋白尾，这些修饰包括：乙酰化、甲基化、磷酸化、泛蛋白化等。组蛋白呈高度碱性，上述修饰可改变组蛋白的荷电情况；这样会对紧凑型结构的染色体产生较大影响。通常，呈紧凑型结构的染色体不易与蛋白因子结合并阻止蛋白因子与DNA的后续反应。乙酰化后的组蛋白尾与基因组DNA反应的Km值远远高于非乙酰化组蛋白尾。亦即：乙酰化组蛋白与DNA反应减弱，从而有利于蛋白因子与DNA结合,寻找到相对应的DNA元件。通常，高水平乙酰化组蛋白有利于转录激活，低水平乙酰化组蛋白导致转录抑制／沉默。已知乙酰化组蛋白可以结合蛋白中高度保守序列─bromodomain,含bromodomain的蛋白大都为转录激活蛋白因子。甲基化组蛋白可以结合chromodomain,这一结构域常见于转录抑制蛋白因子中。除乙酰化外，其它组蛋白修饰如：磷酸化、甲基化和泛酸化也与特异性基因转录调节有关。
此外，不同的组蛋白修饰之间相互作用,如:组蛋白H3S10磷酸化可以促进H4K14的乙酰化,H4R3甲基化可增强H4K8乙酰化等。
上述证据倾向于支持这种观点：每种组蛋白修饰可以被其它蛋白或蛋白结构域所解读，从而引发不同的生物学过程。2000年, David Allis 首次提出了”组蛋白密码”假说，该假说认为不同形式修饰的组蛋白可以作为特定的信号被蛋白因子识别，这些蛋白因子在包含相应组蛋白修饰的基因组区域实施其功能。
到目前为止，所有与修饰后组蛋白结合的蛋白鉴定仅限于生物化学方
法，而在基因组范围内的筛选工作还很少，有报道在细胞内表达异源性的蛋白激酶，用双杂合试验进行翻译后修饰特异性结合蛋白的筛选。如：在酵母中表达酪氨酸激酶或在大肠杆菌中表达哺乳动物丝氨酸／苏氨酸激酶，以使底物磷酸化后再用双杂合试验来进行筛选。由于磷酸化过程依赖于传统的酶－底物反应（反式作用），如果外源性的酶难以从宿主细胞中众多的蛋白中识别特异蛋白底物则接下来的筛选可能失败。因此，为提高底物磷酸化的效率，增加“诱饵”蛋白的特异性就必须过量表达激酶，而这样可使宿主细胞中蛋白被不当修饰导致细胞生长停滞。
为使细胞内酵母双杂合试验“诱饵”蛋白能特异地、持续修饰，我们将Gcn5 HAT结构域和组蛋白H3尾相连，这样酶与底物的物理性连接可能使“自体催化”现象发生在一个蛋白分子内而产生高效率的乙酰化修饰。我们将组蛋白H3尾（1-59位氨基酸）与Gcn5(18-252位氨基酸)野生型相连,再连接Gal4DNA结合结构域和HA；用无HAT 活性的Gcn5 变异体作为对照。将含有上述融合蛋白ORF的质粒和载体对照分别转入酵母细胞中(PJ69-4α)，用抗HA和抗组蛋白H3K9/K14乙酰化抗体检测酵母全细胞提取液中人工融合蛋白的工作情况。结果显示与Gcn5 野生型相连的组蛋白H3/H4被乙酰化,而与变异体Gcn5 F221A连接的组蛋白H3却无乙酰化。这个现象在其它嵌合蛋白中也得到了印证，如将GST 取代HA,质粒在大肠杆菌中表达，也得到同样结果。重要的是，用抗非乙酰化H3抗体检测在酵母细胞中表达的融合蛋白，与Gcn5野生型相连接的组蛋白H3的信号很弱,表明Gcn5野生型自体催化效率很高。这些结果说明以下问题：
1、Gcn5在融合蛋白中,尽管有Gal4 DBD和HA的存在,仍然能够催化其连接的底物。如此，其它HAT也有可能在类似的融合蛋白中起作用。
2、Gcn5在融合蛋白中仍具有底物特异性。
3、对于Gcn5 HAT催化活性丧失的变异型(F221A)，尽管连接了底物，但仍然不能行使转乙酰基的功能；因此，这一重组体可以用来作为阴性对照以排除与组蛋白乙酰化无关的反应。
4、尽管在酵母细胞内有多种HAT,其中至少两种HAT可以乙酰化组蛋白H3；但没有一种HAT可以催化融合蛋白内的组蛋白H3。其中的原因可能是正常情况下细胞内的HAT被转录因子结合到特异的DNA位置，因此内源性
的HAT催化融合蛋白中组蛋白H3乙酰化的可能性甚小。
5、酵母细胞内至少有5种确定的组蛋白脱乙酰化酶(HDACs)以及数种HDAC同源酶,而本试验的结果表明在上述酶同时存在的情况下,融合蛋白的乙酰化作用占主导地位。
总之，对于酵母细胞双杂合筛选试验，融合蛋白GAL4DBD-H3-Gcn5 wt-HA可以作为高特异性、高催化效率的自体催化“诱饵”蛋白；含有Gcn5 野生型的融合蛋白可以用来检测乙酰化结合蛋白，而含有Gcn5F221A的融合蛋白可以筛除与组蛋白乙酰化无关的反应。
我们还构建了GAL4DBD-H4-Gcn5 wt-HA和GAL4DBD-H4-Gcn5 F221A-HA重组体,试验表明: GAL4DBD-H4-Gcn5 wt-HA 融合蛋白有自体乙酰化现象(H4K8)而 GAL4DBD-H4-Gcn5F221A-HA则无。
为检测融合蛋白能否用于酵母双杂合?
Core histones H2A,H2B,H3 and H4 are the essential proteins for chromatin organization and cell viability. DNA-templated nuclear activities, including transcription, replication, recombination, and repair, all take place within the chromatin. Two molecules of each of the core histones are wrapped around by about 150 base pairs of DNA to form a nucleosome. Formation of nucleosomes requires extensive histone-histone and histone-DNA interactions occurred mainly within the central histone-fold domain of each core histone. The N' termini of all core histones are "flexible tails" which protrude from the nucleosomal core particles and are largely unstructured. The yeast cells without histones H3 and H4, or H2A and H2B N' termini are not viable ,they die at G2/M phase of the cell cycle.
