Activation of Transcriptional Activities of AP-1 and SRE by a New Zinc
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http://www.paper.edu.cn 1 Biochemical and Biophysical Research Communications 339 (2006) 1155–1164 www.elsevier.com/locate/ybbrc 2 Activation of transcriptional activities of AP-1 and SRE by a new q 3 zinc-finger protein ZNF641 1 1 4 Xingzhu Qi , Yongqing Li , Jing Xiao, Wuzhou Yuan, Yan Yan, Yuequn Wang, 5 Shuyuan Liang, Chuanbing Zhu, Yinduan Chen, Mingyao Liu *, Xiushan Wu * 6 The Center for Heart Development, College of Life Sciences, Hunan Normal University, Changsha, 410081 Hunan, PR China 7 Received 13 November 2005 8 9 Abstract 10 Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface 11 receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that 12 zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and character- 13 ization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9 kb, encoding 438 amino acids in the nucleus. 14 The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates 15 that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B- 16 ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between 17 KRAB box and C2H2-type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive 18 regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE. 19 Ó 2005 Elsevier Inc. All rights reserved. 20 Keywords: ZNF641; KRAB motif; Linker; Transcriptional suppressor; Heart development; MAPK signaling pathway; SRE; AP-1 21 22 Transcriptional regulation of gene expression is medi- zinc-finger proteins. These proteins contain two or more 32 23 ated primarily by sequence-specific DNA-binding tran- C2H2-type zinc-fingers that are separated by a conserved 33 24 scription factors that are composed of a DNA-binding consensus sequence, T/SGEKPY/FX. It has been estimat- 34 25 domain and one or more separable effector domains that ed that the human genome contains 564–706 C2H2-type 35 26 play an important role in activating or repressing initiation zinc-finger genes [5,6]. The C2H2 zinc-finger motif corre- 36 27 of transcription [1–3]. Zinc-finger gene family belongs to sponds to the consensus sequence: Tyr/Phe-X-Cys-X2, 4- 37 28 one of the biggest families of transcription factors and Cys-X3-Phe-X5-Leu-X2-His-X3, and 4-His-Thr-Gly-Glu- 38 29 can be divided into many subclasses based on the number Lys-Pro, where Xn, m denote the presence of n or m amino 39 30 and type of zinc-fingers they contain [4]. The family of acids between conserved residues; the cysteines and histi- 40 31 Kru¨ppel-like proteins is one of the largest families of dines coordinating the zinc are in bold and the conserved 41 amino acids forming the link between consecutive fingers 42 are underlined [7,8]. The C2H2 type zinc finger motif is ini- 43 q Abbreviations: DMEM, DulbeccoÕs modified EagleÕs medium; DAPI, tially found in the TFIII, a transcription factor of Xenopus, 44 40,60-diamidino-2-phenylindole hydrochloride; MAPK, mitogen-activated and subsequently in the Kru¨ppel of Drosophila [9]. Since 45 protein kinase; MAPKK, MKK or MEK, MAPK kinase; MAPKKK or then, this motif has been found in many proteins with tran- 46 MEKK, a MAPKK kinase or MEK kinase; SRE, serum response scriptional regulatory functions, e.g., in transcription fac- 47 element; AP-1, activation protein 1. * Corresponding authors. Fax: +86 0731 8615078. tor Sp1 and yeast regulatory gene GAL4 [10], in the 48 E-mail address: [email protected] (M. Liu). WilmsÕ tumor suppressor gene (WT1) [11], and in Gli-3 49 1 These authors contributed equally to the work. that is implicated in Greig syndrome [12]. Most studied 50 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.124 转载 中国科技论文在线 http://www.paper.edu.cn 1156 X. Qi et al. / Biochemical and Biophysical Research Communications 339 (2006) 1155–1164 51 C2H2 zinc finger proteins were found to bind to specific Sequencer (ABI PRISM) according to manufacturerÕs procedures. The 89 52 DNA sequences and to be involved in the transcriptional sequence obtained was subjected to human homology searching against 90 expressed sequence tag (EST) database using Blastn. To confirm the 91 53 regulation of gene expression [7,8]. Members of the Kru¨p- cDNA sequence from the database, one pair of gene-specific primers 92 54 pel-like zinc finger family can function as activators and/or A2sen and A2ant (Table 1) was designed based on the sequences of three 93 55 repressors of gene transcription and regulate embryonic ESTs (BX433165, BX444194, and BX420623) for PCR. The heart cDNA 94 56 development as well as a variety of physiological processes library was used as template. Amplification was carried out at 94 °C, 95 57 in the adult [13]. Recently, studies focusing on C H type 4 min; 94 °C, 30 s, 59 °C, 30 s, and 72 °C, 2 min for 30 cycles; then 72 °C, 96 2 2 97 58 zinc finger genes have suggested their unique involvement 8 min. The band was obviously excised, cloned into PMD18T–vector (TAKARA), and sequenced. 