Báo cáo khoa học: Novel repressor of the human FMR1 gene ) identification ¨ of p56 human (GCC)n-binding protein as a Kruppel-like transcription factor ZF5 ppt

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Novel repressor of the human FMR1 gene)identificationof p56 human (GCC)n-binding protein as a Kru¨ppel-liketranscription factor ZF5Sergey V. Orlov1,2, Konstantin B. Kuteykin-Teplyakov1*, Irina A. Ignatovich3, Ella B. Dizhe1,Olga A. Mirgorodskaya3, Alexander V. Grishin2, Olga B. Guzhova1, Egor B. Prokhortchouk4,Pavel V. Guliy1,2and Andrej P. Perevozchikov1,21 Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, St Petersburg, Russia2 Department of Embryology, St Petersburg State University, Russia3 Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia4 Department of Molecular Basis of Medicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, RussiaA variety of human diseases have been associated withexpansion of CGG ⁄ CCG triplet repeat tracts withinthe human genome [1–3]. To date, five human chromo-somal folate-sensitive fragile sites associated withexpansion of 5¢-(CGG)n-3¢ trinucleotide repeats havebeen characterized at the molecular level: FRAXA,FRAXE, FRAXF, FRA16A, and FRA11B. Expansionat the FRAXA locus results in fragile X-mental retar-dation syndrome, whereas expansion at the FRAXElocus leads to mild mental retardation, and theFRA11B locus has been implicated in Jacobsen’ssyndrome [3,4]. In the case of the FRAXA locus,the (GCC)ntriplet repeat amplification has occurredwithin the 5¢-UTR of the fragile X-mental retarda-tion 1 (FMR1) gene [5]. Expansions of n > 200(n < 70 is normal) followed by cytosine methylationinactivate the FMR1 gene. The FMR1 gene productis an RNA-binding protein that associates withKeywordsFMR1; fragile X syndrome; (GCC)n; tripletrepeats; ZF5; zinc finger transcription factorsCorrespondenceS. V. Orlov, Department of Biochemistry,Institute of Experimental Medicine, RussianAcademy of Medical Sciences, 197376,Acad. Pavlov Street 12, St Petersburg,RussiaFax: +7 812 234 0310Tel: +7 812 346 0644E-mail: serge@iem.sp.ruPresent address*Department of Molecular Neurobiochemistry,Ruhr-University, Bochum 44801,Germany(Received 1 May 2007, revised 20 July2007, accepted 24 July 2007)doi:10.1111/j.1742-4658.2007.06006.xA series of relatively short (GCC)ntriplet repeats (n ¼ 3–30) located withinregulatory regions of many mammalian genes may be considered as puta-tive cis-acting transcriptional elements (GCC-elements). Fragile X-mentalretardation syndrome is caused by an expansion of (GCC)ntriplet repeatswithin the 5¢-untranslated region of the human fragile X-mental retarda-tion 1 (FMR1) gene. The present study aimed to characterize a novelhuman (GCC)n-binding protein and investigate its possible role in the regu-lation of the FMR1 gene. A novel human (GCC)n-binding protein, p56,was isolated and identified as a Kru¨ppel-like transcription factor, ZF5, byMALDI-TOF analysis. The capacity of ZF5 to specifically interact with(GCC)ntriplet repeats was confirmed by the electrophoretic mobility shiftassay with purified recombinant ZF5 protein. In cotransfection experi-ments, ZF5 overexpression repressed activity of the GCC-element contain-ing mouse ribosomal protein L32 gene promoter. Moreover, RNAinterference assay results showed that endogenous ZF5 acts as a repressorof the human FMR1 gene. Thus, these data identify a new class of ZF5targets, a subset of genes containing GCC-elements in their regulatoryregions, and raise the question of whether transcription factor ZF5 is impli-cated in the pathogenesis of fragile X syndrome.AbbreviationsCAST, cyclic amplification and selection of targets; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; NFjB, nuclear factor kappa B; siRNA, small interfering RNA.4848 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government workspolyribosomes as part of large messenger