Histone tails are subject to multiple post-translational modifications which include acetylation, methylation, phosphorylation, ubiquitylation , sumolation etc.Many of these modifications may change the ionic charge of the highly basic histones, which have substantial effects on the compact structure of chromatin. Usually the compact structure of chromatin restricts the binding and progression of protein factors. The acetylated histone tail peptide interacts with general DNA at much more higher Km than that of unacetylated ,that means a weakened DNA histone binding interaction may allow for better access of DNA binding and processing factors to find their cognate DNA elements. Generally speaking, histone hyper- and hypo-acetylation each triggers transcriptional activation and repression / silence, respectively. Acetylated histones have been shown to be bound by a highly conserved motif, bromodomain,that is found in many transcriptional activators. Likewise, methylated histones recruit chromodomain, that is frequently found to be part of transcriptional repressors. In addition to acetylation, the other modification pattern including phosporylation, methylation
and ubiquitylation were found to be involved in gene specific regulation.
In addition, different histone modifications may influence each other. for instance, acetylation at lysine 14 of H3 by several histone acetyltransferases (HAT) has been shown to be facilitated by phosphorylation at serine 10, and there is same case between arginine 3 methlylation of H4 and lysine 8 acetylation.
These evidences has caused scientists to favor the view that each histone modification pattern may be read by other proteins or protein modules, and thus followed different biological procedures. In 2000, David Allis proposed the "histone code" hypothesis which suggests that differentially modified histones may act as specific signals that are interpreted by the binding of selective factors. Such factors thus perform certain functions at the genomic loci bearing the corresponding histone modifications.
