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 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 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 80 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). A4 containing EcoRI and SalI restrictions, respectively (Table 1). The 133 88 The transformants were randomly chosen and sequenced with 3771 DNA amplified DNA fragment was subcloned into pMD18-T, after cleaving 134

Table 1 Sets of specific oligonucleotide primers Primer Orientation Nucleotide sequence A1sen Sense 50-GTNCANTTMMGNGAMGTNGCNGT-30 A1ant Antisense 50-CCANGGMTCMTCNCCMTGMTCNAG-30 A2sen Sense 50-TGAGGATCCGGAAATGCAAGCTGAGGA-30 A2ant Antisense 50-CGGGGATCCGATTTCAGAAGACAGATG-30 50-RACEsen Sense 50-GCAACGGTCCGAACCTCAT-30 50-RACEant Antisense 50-GCTGCAGACCCAAATTGTGAC-30 30-RACEsen UNCORRECTEDSense PROOF50-GCAGGTTCAAAGCCCATCT-30 30-RACEant Antisense 50-AACCTCTGGCGAAGAAGTCC-30 A3 Sense 50-GGCGAATTCGAAATGCAAGCTGAGGAC-30 A4 Antisense 50-CGCGTCGACAGAAGACAGATGTTCCTC-30 A5 Antisense 50-TGGGTCGACTCTGTCTGAGAGGGGTCC-30 A6 Antisense 50-CGAGTCGACCCCCTCATGTTCACTTCC-30 A7 Antisense 50-CCTGTCGACCCCACATTCAGTGCACAC-30 A8 Sense 50-ACCGAATTCGAACATGAGGGGGATACC-30 A9 Sense 50-GCGGAATTCGACCCCCAGGACTTAGA-30 A10 Antisense 50-GACGTCGACTGTGTGGGGTCTTAACAGG-30 中国科技论文在线 http://www.paper.edu.cn