50-RACE and 30-RACE were performed 98 59 in the regulation of embryogenesis [14–17] and in a variety using SMART-RACE cDNA Amplification Kit (Clontech). 50 Gene- 99 60 of diseases [18,19]. specific reverse primers (50-RACEsen and 50-RACEant) and 30 gene-spe- 100 61 With the aim of identifying genes involved in human cific reverse primers (30-RACEsen and 30-RACEant) were designed using 101 62 heart development and diseases, we isolated a novel Primer Premier 5.0 for 50-RACE and 30-RACE reactions, respectively. 102 103 63 KRAB/C H -type zinc-finger gene named ZNF641 from The products were then cloned into PMD18T-vector and sequenced. 2 2 Sequence analysis was performed using the DNASTAR program and 104 64 heart cDNA library in this study. ZNF641 encodes a BLAST program from NCBI. Pfam 9.0 was used to analyze genomic 105 65 zinc-finger protein with five different C2H2 type zinc fin- structure and the protein domain, respectively. The full-length sequence of 106 66 gers and a KRAB-A box. Northern blot analysis indi- ZNF641 was submitted to GenBank. 107 67 cates that ZNF641 is expressed in most of the Northern blot hybridization. The ZNF641 cDNA was labeled with 108 32 109 68 examined adult tissues, at a high level in skeletal muscle. [a- p]dCTP by using a Random Primer Labeling Kit (TaKaRa). An adult human Northern blot containing mRNA from a variety of adult 110 69 Transfection of GAL4–ZNF641 into COS-7 cells sug- tissues purchased from CLONTECH Company was hybridized 111 70 gests that ZNF641 may be a transcriptional activator. sequentially to the radiolabeled ZNF641 cDNA probe and b-actin 112 71 Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cDNA probe (CLONTECH). Hybridization was carried out with 5· 113 72 cells activates the transcriptional activities of AP-1 and SSC, 5· DenhardtÕs, 10% dextran sulfate, and denatured human DNA, 114 115 73 SRE. Deletion analysis of different domains indicates at 65 °C overnight. After hybridization, the blots were washed three times at 65 °Cin2· SSC containing 0.1% SDS for 5 min and twice in 116 74 that the linker between KRAB box and C2H2 type zinc 0.1· SSC and 0.1% SDS at 65 °C for 15 min, and then subjected to 117 75 fingers of ZNF641 represents the basal activation region. autoradiography at À80 °C. The blots were stripped by being incubated 118 76 Together, the results suggest that ZNF641 protein acts as for 10 min in 0.1· SSC and 0.5% SDS at 95 °C. The membranes were 119 77 an active regulator in the MAPK signaling pathway to reprobed with radiolabeled b-actin cDNA as an indicator of mRNA 120 121 78 mediate cellular functions. loading. Plasmid construction. The following plasmids were constructed and 122 used for mammalian cell transfections. Escherichia coli DH5a was used as 123 79 Materials and methods recipient for all transformations. The enzymes were obtained from 124 TAKARA and used as recommended by the manufacturer. (i) Con- 125 80 Full-length ZNF641 cDNA cloning and bioinformatics analysis. PCR struction of pEGFP-N1-ZNF641: to generate a fusion protein of ZNF641 126 81 was performed on a PCRSPRINT reactor (Thermo Hybaid) with one pair with enhanced green fluorescent protein (EGFP), the coding region of 127 82 of degenerated oligonucleotide primers A1sen and A1ant (Table 1) cor- ZNF641 was subcloned into the BglII and SalI sites of pEGFP-N1 vector 128 83 responding to the amino acid sequence of KRAB domain, a highly con- in-frame with the GAC codon instead of the TGA stop codon in the 129 84 served consensus sequence of Krupple-like type zinc-finger genes. A PCR ZNF641 coding sequence. (ii) Construction of pGAL4-ZNF641 and five 130 85 was performed using the heart cDNA library as the template according to pGAL4-ZNF641 mutants (deletion fragments): the DNA fragment con- 131 86 standard procedures. The amplification products were separated by aga- taining the coding region of ZNF641 was amplified with primers A3 and 132 87 rose gel and the bands were cloned into PMD18T–vector (TAKARA).