Thus far, all modified histone-binding proteins were identified by biochemical methods. Genome-wide screening for proteins possessing affinity toward specifically modified histones needs to be done. To identify the modified histone binding proteins, previous strategies have been based on the ectopic expression of protein kinases in the two-hybrid hosts which normally lack such enzymes. For example, tyrosine kinases and mammalian serine/threonine kinases were respectively expressed in yeast and E. coli for two-hybrid screens. The substrate proteins to be screened in these assays were phosphorylated and hence allowed the detection of interactions involving these phosphorylation events. Major concerns of these methods include the efficiency and specificity with which the bait protein to be screened is modified. Phosphorylation relies on a typical trans reaction between an enzyme and its substrates. If the foreign enzyme cannot identify the hybrid protein in the sea of host cellular proteins for efficient modification, the subsequent screen is likely to be unsuccessful. The efficiency of modification may be increased by overexpressing the enzyme, but inadvertent modifications of host protein
许多翻译后的修饰可以发生在组蛋白尾，这些修饰包括：乙酰化、甲基化、磷酸化、泛蛋白化等。组蛋白呈高度碱性，上述修饰可改变组蛋白的荷电情况；这样会对紧凑型结构的染色体产生较大影响。通常，呈紧凑型结构的染色体不易与蛋白因子结合并阻止蛋白因子与DNA的后续反应。乙酰化后的组蛋白尾与基因组DNA反应的Km值远远高于非乙酰化组蛋白尾。亦即：乙酰化组蛋白与DNA反应减弱，从而有利于蛋白因子与DNA结合,寻找到相对应的DNA元件。通常，高水平乙酰化组蛋白有利于转录激活，低水平乙酰化组蛋白导致转录抑制／沉默。已知乙酰化组蛋白可以结合蛋白中高度保守序列─bromodomain,含bromodomain的蛋白大都为转录激活蛋白因子。甲基化组蛋白可以结合chromodomain,这一结构域常见于转录抑制蛋白因子中。除乙酰化外，其它组蛋白修饰如：磷酸化、甲基化和泛酸化也与特异性基因转录调节有关。
此外，不同的组蛋白修饰之间相互作用,如:组蛋白H3S10磷酸化可以促进H4K14的乙酰化,H4R3甲基化可增强H4K8乙酰化等。
上述证据倾向于支持这种观点：每种组蛋白修饰可以被其它蛋白或蛋白结构域所解读，从而引发不同的生物学过程。2000年, David Allis 首次提出了”组蛋白密码”假说，该假说认为不同形式修饰的组蛋白可以作为特定的信号被蛋白因子识别，这些蛋白因子在包含相应组蛋白修饰的基因组区域实施其功能。
到目前为止，所有与修饰后组蛋白结合的蛋白鉴定仅限于生物化学方
法，而在基因组范围内的筛选工作还很少，有报道在细胞内表达异源性的蛋白激酶，用双杂合试验进行翻译后修饰特异性结合蛋白的筛选。如：在酵母中表达酪氨酸激酶或在大肠杆菌中表达哺乳动物丝氨酸／苏氨酸激酶，以使底物磷酸化后再用双杂合试验来进行筛选。由于磷酸化过程依赖于传统的酶－底物反应（反式作用），如果外源性的酶难以从宿主细胞中众多的蛋白中识别特异蛋白底物则接下来的筛选可能失败。因此，为提高底物磷酸化的效率，增加“诱饵”蛋白的特异性就必须过量表达激酶，而这样可使宿主细胞中蛋白被不当修饰导致细胞生长停滞。
为使细胞内酵母双杂合试验“诱饵”蛋白能特异地、持续修饰，我们将Gcn5 HAT结构域和组蛋白H3尾相连，这样酶与底物的物理性连接可能使“自体催化”现象发生在一个蛋白分子内而产生高效率的乙酰化修饰。我们将组蛋白H3尾（1-59位氨基酸）与Gcn5(18-252位氨基酸)野生型相连,再连接Gal4DNA结合结构域和HA；用无HAT 活性的Gcn5 变异体作为对照。将含有上述融合蛋白ORF的质粒和载体对照分别转入酵母细胞中(PJ69-4α)，用抗HA和抗组蛋白H3K9/K14乙酰化抗体检测酵母全细胞提取液中人工融合蛋白的工作情况。结果显示与Gcn5 野生型相连的组蛋白H3/H4被乙酰化,而与变异体Gcn5 F221A连接的组蛋白H3却无乙酰化。这个现象在其它嵌合蛋白中也得到了印证，如将GST 取代HA,质粒在大肠杆菌中表达，也得到同样结果。重要的是，用抗非乙酰化H3抗体检测在酵母细胞中表达的融合蛋白，与Gcn5野生型相连接的组蛋白H3的信号很弱,表明Gcn5野生型自体催化效率很高。这些结果说明以下问题：
1、Gcn5在融合蛋白中,尽管有Gal4 DBD和HA的存在,仍然能够催化其连接的底物。如此，其它HAT也有可能在类似的融合蛋白中起作用。
2、Gcn5在融合蛋白中仍具有底物特异性。