ribonucleo-protein complex, modulating the translation of itsRNA ligands [6]. FMR1 protein is thought to beinvolved in RNA interference machinery [7–9].The molecular mechanisms responsible for the insta-bility of (GCC)ntriplet repeats remain largelyunknown. Several models have been suggested, includ-ing DNA polymerase slippage [10–12], formation ofunusual secondary structures by contiguous (GCC)ntracts [13,14], and interactions of (GCC)ntripletrepeats with DNA-binding proteins [15,16]. (GCC)ntriplet repeats are not restricted to folate-sensitive frag-ile sites in mammalian genomes [4,17]. There are rela-tively short (GCC)ntriplet repeats (n ¼ 3–30) locatedwithin regulatory regions of many mammalian genesthat may operate as cis-acting transcriptional elements(GCC-elements) [17]. We previously characterizedGCC-elements within the promoter of the gene thatencodes mouse ribosomal protein L32 (rpL32) [18–20]and within the 5¢-UTR of the human very low densitylipoprotein receptor gene (VLDLR) [21]. Such GCC-elements are transcriptionally active and can interactspecifically with human and mouse nuclear proteins.To date, only one (GCC)n-binding protein, p20, hasbeen characterized [16,18,22]. The p20 protein was firstdetected in HeLa cells as a protein interacting with theGCC-element of rpL32 [18], and then purified accord-ing to its ability to interact specifically with (GCC)ntriplet repeats within the 5¢-UTR of the human FMR1gene; the corresponding gene was cloned [16,22]. Puri-fied p20 (20 kDa) was found not to be homologous toany known DNA-binding proteins and was designatedCGG triplet repeat binding protein 1 (CGGBP1). Sub-sequently, in cotransfection experiments, CGGBP1 hasbeen demonstrated to repress the activity of the FMR1[23], VLDLR [21], and rpL32 (K. B. Kuteykin-Teplya-kov, E. B. Dizhe, S. V. Orlov & A. P. Perevozchikov,unpublished results) genes. Thus, DNA-binding pro-teins that interact with (GCC)nrepeats may beinvolved in stabilization ⁄ destabilization of the tripletrepeats [16,22] and ⁄ or transcriptional regulation ofGCC-triplet containing genes [19–21]. Hence, in ourprevious studies, we proposed the existence of otheryet uncharacterized mammalian (GCC)n-binding pro-teins [19–21]. In the present study, we report the iso-lation of a novel (GCC)n-binding protein p56 fromhuman hepatoma HepG2 cells that interacts specifi-cally with the GCC-elements of the rpL32 and FMR1genes. This protein was identified herein to be aKru¨ppel-like Zn-finger transcription factor ZF5.Mammalian ZF5 protein was first identified inmouse as a transcriptional repressor of the murinec-myc gene with a molecular mass of approximately52 kDa. It was subsequently shown to repress the her-pes simplex virus thymidine kinase gene promoter [24].ZF5 homologues have since been cloned in humansand in chicken [25–27]. Vertebrate ZF5 proteins arehighly conserved and contain five Kru¨ppel-like Zn-fin-gers at the C-terminus. They also have a hydrophobicBTB ⁄ POZ domain (124 N-terminal amino acid resi-dues), which is responsible for protein–protein inter-actions, and a nuclear localization signal [25,26]. TheDNA-binding domain of ZF5 consists of the third andforth Zn-fingers [28]. Cyclic amplification and selectionof targets (CAST) assays performed independently bytwo groups confirmed the GC-rich content of ZF5binding sites with a common core sequence5¢-GCGCG-3¢ [28,29]. Interestingly, none of the ZF5sequences isolated by CAST assays contained (GCC)ntriplet repeats.Several novel ZF5 targets have been described[29,30]. Transcription factor ZF5 has been reported tohave both positive and negative effects on target genetranscription. Moreover, in silico content analysis ofthe core promoter regions of human genes revealed thepresence of ZF5 consensus sites within approximately60% of human gene core promoters [31]. In spite ofsignificant over-representation of these sites in