X. Qi et al. / Biochemical and Biophysical Research Communications 339 (2006) 1155–1164 1157

135 with EcoRI and SalI from pMD18T-ZNF641, the DNA fragment was Results and discussion 203 136 subcloned into expression vector pCMV-BD that contained the coding 137 region of the GAL4 DNA-binding domain (DBD) to create a fusion Identification and sequence analysis of ZNF641 204 138 protein between GAL4 DBD and ZNF641. Five deletion fragments 139 (encoding amino acids 1–132, 1–195, 1–378, 192–438, and 171–265) were 140 amplified by PCR from the pMD18T-ZNF641 plasmid with five pairs of In an effort to understand the role of KRAB/C2H2 zinc 205 141 primers A3 and A5, A3 and A6, A3 and A7, A8 and A4, and A9 and A10. finger transcription factors in heart development and in cell 206 142 All of the sense primers (A3, A8, and A9) contain EcoRI restrictions and signaling pathway, we have performed a screen of heart 207 143 all of the antisense primers (A4, A5, A6, A7, and A10) contain SalI cDNA library that was constructed from a 20-week-old 208 144 restrictions (Table 1). Subsequently, these DNA fragments were cloned in- 145 frame into expression vector pCMV-BD that contained the coding region human embryonic heart. We used oligonucleotide primers 209 146 of the DBD to create five fusion proteins after cleaving from pMD18T- A1sen and A1ant (Table 1) based on conserved sequences 210 147 ZNF641 mutants. (iii) Construction of pCMV-tag2B-ZNF641: the of Kru¨pple-like gene family to drive homologue PCR 211 148 expression plasmid for FLAG epitope-tagged ZNF641, pCMV-tag2B- amplification. The PCR was performed using the heart 212 149 ZNF641, was constructed by inserting ZNF641 ORF downstream of the cDNA library as the template and amplification products 213 150 FLAG epitope sequence (MDYKDDDDK) in a pCMV-tag2B expression 151 vector. The ZNF641 ORF was isolated from pGAL4-ZNF641. All of the were cloned into pMD18T-vector and sequenced. The 214 152 transformed cells were selected by kanamycin resistance, and all of the sequences obtained were subjected to human homology 215 153 recombination plasmids constructed were cleaved with enzymes and the searching against expressed sequence tag (EST) database 216 154 inserted fragments of each clone were sequenced. using Blastn. An EST BX433165 was found to match the 217 155 Cell culture and subcellular localization analysis. COS-7 cells were cDNA clone. A number of ESTs representing the same 218 156 cultured in DMEM (DulbeccoÕs modified EagleÕs medium; Gibco-BRL) 157 which was supplemented with 10% fetal calf serum (FCS) in a humidified novel gene were identified in a further search. The partial 219 158 atmosphere of 95% air and 5% CO2. Sixteen to twenty hours prior to cDNA sequence of this novel gene was assembled from 220 159 transfection, growing cells were seeded (105 cells/ml) on sterile microscope ESTs, including BX420623, BX444194, BI668274, 221 160 cover-glasses placed in a 35 mm Petri dish. To investigate the subcellular CV024042, BP277781, AW504412, BI753433, CN479787, 222 161 PROOF localization of ZNF641, COS-7 cells were transfected with pEGFP-N1- CN106928, and CD106346. To confirm the cDNA 223 162 ZNF641 using LipofectAMINE (Invitrogen) according to the method 163 described before [20]. Forty-eight hours after transfection, cells were fixed sequence from the database, one pair of gene-specific prim- 224 164 with 4% paraformaldehyde for 15 min and nuclear fractions were stained ers A2sen and A2ant (Table 1) was designed based on the 225 165 with 40,60-diamidino-2-phenylindole hydrochloride (DAPI). Subcellular sequences of three ESTs (BX433165, BX444194, and 226 166 localization of the EGFP–ZNF641 fusion proteins was detected using BX420623) for PCR. The heart cDNA library was used 227 167 fluorescence microscopy. as template and a 1343 bp PCR product was obtained 228 168 Transient transfection and reporter gene assays. For luciferase assays, 169 using LipofectAMINE (Invitrogen) reagent according to the same and confirmed to be the cDNA sequence of this new gene. 229 170 method [20], COS-7 cells were transfected with 2 lg of each GAL4– To obtain the full-length cDNA, we performed 50-rapid 230 171 ZNF641 fusion protein plasmid, 2 lg of L8G5-Luciferase reporter amplification of cDNA ends (50-RACE) and 30-RACE, and 231 172 plasmid which contains eight LexA-binding sites upstream of five copies obtained a 559 bp DNA for 50-RACE fragment and 596 bp 232 173 of the GAL4-UAS site, and 0.1 lg pCMV-b-gal, in the presence or 30-RACE fragment. Analysis of these two cDNAs con- 233 174 absence 0.1 lg LexA-VP16 activator plasmid. COS-7 cells were co- 175 transfected with 2 lg pSRE-Luc, and 2 lg pCMV-Tag2B-ZNF641 or firmed that they were cDNA fragments from the novel 234 176 2 lg pCMV-Tag2B vector to investigate the effect of ZNF641 on the gene. The full-length new KRAB/C2H2 zinc-finger gene 235 177 transcriptional activity SRE. To examine the effect of ZNF641 on the was named ZNF641 as approved by the Human Gene 236 178 transcriptional activity of AP-1, cells were co-transfected with 2 lg Nomenclature Committee. The nucleotide sequence data 237 179 pAP-1-Luc and 2 lg pCMV-Tag2B-ZNF641 or 2 lg pCMV-Tag2B reported here are available in GenBank with Accession 238 180 vector with LipofectAMINE. Forty-eight hours later, cells were har- 181 vested and the luciferase activity assay was performed according to the No. AY842285. 239 182 protocols of Stratagene. Each experiment was performed in triplicate The ZNF641 gene consists of an open-reading frame 240 183 and each assay was repeated at least three times. The means of the data (ORF) of 1317 bp extending from the first ATG codon 241 184 from three individual transfected wells are presented after normalization at nucleotide 717 to a termination TGA at 2033, a 242 185 for b-galactosidase. 716 bp 50-untranslated region (UTR), and a 2870 bp 30- 243 186 Five GAL4–ZNF641 mutants (deletion fragments) were constructed as 187 described above. COS-7 cells were transfected with five GAL4–ZNF641 UTR with a consensus polyadenylation signal (aataaa) 244 188 mutants according to the method above. (Fig. 1). The deduced ZNF641 protein has 438 amino 245 189 Sequence comparisons and phylogenetic tree analysis. Comparison of acids (Figs. 1A and B) with a calculated molecular mass 246 190 the amino acid sequences of KRAB and the linker motif among of 49.5 kDa. ZNF641 is assigned to human chromosome 247 191 ZNF641 and its homologues was performed using the MegAlign pro- 12q13.11 according to the mapping information in NCBI 248 192 gram of DNASTAR. Phylogenetic tree analysis of amino acid 193 sequences deduced from ZNF641UNCORRECTEDDNA sequences was also performed and spans approximately 10.45 kb on the genome. 249 194 using the MegAlign program of DNASTAR. The clustal method was ZNF641 gene contains six exons and five introns on 250 195 chosen to correct the distances for multiple substitutions at a single site. the genome (Fig. 1B). A summary of the various sizes 251 196 GenBank accession numbers of previously known and novel C2H2 type of the exons and introns, and the sequence of the splice 252 197 zinc finger gene sequences used for these analyses are XP_543709 (Canis junctions is shown in Table 2. The exon–intron bound- 253 198 familiaris (dog)), XP_582187 (Bos taurus (cow)), XP_509030 (Pan 199 troglodytes (chimpanzee)), NP_776130 (Mus musculus), XP_235625 aries conform to the consensus splicing signals, where 254 200 (Rattus norvegicus), ZNF202 (Homo sapiens), and ZFP316 (Mus mus- there are a gt and an ag dinucleotide at the 50-donor 255 201 culus), ZNF213 (Homo sapiens), ZNF641 (Homo sapiens), and ZNF264 and 30-acceptor sites, respectively. Analysis of ZNF641 256 202 (Homo sapiens). protein using the SMART program indicates that amino 257 中国科技论文在线 http://www.paper.edu.cn