3、对于Gcn5 HAT催化活性丧失的变异型(F221A)，尽管连接了底物，但仍然不能行使转乙酰基的功能；因此，这一重组体可以用来作为阴性对照以排除与组蛋白乙酰化无关的反应。
4、尽管在酵母细胞内有多种HAT,其中至少两种HAT可以乙酰化组蛋白H3；但没有一种HAT可以催化融合蛋白内的组蛋白H3。其中的原因可能是正常情况下细胞内的HAT被转录因子结合到特异的DNA位置，因此内源性
的HAT催化融合蛋白中组蛋白H3乙酰化的可能性甚小。
5、酵母细胞内至少有5种确定的组蛋白脱乙酰化酶(HDACs)以及数种HDAC同源酶,而本试验的结果表明在上述酶同时存在的情况下,融合蛋白的乙酰化作用占主导地位。
总之，对于酵母细胞双杂合筛选试验，融合蛋白GAL4DBD-H3-Gcn5 wt-HA可以作为高特异性、高催化效率的自体催化“诱饵”蛋白；含有Gcn5 野生型的融合蛋白可以用来检测乙酰化结合蛋白，而含有Gcn5F221A的融合蛋白可以筛除与组蛋白乙酰化无关的反应。
我们还构建了GAL4DBD-H4-Gcn5 wt-HA和GAL4DBD-H4-Gcn5 F221A-HA重组体,试验表明: GAL4DBD-H4-Gcn5 wt-HA 融合蛋白有自体乙酰化现象(H4K8)而 GAL4DBD-H4-Gcn5F221A-HA则无。
为检测融合蛋白能否用于酵母双杂合?
Core histones H2A,H2B,H3 and H4 are the essential proteins for chromatin organization and cell viability. DNA-templated nuclear activities, including transcription, replication, recombination, and repair, all take place within the chromatin. Two molecules of each of the core histones are wrapped around by about 150 base pairs of DNA to form a nucleosome. Formation of nucleosomes requires extensive histone-histone and histone-DNA interactions occurred mainly within the central histone-fold domain of each core histone. The N' termini of all core histones are "flexible tails" which protrude from the nucleosomal core particles and are largely unstructured. The yeast cells without histones H3 and H4, or H2A and H2B N' termini are not viable ,they die at G2/M phase of the cell cycle.
Histone tails are subject to multiple post-translational modifications which include acetylation, methylation, phosphorylation, ubiquitylation , sumolation etc.Many of these modifications may change the ionic charge of the highly basic histones, which have substantial effects on the compact structure of chromatin. Usually the compact structure of chromatin restricts the binding and progression of protein factors. The acetylated histone tail peptide interacts with general DNA at much more higher Km than that of unacetylated ,that means a weakened DNA histone binding interaction may allow for better access of DNA binding and processing factors to find their cognate DNA elements. Generally speaking, histone hyper- and hypo-acetylation each triggers transcriptional activation and repression / silence, respectively. Acetylated histones have been shown to be bound by a highly conserved motif, bromodomain,that is found in many transcriptional activators. Likewise, methylated histones recruit chromodomain, that is frequently found to be part of transcriptional repressors. In addition to acetylation, the other modification pattern including phosporylation, methylation
and ubiquitylation were found to be involved in gene specific regulation.