pro-moter regions relative to nonpromoter backgrounddata, these results must be verified experimentally,especially in terms of the relative low complexity ofthe ZF5 consensus sequence. In the present study, wehave shown for the first time that endogenous ZF5protein acts as a repressor of the FMR1 gene inHepG2 cells.ResultsCharacterization of human nuclear proteins thatinteract with a composite cis-acting element ofrpL32 ()24 +11)We have previously shown that the mouse rpL32 genecontains a composite cis-acting element that spans thetranscription start site ()24 +11) [19]; this compositeelement consists of a GCC-element and a pyrimidineblock (Fig. 1A). Prior tissue distribution studies ofmammalian nuclear proteins interacting with therpL32 fragment ()24 +11) revealed similaritiesbetween the DNA–protein complexes formed by therpL32 fragment ()24 +11) with nuclear proteinsfrom human embryonic fibroblasts and HepG2 cells[19]. Electrophoretic mobility shift assay (EMSA) com-petition experiments in the present study indicated thatthe complete rpl32 fragment ()24 +11) has a greateraffinity to nuclear proteins from human fibroblastsSergey V. Orlov et al. A novel human (GCC)n-binding proteinFEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4849than does either the GCC-element or pyrimidine blockalone (Fig. 1B,C). Similar results were obtained withnuclear proteins from HepG2 cells (data not shown).These results suggest that this rpL32 fragment is a syn-ergetic composite cis-acting element.EMSA was conducted with 1,10-phenanthroline (achelator that specifically removes Zn2+from metallo-proteins) [32] to examine whether Zn-finger DNA-bind-ing human proteins are involved in DNA–proteincomplexes formed by the rpL32 fragment. The presenceof 1,10-phenanthroline in the reaction mixture inhibitedformation of DNA–protein complexes (Fig. 1D) and theaddition of Zn2+partially restored complex formation.The relatively weak Zn-chelator EGTA produced lessinhibition of DNA–protein interactions than 1,10-phe-nanthroline. These results suggest that the DNA-bindingactivity of human nuclear proteins interacting withrpL32 fragment ()24 +11) depends on Zn2+, which isconsistent with the supposition that those proteins con-tain Zn-finger DNA-binding domains.Although there are similarities between the DNA–protein complexes formed by the rpL32 fragmentFig. 1. Characterization of human nuclearproteins interacting with the rpL32 fragment()24. . .+11). (A) Composite cis-acting ele-ment of mouse ribosomal protein L32 gene.The numbers indicate position relative tothe rpL32 transcription start point. Thesequences corresponding to the GCC-ele-ment and pyrimidine tract are underlined.(B,C) Results of EMSA competition experi-ments with nuclear proteins from humanembryonic fibroblasts. The competitors andtheir molar excesses are shown above thelanes: (GCC)3-element; rpL32 ()6. +11)-pyrimidine part of the rpL32 fragment()24. . .+11); K–-negative control (withoutnuclear proteins); K+-binding reaction withno competitors; CI and CII refer to the spe-cific DNA-protein complexes I and II formedby the rpL32 fragment ()24. +11); F refersto the free rpL32 fragment ()24. +11). (D)EMSA experiments with chelators specificto bivalent cations: 1, free rpL32 fragment()24. . .+11) without human embryonic fibro-blast nuclear proteins (negative control);2, binding reaction of the rpL32 fragment()24. . .+11) with human embryonic fibro-blast nuclear proteins with no chelators;3, binding reaction with addition of 5 mM1,10-phenanthroline; 4, binding reaction withaddition of 5 mM 1,10-phenanthroline and5mM ZnCl2; 5, binding reaction with addi-tion of 5 mM EGTA. CI and CII refer to thespecific DNA-protein complexes I and IIformed by the rpL32 fragment ()24. +11).(E) Southwestern assay of HepG2 nuclearproteins with the rpL32 fragment()24. . .