1158 X. Qi et al. / Biochemical and Biophysical Research Communications 339 (2006) 1155–1164

Fig. 1. (A) Nucleotide sequence and deduced protein sequence of the human ZNF641 gene. The initiation ATG and termination TGA codons are boxed. Amino acids are identified by their one-letter code. The KRAB box is boxed and the five zinc finger regions are underlined. The nucleotides and amino acids are numbered at the left andUNCORRECTED the right sides, respectively. The putative polyadenylation signal PROOF sequence AATAAA is boxed, and the linker motif is shaded in gray. (B) The structure of ZNF641 gene in genome, the structure of ZNF641 mRNA, and the protein domains of ZNF641. The big green box

indicates the KRAB domain (aa 109–170) and the 5 small boxes show 5 C2H2 zinc fingers (aa 266–286, 294–316, 322–342, 374–394, and 402–422). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

258 acid sequence of ZNF641 contains an N-terminal Krup- Homologies and evolution of ZNF641 262 259 pel-associated box (KRAB) domain (amino acids 109– 260 170) and 5 C2H2 zinc finger motifs that extend to the The KRAB and linker sequences of homologues of 263 261 end of the protein sequence (Fig. 1B). ZNF641 protein including XP_543709 (Canis familiaris), 264 中国科技论文在线 http://www.paper.edu.cn