In addition, different histone modifications may influence each other. for instance, acetylation at lysine 14 of H3 by several histone acetyltransferases (HAT) has been shown to be facilitated by phosphorylation at serine 10, and there is same case between arginine 3 methlylation of H4 and lysine 8 acetylation.
These evidences has caused scientists to favor the view that each histone modification pattern may be read by other proteins or protein modules, and thus followed different biological procedures. In 2000, David Allis proposed the "histone code" hypothesis which suggests that differentially modified histones may act as specific signals that are interpreted by the binding of selective factors. Such factors thus perform certain functions at the genomic loci bearing the corresponding histone modifications.
Thus far, all modified histone-binding proteins were identified by biochemical methods. Genome-wide screening for proteins possessing affinity toward specifically modified histones needs to be done. To identify the modified histone binding proteins, previous strategies have been based on the ectopic expression of protein kinases in the two-hybrid hosts which normally lack such enzymes. For example, tyrosine kinases and mammalian serine/threonine kinases were respectively expressed in yeast and E. coli for two-hybrid screens. The substrate proteins to be screened in these assays were phosphorylated and hence allowed the detection of interactions involving these phosphorylation events. Major concerns of these methods include the efficiency and specificity with which the bait protein to be screened is modified. Phosphorylation relies on a typical trans reaction between an enzyme and its substrates. If the foreign enzyme cannot identify the hybrid protein in the sea of host cellular proteins for efficient modification, the subsequent screen is likely to be unsuccessful. The efficiency of modification may be increased by overexpressing the enzyme, but inadvertent modifications of host protein
引文
James R. Davie and Virginia A. Spencer, Control of histone modifications. J.Cell. Biochem. Suppl. 1999, 32/33:141-148
Patrick A. Grant A tale of histone modifications. Genome Biol. 2001,2：1-6
LeAnn Howe,Darryl Auston,Patrick Grant,et al. Histone H3 Specific Acetytransferases are essential for cell cycle progression. Genes & Dev. 2001， 15:3144-3154
Kevin Struhl， Histone acetylation and transcriptional regulatory mechanisms. Genes & Dev. 1998，12:599-606
Ajiro,K., Histone H2B phosphorylation in mammalian apoptotic cells, An association with DNA fragmentation. J. Biol. Chem. 2000, 275:439-443
Wei Y.,Yu L., Bowen J., et al. Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 1999, 97:99-109
Masayoshi Lizuka and Mitchell Smith, Functional consequences of histone modifications. Curr. Opin. Genet. Dev. 2003, 13:154-160
Wenzheng Zhang, James R.Bone, Diane G.Edmondson, et al. Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. EMBO J. 1998, 17:3155-3167
Brian D.Strahl & David Allis, The laguage of covalent histone modifications. Nature 2000，403：41-45
Christophe Dhalluin, Justin E., Carlson, et al. Structure and ligand of a histone acetyltransferase bromodomain. Science 1999, 399:491-496
Jacobson R.H., Ladurner A.G., King D.S., et al.Structure and function of a human TAFII 250 double bromodomain module. Science 2000,288: 1422-1425
Peter R. Nielsen,Daniel Nietlispach, Helen R. Mott, et al. Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature
2002, 416:103-107
Agalioti T, Chen G. Thanos D, Deciphering the transcriptional histone acetylation code for human gene. Cell 2002, 111:381-392