+11) revealing the p56 and p68bands (arrowheads).A novel human (GCC)n-binding protein Sergey V. Orlov et al.4850 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works()24 +11) with human fibroblast nuclear proteinsand those formed with HepG2 cell nuclear proteins,the latter were found to be much more abundant.Therefore, nuclear proteins from HeG2 cells wereemployed in the subsequent Southwestern assays. Twomajor putative (GCC)n-binding proteins, p56 and p68,were detected by Southwestern assays. (Fig. 1E).Purification of human nuclear proteins thatinteract with a composite cis-acting element ofrpL32 ()24 +11)We designed a four-step purification procedure to iso-late proteins that interact with the rpL32 fragment()24 +11) (Fig. 2A). This approach included a con-sequent preparative EMSA stage, two-step purificationby DNA-affinity chromatography with the rpL32 frag-ment ()24 +11) immobilized on magnetic beads as asubstrate, and DNA-affinity chromatography on anonspecific substrate to remove the remaining nonspe-cific DNA-binding proteins. Figure 2B shows the elu-tion profile of the preparative EMSA of the rpL32fragment ()24 +11) with nuclear proteins fromHepG2 cells. The first peak corresponds to unboundlabeled DNA probe, the second and third peaks corre-spond to DNA–protein complexes formed by therpL32 fragment ()24 +11) with studied proteins.Fractions 21–27, corresponding to latter two peaks,were collected, concentrated by ultrafiltration andchecked for DNA-binding activity by analytical EMSA(Fig. 2C). The slower migrating complex (Complex I)spontaneously converted into the faster migrating one(Complex II) (Fig. 2C). Therefore, fractions 22–25with high DNA-binding activity were pooled, analyzedby SDS electrophoresis (Fig. 2E, lane 1), and subjectedto purification by DNA-affinity chromatography.In the final purification step, the proteins were elutedfrom immobilized rpL32 fragment ()24 +11) ontoimmobilized human lactoferrin binding sites (nonspe-cific DNA substrate). The DNA-binding activity analy-sis results are summarized in Fig. 3D. The final fractioncontaining unbound last stage proteins was analyzed bySDS electrophoresis and compared with proteins boundto nonspecific sorbent (negative control) (Fig. 2E, lanes3 and 2, respectively). Only two proteins, p56 (approxi-mately 56 kDa) and p68 (approximately 68 kDa), thatwere not in the negative control fraction remained in afinal fraction. There were also two bands at 65 kDa and72 kDa that were detected in both the negative controland final fractions. The p56 and p68 proteins in thefinal fraction fit with the rpL32 fragment ()24 +11)-binding proteins detected by Southwestern assay (seeabove). Together, these findings indicate that p56 andp68 are DNA-binding proteins that interact specificallywith composite cis-acting element of rpL32.Identification of rpL32-binding protein p56 byMALDI-TOF assay as a Kru¨ppel-like transcriptionfactor ZF5MALDI-TOF assays were employed to identify thep56 protein. After SDS electrophoresis, the corres-ponding bands from silver-stained gel were cut out,digested by trypsin and subjected to analysis. A typicalrepresentative spectrum fragment containing severalpeaks corresponding to p56-derived peptides is shownin Figure 3A. Identification of p56 protein was per-formed using the Mascot search engine. Despite thepresence of substantial noise, we were able to detectamong the potential candidates one Zn-finger proteinwith a molecular weight of approximately 52 kDa:human Kru¨ppel-like Zn-finger transcription factor ZF5(Fig. 3B). The cDNA gene encoding human ZF5protein has been cloned by two-step RT-PCR andsubcloned into a pBluescript vector. Experiments toidentify the p68 protein are currently in progress.Recombinant fusion protein GST-ZF5-ZF5 purifiedfrom bacterial cells specifically interacts with(GCC)nrepeatsEMSA experiments with a recombinant fusion proteincontaining ZF5 Zn-finger domains linked to bacterialglutathione