X. Qi et al. / Biochemical and Biophysical Research Communications 339 (2006) 1155–1164 1159

Table 2 ZNF641 is a transcriptional activator 306 Genomic structure of the ZNF641 gene Exon Exon Splice donor site Intron Splice acceptor Previous studies have indicated that some members of 307 number size (bp) size (bp) size the Kru¨ppel-like family of transcription factors consist of 308 I 726 gtacccatcccatc 1930 tgaatttgttccag multiple domains that function in transcriptional activa- 309 II 209 gtgagaactcacag 563 tctctccttatcag tion, inhibition of activation, protein–protein interaction, 310 III 92 gtgagctctgcttc 1777 acattttgtttcag DNA binding, and transcriptional repression [21–24].Most 311 IV 130 gtaagcatgacctt 632 tttcccatgagcag V 114 gtaagaacctagaa 671 ccccttctccgcag of the studies have shown that zinc finger protein family 312 VI 3601 members are transcriptional intermediary factors, most of 313 Summary of the size of the exons and introns of the ZNF641 gene based them are corepressors, and few of them are coactivators 314 on comparison of the cDNA and the genomic sequence. Invariant [13]. Although ZNF641 shares conserved regions with 315 nucleotides (ag/gt) are in boldface type. other C2H2 zinc-finger proteins, the potential role of 316 ZNF641 is unknown. 317 As a first step in our understanding of ZNF641 for tran- 318 265 XP_582187 (Bos taurus), NP_776130 (Mus musculus), scriptional activator and/or repressor activity, we used a 319 266 XP_235625 (Rattus norvegicus), and ZNF641 (Homo system in which a luciferase reporter gene was activated 320 267 sapiens) are aligned with each other by using the Meg- by a fusion protein of the LexA DNA-binding domain 321 268 Align program of DNASTAR. The alignment reveals fused to the potent activation domain of the viral co-acti- 322 269 that the KRAB region and the linker motif of ZNF641 vator protein, VP16 (LexAVP16) (Fig. 5) [25]. As shown 323 270 are highly homologous with similar elements in its in Fig. 5, cotransfection of GAL4–ZNF641 fusion protein 324 271 homologous zinc-finger transcription factors (Figs. 2A with Vp16 strongly activated luciferase expression (Fig. 5), 325 272 and B). Further sequence comparison found that zinc suggesting thatPROOF ZNF641 is a potent transcriptional 326 273 fingers in these proteins are highly homologous. These activator. 327 274 results suggest that ZNF641 is a novel member of the 275 zinc-finger family, and these proteins probably are func- ZNF641 activates SRE and AP-1-mediated transcriptional 328 276 tionally similar. We then tried to analyze the evolution- activation 329 277 ary relationship between the ZNF641 protein and the 278 other zinc-finger proteins with phylogenetic tree analysis To investigate the role of ZNF641 in cell signal trans- 330 279 (Fig. 2C). The most closely related proteins of ZNF641 duction, we examined whether ZNF641 was directly or 331 280 are XP_582187 and NP_776130. indirectly involved in the regulation of transcription fac- 332 tors, especially in the signaling pathway. Mitogen-activated 333 281 Expression of the ZNF641 mRNA and subcellular protein kinase (MAPK) signal transduction pathways are 334 282 localization of GFPZNF641 fusion protein the most widespread mechanisms of eukaryotic cell regula- 335 tion [26]. To examine the effect of ZNF641 on this specific 336 283 To characterize the transcript of ZNF641 gene with cell signaling pathway, we performed pathway-specific 337 284 respect to its size and expression distribution, Northern reporter gene assays to measure the modulation of SRE 338 285 blot was performed using ZNF641 cDNA as the probe. and AP-1 by ZNF641 in the cell. First, the effect on the 339 286 A single 4.9 kb ZNF641 mRNA transcript was detected. transcriptional activity of AP-1 by ZNF641 was tested. 340 287 Among the 8 tissues tested at adult stage, the gene As shown in Fig. 6A, expression of ZNF641 significantly 341 288 showed a restricted expression pattern, including high enhanced the AP-1-luciferase activity by approximately 342 289 level expression in skeletal muscle, a moderate expression twofold. Ap-1 is a dimeric transcription factor composed 343 290 in heart, liver, and pancreas, a lower expression in of Jun, Fos or ATF (activating transcription factor) sub- 344 291 placenta, and no expression in brain, lung, and kidney units that bind to a common DNA site, the AP-l-binding 345 292 (Fig. 3). site [27]. We then tested the effect of ZNF641 on the tran- 346 293 To examine the subcellular localization of ZNF641, scriptional activity of SRE. As observed in the SRE-lucif- 347 294 we fused the entire coding sequence of ZNF641 to green erase assays, we found that expression of ZNF641 348 295 fluorescent protein (GFP) to create the pEGFP-N1- activates the endogenous transcriptional activity of SRE 349 296 ZNF641. PEGFP-N1-ZNF641 was introduced into by approximately threefold (Fig. 6B). 350 297 COS-7 cells by transientUNCORRECTED transfection. Forty-eight hours 298 after the transfection, the localization of the fusion pro- ZNF641 contains a novel transcriptional activation 351 299 tein (GFPZNF641) was visualized with epifluorescence domain—the linker motif 352 300 microscope after labeling with DAPI for nuclei. Cells 301 transfected with pEGFP-N1-ZNF641 showed a nuclear Using simple modular architecture research tool 353 302 and cytoplasm fluorescence pattern (Fig. 4A) and DAPI (SMART) analysis showed that ZNF641 protein contains 354 303 binds to DNA (Fig. 4B). The combined image (Fig. 4C) a KRAB box and 5 C2H2 zinc fingers. KRAB box-contain- 355 304 shows that the ZNF641 protein exists predominantly in ing zinc finger proteins in most of studies have been 356 305 the nuclei of the cells. shown to act as potent transcriptional repressors that are 357 中国科技论文在线 http://www.paper.edu.cn

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Fig. 2. (A) Comparison of the amino acid sequences of the KRAB region in ZNF641, XP_543709, XP_582187, NP_776130, and XP_235625. The residues that are highly conserved among these sequences are indicated within the majority sequence. Identical residues have been boxed and are shaded in green. (B) Comparison of the amino acid sequences of linker sequences in ZNF641 and its homologues. The same as (A), identical residues have been boxed and are shaded in green. (C) Unrooted phylogenetic tree analysis of ZNF641 and other Krupple-like zinc finger proteins. Gene names and accession numbers are listed in Materials and methods. All genes originate from mammalian. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

