摘要
心血管系统疾病已经成为威胁人类健康的最大的两个敌人之一。在心血管系统疾病的研究中已发现,某些基因在时空上的表达紊乱可导致先天性心脏病,而对部分心血管系统病症高发人群的研究也证明部分后天形成的心脏病与遗传因素有关。我们研究室进行的大规模筛选克隆人类心脏发育的相关基因工作为理解心脏发育的机理、探索心脏发育中的分子调控机制打下了坚实的基础。
ZNF569就是本文作者在利用生物信息学方法筛选人类心脏发育候选基因的过程中发现的一个人类新基因。ZNF569 cDNA全长4071个碱基,ORF全长2061个碱基,编码一个长686个氨基酸残基的蛋白质。该蛋白包含一个KRAB结构域和18个锌指结构域,在包括小鼠、大鼠、果蝇在内的多个物种中均发现了它的同源蛋白,并且它们之间具有极高的相似度,说明此蛋白所在的亚家族可能具有相当重要的功能。Northern结果显示ZNF569在成人的肝、心脏和胎盘中有高表达,在胰腺和骨骼肌中表达较弱,在胚胎心脏、骨骼肌和肝也有表达,提示该基因可能与心脏发育有关。转录活性分析表明在COS-7细胞中过表达ZNF569蛋白可以使MAPK信号途径的两个下游转录因子AP-1和SRE的转录抑制活性显著下降。ZNF569在核质内的亚细胞定位分析进一步证明了其作为转录因子的作用。对ZNF569的分段report assays分析表明,ZNF569可能通过KRAB框和C2H2锌指结构域行使其抑制转录的作用。综合目前的研究结果来看,ZNF569可以通过MAPK信号途径参与了对细胞生命活动的调控过程。
本文作者还对一个Rho family GTPases交换因子hGEFT进行了
Cardiovascular diseases have become one of the two kinds of most serious diseases that threaten human beings. It is a critical prerequisite to understand the normal progress of cardiovascular development and the mechanisms underlying the causes of this kind of diseases, so it is possible to cure the disease. As it known, mistakes in spatiotemporal expression of critical genes result in congenital cardiovascular diseases. Furthermore, studies on high risk population of cardiovascular disease have also suggested the relationship between noncongenital cardiovascular diseases and genetic factors. In order to promote our understanding for cardiovascular development and to discover the molecular regulatory mechanisms during this progress, our laboratory performed a large scale scanning project to search and clone cardiac developmental candidate genes.In the present study, we report the identification and characterization of a novel KRAB-containing zinc-finger protein, ZNF569, from a human embryonic heart cDNA library. ZNF569 encodes a putative protein of 686 amino acids. The protein is conserved across different species during evolution. Northern blot analysis indicates that ZNF569 is expressed in heart, liver, placenta, muscles, and pancreas and in a very embryonic development-specific stage of human tissues, including heart, pancreas,
引文
[1] 吴秀山等.心脏发育研究.湖南长沙:湖南科学技术出版社,2004。
[2] Bodmer R. The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development, 1993, 118(3):719-29.
[3] Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, Moak JP, Maron BJ, Seidman CE, Seidman JG. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science, 1998, 281(5373):108-11.
[4] Pandur P, Lasche M, Eisenberg LM, Kuhl M. Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature, 2002, 418(6898):636-41.
[5] Wu X, Golden K, Bodmer R. Heart development in Drosophila requires the segment polarity gene wingless. Developmental Biology, 1995,169:619-628.
[6] Wu X, Park M, Gold K, et al. The wingless signaling pathway is directly involved in Drosophila development. Developmental Biology, 1996,177:104-116.
[7] Klug, A., and J. W. Schwabe. Protein motifs 5. Zinc finger [J]. FASEBJ,1995, 9:597-604.
[8] E. J. Bellefroid, D. A. Poncelet, P. J. Lecocq, O. Relevant, and J. A. Martial. The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins[J]. Proc. Natl. Acad Sci, USA, 1991,88(?):3608-3612.
[9] McCarty AS, Kleiger G, Eisenberg D, Smale ST. Selective dimerization of a C2H2 zinc finger subfamily [J] Mol. Cell, 2003,11(2):459-70.