Bannister AJ ,Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromodomain. Nature 2001, 410:120-124
Soren J. Nielsen, Robert Schneider, Uta-Maria Bauer, et al. Rb targets histone H3 methylation and HP1 to promoters. Nature 2001 412:561-565
Berger SL , Histone modifications in transcriptional regulation. Curr. Opin. Genet. Dev. 2002,12:142-148
Smith MM: Centromeres and variant histones: what,where,when and why? Curr. Opin. Cell. Biol. 2002, 14:279-285
Sarah C. R. Elgin & Jerry L. Workman , Chromatin Structure and gene expression, Oxford University Press. Second edition 2000, pp142
Wan-Sheng Lo, Laura Duggan, N. C. Tolga, et al. Snf1-a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 2001,293: 1142 -1146
Hengbin Wang Zhi-Qing Huang, Li Xia, et al.Methylation of histone H4 at arginine 3 facillitating transcriptional activation by nuclear hormone receptor. Science 2001, 293: 853-857
Fields,S & Song,O, A novel genetic system to detect protein-protein interactions. Nature 1989, 340:245-246
Masashi Yamada, Kenji Suzuki, Masaharu Mizutani, et al. Analysis of tyrosine phosphorylation-dependent protein protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system. J Biochem (Tokyo) 2001, 130:157-165
Haiming Cao, William E. Courchesne, and Cynthia Dorley Mastick A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of Caveolin-1 on Tyrosin 14 . J Biol Chem 2002, 277: 8771-8774
Petr Uetz, Loic Giot, Gerard Cagney, Traci A. Mansfield,et al. A
comprehensive analysis of protein -protein interactions in Saccharomyces cerevisiae. Nature 2000,403: 623-627
Helena Santos-Rosa,Ester Valls, TonyKouzarides et al. Mechanisms of P/CAF auto-acetylation. Nucleic Acids Res. 2003,31:4285-4292
Andreas G. Ladurner,Carla Inouye, Rajan Jain, et al. Bromodomain mediate an acetyl-histone encoded antisilencing function at heterochromatin boundaries. Mole. Cell 2003, 11: 365-376
Luger, K., Mader, A.W. Richmond, R.K.Sargent,D.F.,et al. Crystal structure of the nucleosome core particle at 2.8A resolution. Nature 1997,389:251-260
Sameez Hasan ,Michael O. Hottiger Histone acety trasnfereases, a role in DNA repair and DNA replication. J.Mol. Med. 2002, 80:463-474
Michael Grunstein, Histone acetylation in chromatin structure and transcription. Nature,1997, 389:349-352
McGhee, J.D. and Felsenfeld G., Nucleosome structure. Annu. Rev. Biochem. 1980 49:1115-1156
Gross, D.A., and Garrard W.T., Nucleosome hypersensitive sites in chromatin. Annu. Rev. Biochem. 1988, 57:159-197
Noton, V.G., Marvin K.W., Yau, P., et al.Nucleosome linking number change comtrolled by acetylation of histone H3 and H4. J. Biol. Chem. 1990, 265:19848-19852
Lee D., Hayes J.J., Pruss D., et al. A positive role for histone acetylation in transcription factor access to DNA. Cell 1993, 72:73-84
Christophe Dhalluin, Justin E. Carlson, Li Zeng, et al.Structure and ligand of a histone acetyltransferase bromodomain. Nature 1999, 399: 491-496
Syntichaki P., Topalidou I., Thireos G., The Gcn5 bromodomain co-ordinates nucleosome remodeling. Nature 2000, 404:414-417
Min-Hao Kuo, Jianxin Zhou, Per Jambeck, et al. Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. Genes & Dev. 1998, 12:627-639
Min-Hao Kuo, James E. Brownell, Richard E. Sobel,et al. Transcription-linked acetylation by Gcn5p of histone H3 and H4 at specific lysines. Nature 1996 383:269-272
Philip James, John Halladay and Elizabeth A. Craig， Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 1996,144: 1425-1436
Michael F. Henry and Pamela A. Silver， A novel methyltransferase (Hmt1p)modifies poly(A)+-RNA-binding proteins. Mol. Cell. Biol. 1996, 16:3668-3678