transferase (GST-ZF5) were performed totest the capacity of ZF5 to interact with the rpL32fragment ()24 +11) and to examine the possible(GCC)n-binding activity of ZF5. The recombinant pro-tein was isolated from bacterial cells by glutathioneaffinity chromatography (Fig. 4). Recombinant GST-ZF5 protein efficiently recognized the ZF5 consensussequence (Fig. 4A, lanes 2 and 6). Molar excesses ofunlabeled ZF5 binding site depleted the correspondingbands in a dose-dependent manner (Fig. 4A, lanes3–5), indicating a good specificity of the DNA–proteininteractions. Interestingly, the unlabeled rpL32 frag-ment ()24 +11) depleted the corresponding bandeven more efficiently (Fig. 4A, lanes 7–9).Similar results were obtained in reciprocal EMSAexperiments (Fig. 4B). Recombinant GST-ZF5 pro-tein specifically interacted with the rpL32 frag-ment ()24 +11). Moreover, the rpL32 fragment()24 +11) was a more effective competitor than theZF5 binding site (Fig. 4B, lanes 2–4 and 6–8, respec-tively). The irrelevant lactoferrin binding site (negativecontrol) did not disrupt the corresponding DNA–pro-tein complex (Fig. 4B, lanes 10–12). Therefore, theseSergey V. Orlov et al. A novel human (GCC)n-binding proteinFEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4851Fig. 2. Purification of HepG2 nuclear proteins interacting with the rpL32 fragment ()24. . .+11). (A) Summary of purification scheme. (B) Pre-parative EMSA elution profile. (C) Testing of the rpL32 fragment ()24. +11)-binding activity of 21st to 27th fractions obtained on a prepara-tive EMSA purification stage. (D) EMSA testing of the rpL32 fragment ()24 +11)-binding activity in the samples after DNA affinitychromatography: 1, negative control (without proteins); 2, positive control (EMSA with HepG2 crude nuclear extracts); 3, the proteins elutedfrom the immobilized rpL32 ()24 .+11) fragment (first round of DNA affinity chromatography); 4, the proteins eluted after second round ofDNA affinity chromatography; 5, proteins not bound to immobilized lactoferrin binding site (final fraction). (E) Analysis of fractions obtainedfrom the preparative EMSA and DNA affinity chromatography stages by SDS electrophoresis: 1, pooled fractions 22–25 obtained from thepreparative EMSA stage; 2, the proteins bound by the lactofferin binding site in the last purification stage; 3, unbound proteins obtained fromthe last stage of purification (final fraction). CI and CII, DNA-protein complexes I and II, respectively. C¢, Nonspecific DNA–protein complexes;F, free rpL32 fragment ()24. +11), p56 and p68- purified proteins specifically interacting with the rpL32 fragment ()24. +11).A novel human (GCC)n-binding protein Sergey V. Orlov et al.4852 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government worksFig. 3. Identification of p56 polypeptide as Kru¨ppel-like transcription factor ZF5 by MALDI-TOF assay. (A) Fragment of MALDI-TOF spectrum ofp56-derived peptides. (B) Mascot search results. Peptides with matched mass values are listed with their locations in the total ZF5 protein.Sergey V. Orlov et al. A novel human (GCC)n-binding proteinFEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4853data provide a robust demonstration of the capacity ofZF5 to interact specifically with rpL32 composite cis-acting element. Furthermore, the affinity of recombi-nant GST-ZF5 for the rpL32 fragment ()24 +11) ishigher than its affinity for a classic ZF5 binding site.EMSA experiments with a labeled GCC-elementprobe or pyrimidine motif probe were conducted todetermine whether the ZF5 binds to the GCC-elementor pyrimidine motif of rpL32 composite cis-acting ele-ment. The pyrimidine part of the rpL32 fragmentbound nuclear proteins from HepG2 cells, but did notinteract with recombinant GST-ZF5 (Fig. 4C). Mean-while, a (GCC)9sequence corresponding to a 5¢-UTRfragment of the human FMR1 gene (normal allele)bound specifically to GST-ZF5. Molar excessesof unlabeled rpL32 fragment ()24 +11), (GCC)9sequence, or ZF5 binding site, but not lactoferrin bind-ing site, efficiently disrupted (GCC)9⁄ ZF5 complexes(Fig. 4D,E). These data demonstrate unequivocallythat ZF5 transcription factor interacts specifically withFig. 