[10] Kaczynski J, Cook T, Urrutia R. Spl-and Kruppel-like transcription factors [J]. Genome Biol, 2003, 4(2):206.
[11] Roger R. Beerli and Carlos F. Barbas. Engineering polydactyl zinc-finger transcription factors[J], nature biotechnology, 2002,20(3):135-141.
[12] Tucker C, James R and Amy J. All in the family: the BTB/POZ, KRAB, and SCAN Domains[J]. Molecular and cellular biology, 2001,21 (3):3609-3615.
[13] S. G. Tevosian, A. E. Deconinck, M. Tanaka, M. Schinke, S. H. Litovsky, S. lzumo, Y. Fujiwara, S. H. Orkin, FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium[J]. Cell, 2000,101(3): 729-39.
[14] Dai KS, Liew CC. Chromosomal, in silico and in vitro expression analysis of cardiovascular-based genes encoding zinc finger proteins[J] J Mol Cell Cardiol, 2000,31(9): 1749-69.
[15] Morris,J. E, E J. Rauscher, B. Davis, M. Klemsz, D. Xu, D. Tenen, and R. Hromas. The mye -loid zinc finger gene MZF-1 regulates the CD34 promoter in vitro[J].Blood, 1995,186(2):3640-3647.
[16] Sander, T. A., A. L. Haas, M. J. Peterson, and J. E Morris. Identification of a novel SCAN box-related protein that interacts with MZFlB[J].J. Biol. Chem, 2000,275(2):12857-12867.
[17] Morris, J. E, R. Hromas, and E J. Rauscher. Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core[J]. Mol. Cell. Biol,1994, 14(1):1786-1795.
[18] R.Schuh, U.Aicher, S.Cote, A.Preiss, D.Maier, E.Seifert, U. Nauber, C.Schroder, R.Kemler, H.Jackle.A conserved family of nuclear proteins containing structural elements of the finger protein encoded by kriipple, a Drosophila segmentation gene [J] . Cell, 1986, 47 (6) :1025-1032.
[19] R. Bodmer, T.V. Venkatesh, Heart development in Drosophila and vertebrates: conservation of molecular mechanisms[J].Dev.Genet,1998,22(3):181-186.
[20] X. Wu, K. Golden, R. Bodmer.Heart development in Drosophila requires the segment polarity gene wingless[J].Dev. Biol, 1995,169(1): 619-628.
[21] Zhu H, Nguyen V T,Brown A B, et al.A novel, tissue-restricted zinc finger protein (HF-1b) binds to the cardiac regulatory element (HF-1b/MEF-2) in the rat myosin light-chain 2 gene [J]. Mol Cell Biol, 1993,13 (2): 4432-4444.
[22] Kaimei Luo, Wuzhou Yuan, Xiushan Wu, et al. Expression of a novel Krüpple-like zinc-finger gene,ZNF382, in human heart [J].Biochemical and Biophysical Research Communications,2002, 299(1):606-612
[23] Liang Zhou, Chuanbing Zhu, Mingyao Liu, et al. Identification and characterization of two novel zinc finger genes,ZNF359 and ZNF28, in human development[J]. Biochemical and Biophysical Research Communications, 2002,295(l):862-868.
[24] Berg,J.M. Zinc fingers and other metal-binding domains. Elements for interactions between macromolecules. J. Biol. Chem, 1990,265(12).6513-6516
[25] Attar, R. M., and M. Z. Gilman. Expression cloning of a novel zinc finger protein that binds to the c-fos serum response element. Mol. Cell. Biol, 1992,12: 2432-2443.
[26] Pavletich, N.P., Pabo, CO., Crystal structure of a five-finger GLIDNA complex: new perspectives on zinc fingers. Science, 1993, 261:1701-1707.
[27] Dang, D.T., Pevsner, J., Yang, V.W, The biology of the mammalian Kruppel-like family of transcription factors. Int. J. Biochem. Cell Biol, 2000,32:1103-1121.
[28] L.C. Wu. ZAS: C2H2 zinc finger proteins involved in growth and development, Gene Expr, 2002,10:137- 152.
[29] S. S. Kim, Y. M. Chen, E. O'Leary, R. Witzgall, M. Vidal, J.V. Bonventre, A novel member of the RING finger family, KRIP-1, associated with the KRAB-A transcriptional repressor domain of zinc finger proteins, Proc Natl Acad Sci, 1996, 93:15299-15304.