358 dependent on DNA-binding [28–30]. However, our experi- GAL4 DBD directs proteins to the nucleus, alleviating 368 359 ments showed that full-lengthUNCORRECTED ZNF641 can activate the the concern that PROOF deletion mutants might disrupt the natural 369 360 activity on a GAL4-driven promoter when fused to the nuclear localization signal of a protein. Then, we tested the 370 361 GAL4 DBD. In order to further identify potential novel ability of these truncated GAL4–ZNF641 fusion proteins 371 362 transcriptional regulatory domains in ZNF641, a series of to regulate the activity of the GAL4 site-driven luciferase 372 363 plasmids containing various portions of ZNF641 cDNA reporter gene. Full-length ZNF641 (aa 1–438) fused to 373 364 joined to the DBD of yeast transcription factor GAL4 were the GAL4 DBD transactivated the reporter gene modestly 374 365 constructed (Fig. 7). Because GAL4 fusion proteins are of (proximately 65%). Amino acids 1–132 which contain por- 375 366 yeast origin, they have the advantage of little or no back- tion of KRAB box repress the LexA-VP16-luciferase activ- 376 367 ground interference in mammalian cells. In addition, ity strongly. Amino acids 1–195 containing KRAB box and 377 中国科技论文在线 http://www.paper.edu.cn

X. Qi et al. / Biochemical and Biophysical Research Communications 339 (2006) 1155–1164 1161

Fig. 3. Northern blot analysis of ZNF641 in various human adult tissues, especially skeletal muscle. The RNA filters were hybridized with [a-32]P random-labeled cDNA probe, which contains the coding sequence of ZNF641. b-Actin was used as a control for the equivalent amount of loaded mRNA (total RNA) in each other. A band at 4.9 kb was detected.

378 portion of linker motif repress luciferase activity by 379 approximately 50% (Fig. 7), suggesting that the conserved 380 KRAB box in ZNF641 protein may be a transcriptional 381 repression domain and consistent with the traditional view- Fig. 5. COS-7 cells were transiently transfected with the pL8G5-luc 382 point that KRAB box was identified as a repressor [28–30]. reporter along withPROOF the indicated LexA or GAL4 mammalian expression 383 Amino acids 192–438 containing five zinc fingers also vectors. GAL4–ZNF641 was co-transfected with LexA-VP16. Relative luciferase activity was normalized for transfection efficiency by 384 repress the luciferase activity by approximately 50% co-transfection with GFP. Schematic of LexA–GAL–ZNF641-driven 385 (Fig. 7). In contrast, deletion of portions of C2H2 zinc fin- luciferase-reporter construct. LexA-VP16 represents an activator which 386 gers from full-length ZNF641 (amino acids 1–378) resulted can enlarge the activity activated by ZNF641 in this context. The results of 387 in an 80% increase in transcriptional activation (Fig. 7), transiently transfection are schematized. The data are means of three 388 suggesting that the full-length ZNF641 and the region from repeats in a single transfection experiment. Each transfection experiment was performed at least three times. 389 amino acids 1 to 378 (which contains a whole linker motif 390 between KRAB box and C2H2 zinc finger region) may act 391 as transcriptional activator. Therefore, we speculated that as the hypoalphalipoproteinemia susceptibility gene, 405 392 the linker motif may play an important role in transcrip- ZNF202, which encodes a transcriptional repressor that 406 393 tional activation. To demonstrate that the linker motif binds to elements found in genes involved in lipid metabo- 407 394 plays a role in transcriptional activation, plasmid encoding lism [31]. ZNF215, a Beckwith–Wiedemann syndrome-as- 408 395 amino acids 171–265 of ZNF641 was constructed and co- sociated gene [32]; and ZNF213, which is linked with 409 396 transfected with pCMV-b-gal and pL8G5 as described familial Mediterranean fever [33]. Although animal mod- 410 397 before. As shown in Fig. 7, the linker motif (amino acid els, especially the mouse, have provided us with much of 411 398 171–265) strongly activated the transcriptional activity our knowledge of early mammalian development, gene reg- 412 399 and increased the luciferase activity of the reporter gene ulation, and diseases, there are vital differences between 413 400 by approximately threefold as compared to VP-16 viral-ac- mouse and human. It is therefore very meaningful to sys- 414 401 tivating protein (Fig. 7). tematically search for more C2H2 type zinc finger genes 415 402 Recently, studies focusing on the C2H2 type zinc finger that are relative to gene regulation, diseases, and heart 416 403 genes have suggested their extensive involvement in devel- development of human being. The KRAB domain in 417 404 opment, gene regulation, diseases, and embryogenesis, such KRAB/C2H2 type zinc finger is an evolutionarily con- 418