Vilius Stribinskis, Guo-Jian Gao, Steven R. Ellis et al.Rpm2, the protein subunit of mitochondrial RNase P in Saccharomyces cerevisiae, also has a role in the translation of mitochondrially encoded subunits of cytochrome c oxidase. Genetics 2001, 158: 573-585,
Vilius Stribinskis, Guo-Jian Gao, Steven R. Ellis et al. Rpm2p: separate domains promote tRNA and Rpm2 maturation in Saccharomyces cerevisiae miyochondria. Nucleic Acids Res. 2001,29:3631-3637
Lisete Fernandes, Claudina Rodrigues-Pousada and Kevin Struhl, Yap, a Novel family of eight bZIP proteins in Saccharomyces cerevisiae with Distinct biological functions. Mol. Cell. Biol. 1997, 17:6982-6993
Ken-ichi?Noma and Shiv I. S.?Grewal， Histone H3 lysine 4 methylation is mediated by Set1 and promotes maintenance of active chromatin states in fission yeast. Proc. Natl. Acad. Sci. USA,2002, 99:16438-16445
Nevan J.,Krogan, Minkyu Kim,et al. Methylation of histone H3 by Ste2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol.Cell. Biol. 2003, 23:4207-4218
Patrick A. Grant A tale of histone modifications. Genome Biol. 2001,2：1-6
LeAnn Howe,Darryl Auston,Patrick Grant,et al. Histone H3 Specific Acetytransferases are essential for cell cycle progression. Genes & Dev. 2001， 15:3144-3154
Kevin Struhl， Histone acetylation and transcriptional regulatory mechanisms. Genes & Dev. 1998，12:599-606
Ajiro,K., Histone H2B phosphorylation in mammalian apoptotic cells, An association with DNA fragmentation. J. Biol. Chem. 2000, 275:439-443
Wei Y.,Yu L., Bowen J., et al. Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell 1999, 97:99-109
Masayoshi Lizuka and Mitchell Smith, Functional consequences of histone modifications. Curr. Opin. Genet. Dev. 2003, 13:154-160
Wenzheng Zhang, James R.Bone, Diane G.Edmondson, et al. Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. EMBO J. 1998, 17:3155-3167
Brian D.Strahl & David Allis, The laguage of covalent histone modifications. Nature 2000，403：41-45
Christophe Dhalluin, Justin E., Carlson, et al. Structure and ligand of a histone acetyltransferase bromodomain. Science 1999, 399:491-496
Jacobson R.H., Ladurner A.G., King D.S., et al.Structure and function of a human TAFII 250 double bromodomain module. Science 2000,288: 1422-1425
Peter R. Nielsen,Daniel Nietlispach, Helen R. Mott, et al. Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature
2002, 416:103-107
Agalioti T, Chen G. Thanos D, Deciphering the transcriptional histone acetylation code for human gene. Cell 2002, 111:381-392
Bannister AJ ,Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromodomain. Nature 2001, 410:120-124
Soren J. Nielsen, Robert Schneider, Uta-Maria Bauer, et al. Rb targets histone H3 methylation and HP1 to promoters. Nature 2001 412:561-565
Berger SL , Histone modifications in transcriptional regulation. Curr. Opin. Genet. Dev. 2002,12:142-148
Smith MM: Centromeres and variant histones: what,where,when and why? Curr. Opin. Cell. Biol. 2002, 14:279-285
Sarah C. R. Elgin & Jerry L. Workman , Chromatin Structure and gene expression, Oxford University Press. Second edition 2000, pp142
Wan-Sheng Lo, Laura Duggan, N. C. Tolga, et al. Snf1-a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 2001,293: 1142 -1146
Hengbin Wang Zhi-Qing Huang, Li Xia, et al.Methylation of histone H4 at arginine 3 facillitating transcriptional activation by nuclear hormone receptor. Science 2001, 293: 853-857
Fields,S & Song,O, A novel genetic system to detect protein-protein interactions. Nature 1989, 340:245-246