[30] J. R. Friedman, W. J. Fredericks, D. E. Jensen, D. W. Speicher, X. P. Huang, E. G. Neilson, F. J. Rauscher, KAP-1, a novel corepressor for highly conserved KRAB repression domain, Genes Dev, 1996,10: 2067-2078.
[31] J.F. Margolin, J.R. Friedman, W.K. Meyer, H. Vissing, H.J. Thiesen, F.J, Rauscher Kruppel-associated boxes are potent transcriptional repression domains, Proc Natl Acad Sc, i\ 994, 91:4509-4513.
[32] R. Witzgall, E. O'Leary, A. Leaf, D. Onaldi, J.V .Bonventre, The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression, Proc Natl Acad Sci, 1994, 91:4514-4518.
[33] C. Mark, M. Abrink, L. Hellman, Comparative analysis of KRAB zinc finger proteins in rodents and man: evidence for several evolutionarily distinct subfamilies of KRAB zinc finger genes, DNA Cell Biol, 1999,18:381-396.
[34] Irving EA, Bamford M. Role of mitogenand stress-activated kinases in ischemic injury. J. Cereb. Blood Flow Metab, 2002,22: 631 -647.
[35] Hirai S, Izawa M, Osada S, Spyrou G, Ohno S. Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK. Oncogene, 1996,12: 641 -650.
[36] Atfi A, Buisine M, Mazars A, Gespach C. Induction of apoptosis by DPC4, a transcriptional factor regulated by transforming growth factor-beta through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling pathway. J. Biol. Chem, 1997, 272: 24731 -24734.
[37] Zhang Y, Zhou L, Miller CA. A splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous system. Proc. Natl. Acad. Sci. USA, 1998, 95: 2586 -2591.
[38] Lisovsky M, Itoh K, Sokol SY. Frizzled receptors activate a novel JNK-dependent pathway that may lead to apoptosis. Curr. Biol., 2002,12: 53-58.
[39] New L and Han J. The p38 MAP kinase pathway and its biological function. Trends Cardiovasc. Me., 1998, 8:220 -229.
[40] Stein B, Yang MX, Young DB, Janknecht R, Hunter T, Murray BW, Barbosa MS. p38-2, a novel mitogen-activated protein kinase with distinct properties. J. Biol. Chem., 1997,272:19509-19517.
[41] Beyaert R, Cuenda A, Vanden Berghe W, Plaisance S, Lee JC, Haegeman G, Cohen P. and Fiers W. The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor. EMBOJ, 1996,15:1914 -1923.
[42] Huot J, Houle F, Marceau, F and Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ. Res.1997,80:383-392.
[43] Zhu T, and Lobie P E, Janus, kinase 2-dependent activation of p38 mitogen-activated protein kinase by growth hormone. J. Biol. Chem., 2002,275:2103-2114.
[44] Xia Y, et al. MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration. Proc. Natl Acad. Sci. USA, 2002, 97: 5243-5248.
[45] Yujiri T, Sather S, Fanger CR & Johnson GL. Role of MEKK1 in cell survival and activation of JNK and ERK pathways de(?)ned by targeted gene disruption. Science, 1998, 282: 1911-1914.
[46] Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature, 2001,410:37-40.
[47] Treisman R. Regulation of transcription by MAP kinase cascades. Curr. Opin. Cell Biol, 1996, 8: 205-215.
[48] Kallunki T, Deng T, Hibi M & Karin, M. c-Jun can recruit JNK to phosphorylate dimerization partners via specific docking interactions. Cell, 1996, 87: 929-939.
[49] Shaw PE, Frasch S and Nordheim A. Repression of c-fos transcription is mediated through p67SRF bound to the SRE. The EMBO Journal,1989, 8: 2567-2574.
[50] Han J, Jiang Y, Li Z, Kravchenko VV & Ulevitch RJ. Activation of the transcription factor MEF2C by the MAP kinase p38 in in(?)ammation. Nature, 1997, 386: 296:299.
[51] Winzen R, et al. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J., 1999, 19: 6742-6753.
[52] Lasa M, et al. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade. Mol.