UNCORRECTED

Fig. 4. ZNF641 is expressed predominantly in cell nucleus. (A) COS-7 cells expressing EGFP-ZNF641. (B) The nucleus of cells stained with DAPI. (C) The combined image of (A) and (B). 中国科技论文在线 http://www.paper.edu.cn

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ome are estimated to contain the KRAB domain [34]. The 424 KRAB domain has been shown to function as a repressor 425 of transcription through protein–protein interaction [28– 426 30]. In this study, we report the identification of ZNF641, 427 a novel zinc finger transcription factor, functioning as an 428 activator of transcription not reported previously. The 429 region of ZNF641 spanning amino acids 109–170 shows 430 homology to the KRAB domain of other zinc finger pro- 431 teins (Fig. 2A). The KRAB-A box of ZNF641 consists of 432 about 41 amino acid residues, including a conserved motif: 433 DV (at position 5–6), which have been shown to be impor- 434 tant for repression and interaction with TIF1h (also named 435 KAP-1, KRIP-1) [35–37]. Five C2H2 zinc finger motifs of 436 ZNF641 extend to the C-terminal portion of the protein 437 sequence (Fig. 1B). Each finger motif conforms closely to 438 the consensus sequence X2CX2CXKXFX4HLR(S)HX3H. 439 Both the first and the second zinc finger units, and the 440 fourth and the fifth zinc finger units are connected by high- 441 ly conserved H/C linkers TGE(K/R)P, strongly suggesting 442 a role in DNA binding. The conserved Cys and His resi- 443 dues in C2H2 zinc finger are bound to a tetrahedrally coor- 444 dinated zinc ion, resulting in organization of the key amino 445 acid residues into a structure capable of binding to target 446 DNA [38]. Taken together, these results suggest that 447 ZNF641 belongs to the KRAB/C2H2 subfamily of zinc fin- 448 ger proteins. 449 Fig. 6. Overexpression of ZNF641 activates transcriptional activities of Using transient transfection and reporter assays, we 450 SER and AP-1 in COS-7 cells. (A) Co-transfection of pCMV-Tag2B- have shown that ZNF641 has a transcriptional activation 451 ZNF641 and pAP-1–Luc activates the AP-1 activation in the reporter function and activates transcriptional activities of SRE 452 assay. (B) Activation of SRE transcriptional activity by expression of and AP-1. MAPK signal transduction pathways are the 453 ZNF641. COS-7 cells transfected with individual reporter plasmid and the most widespread mechanisms of eukaryotic cell regulation 454 corresponding plasmids shown in the figure. The data are means of three repeats in a single transfection experiment. Each transfection experiment [26]. It is initiated in cardiac myocytes by G protein cou- 455 was performed at least three times. pled receptors (GPCR) and growth factor receptors. 456 MAPK pathways consist of four major groupings and 457 419 served domain of about 75 amino acids that are subdivided numerous related proteins that constitute interrelated sig- 458 420 into an A box and a B box, usually coded for by separate nal transduction cascades activated by stimuli such as 459 421 exons. The A box is present in every KRAB domain, while growth factors, stress, cytokines, and inflammation. The 460 422 the B box is not always included [34]. Approximately one- four major groupings are the Erk, JNK or SAPK, p38, 461 423 third of all C2H2 type zinc finger genes in the human gen- and the Big MAPK or ERK5 cascades. Signals from cell 462

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Fig. 7. ZNF641 contains an activation domain. A number of ZNF641 serial deletion mutations fused to GAL4 were created. A schematic of GAL4 DBD fused to full-length ZNF641, three deletion mutations in the 30 end of ZNF641, one deletion mutation in the 50 end of ZNF641, and one deletion mutation with the linker motif only are shown. Relative luciferase activity of GAL4 DBD–ZNF641 and different deletion mutants of ZNF641 with or without Vp16 are shown in the bar graph (means ± SE; n = 3 experiments). 中国科技论文在线 http://www.paper.edu.cn

X. Qi et al. / Biochemical and Biophysical Research Communications 339 (2006) 1155–1164 1163

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