Masashi Yamada, Kenji Suzuki, Masaharu Mizutani, et al. Analysis of tyrosine phosphorylation-dependent protein protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system. J Biochem (Tokyo) 2001, 130:157-165
Haiming Cao, William E. Courchesne, and Cynthia Dorley Mastick A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of Caveolin-1 on Tyrosin 14 . J Biol Chem 2002, 277: 8771-8774
Petr Uetz, Loic Giot, Gerard Cagney, Traci A. Mansfield,et al. A
comprehensive analysis of protein -protein interactions in Saccharomyces cerevisiae. Nature 2000,403: 623-627
Helena Santos-Rosa,Ester Valls, TonyKouzarides et al. Mechanisms of P/CAF auto-acetylation. Nucleic Acids Res. 2003,31:4285-4292
Andreas G. Ladurner,Carla Inouye, Rajan Jain, et al. Bromodomain mediate an acetyl-histone encoded antisilencing function at heterochromatin boundaries. Mole. Cell 2003, 11: 365-376
Luger, K., Mader, A.W. Richmond, R.K.Sargent,D.F.,et al. Crystal structure of the nucleosome core particle at 2.8A resolution. Nature 1997,389:251-260
Sameez Hasan ,Michael O. Hottiger Histone acety trasnfereases, a role in DNA repair and DNA replication. J.Mol. Med. 2002, 80:463-474
Michael Grunstein, Histone acetylation in chromatin structure and transcription. Nature,1997, 389:349-352
McGhee, J.D. and Felsenfeld G., Nucleosome structure. Annu. Rev. Biochem. 1980 49:1115-1156
Gross, D.A., and Garrard W.T., Nucleosome hypersensitive sites in chromatin. Annu. Rev. Biochem. 1988, 57:159-197
Noton, V.G., Marvin K.W., Yau, P., et al.Nucleosome linking number change comtrolled by acetylation of histone H3 and H4. J. Biol. Chem. 1990, 265:19848-19852
Lee D., Hayes J.J., Pruss D., et al. A positive role for histone acetylation in transcription factor access to DNA. Cell 1993, 72:73-84
Christophe Dhalluin, Justin E. Carlson, Li Zeng, et al.Structure and ligand of a histone acetyltransferase bromodomain. Nature 1999, 399: 491-496
Syntichaki P., Topalidou I., Thireos G., The Gcn5 bromodomain co-ordinates nucleosome remodeling. Nature 2000, 404:414-417
Min-Hao Kuo, Jianxin Zhou, Per Jambeck, et al. Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. Genes & Dev. 1998, 12:627-639
Min-Hao Kuo, James E. Brownell, Richard E. Sobel,et al. Transcription-linked acetylation by Gcn5p of histone H3 and H4 at specific lysines. Nature 1996 383:269-272
Philip James, John Halladay and Elizabeth A. Craig， Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 1996,144: 1425-1436
Michael F. Henry and Pamela A. Silver， A novel methyltransferase (Hmt1p)modifies poly(A)+-RNA-binding proteins. Mol. Cell. Biol. 1996, 16:3668-3678
Vilius Stribinskis, Guo-Jian Gao, Steven R. Ellis et al.Rpm2, the protein subunit of mitochondrial RNase P in Saccharomyces cerevisiae, also has a role in the translation of mitochondrially encoded subunits of cytochrome c oxidase. Genetics 2001, 158: 573-585,
Vilius Stribinskis, Guo-Jian Gao, Steven R. Ellis et al. Rpm2p: separate domains promote tRNA and Rpm2 maturation in Saccharomyces cerevisiae miyochondria. Nucleic Acids Res. 2001,29:3631-3637
Lisete Fernandes, Claudina Rodrigues-Pousada and Kevin Struhl, Yap, a Novel family of eight bZIP proteins in Saccharomyces cerevisiae with Distinct biological functions. Mol. Cell. Biol. 1997, 17:6982-6993
Ken-ichi?Noma and Shiv I. S.?Grewal， Histone H3 lysine 4 methylation is mediated by Set1 and promotes maintenance of active chromatin states in fission yeast. Proc. Natl. Acad. Sci. USA,2002, 99:16438-16445
Nevan J.,Krogan, Minkyu Kim,et al. Methylation of histone H3 by Ste2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol.Cell. Biol. 2003, 23:4207-4218