4. Interactions of recombinant GST-ZF5 with GCC-element of the rpL32 promoter. (A) EMSA using the ZF5 binding site as a probe. (B)EMSA using the rpL32 fragment ()24. +11) as a probe. (C) EMSA using the pyrimidine site of rpL32 ()6 +11) as a probe. (D,E) EMSAusing a (GCC)9-element as a probe. K–, without any proteins; K+, EMSA with purified recombinant GST-ZF5 without competition; HepG2,EMSA with nuclear proteins from HepG2 cells. Competitors are shown above each image. Triangles above images represent the increasingamounts of competitor applied. C, Specific DNA–protein complex; F, free DNA probe.A novel human (GCC)n-binding protein Sergey V. Orlov et al.4854 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works(GCC)ntriplet repeats and, hence, may be considereda novel (GCC)n-binding protein.ZF5 overexpression leads to repression of rpL32promoter that contains GCC-elementThe capacity of ZF5 transcription factor to interactwith GCC-elements suggests that it may regulate rpL32gene expression. To investigate this possibility, we con-structed a eukaryotic expression vector containing theZF5 coding region under the control of the humancytomegalovirus early gene promoter (pCMVZF5) andperformed cotransfection experiments. When HepG2cells were transiently cotransfected with a pCMVZF5expression vector and pL32luc plasmid containing afirefly luciferase reporter gene under the control ofrpL32 promoter, ZF5 overexpression strongly down-regulated rpL32 promoter activity in dose-dependentmanner (Fig. 5A) relative to its impact on the activityof a synthetic minimal promoter containing five tandemcis-acting elements for transcription factor nuclear fac-tor kappa B NFeˆ B (negative control) (Fig. 5B). Theseresults demonstrate that human ZF5 can regulate tran-scription of genes that contain (GCC)ntriplet repeatswithin their regulatory regions.Endogenous ZF5 down-regulates expression ofthe FMR1 gene in HepG2 cellsRNA interference was used to test whether ZF5 isinvolved in regulation of the human FMR1 gene.Three small interfering RNAs (siRNAs) matching dif-ferent regions of ZF5 mRNA were selected based ongeneral rules for siRNA design [43]. The efficiency ofZF5 down-regulation in HepG2 cells by siRNA trans-fection was estimated by semiquantitative RT-PCR.HepG2 cells were transfected with the most activesiRNA (matched bases 945–963 of human ZF5mRNA; accession number D89859). The amount ofFMR1 mRNA in siRNAZF5transfected cells har-vested after 48 h was accessed by semiquantitativeRT-PCR and compared with the mRNA from non-transfected cells and cells transfected with controlsiRNA that was not homologous to any humanmRNAs (Fig. 6A,B). ZF5 down-regulation increasedFMR1 mRNA levels in HepG2 cells; transfection ofHepG2 cells with control siRNA did not influenceFMR1 mRNA levels. It was suggested previously thatFMR1 protein may be involved in the RNA interfer-ence machinery [7–9]. Thus, accumulation of FMR1mRNA may be due to stimulation of FMR1 geneexpression in response to activation of RNA interfer-ence machinery in the cells trasfected by siRNAZF5but not to down-regulation of ZF5. To test thisassumption, we transfected HepG2 cells by siRNAfor human grp58 gene [44]. siRNAgrp58down-regu-lated expression of grp58 gene but did not affectFMR1 mRNA (Fig. 6C). This, these data indicatethat endogenous transcription factor ZF5 is a repres-sor of the human FMR1 gene.DiscussionCGGBP1 is the only DNA-binding protein describedto date that specifically interacts with (GCC)nrepeats[16,22]. Two observations led us to hypothesize thatother (GCC)n-binding proteins exist and to search forFig. 5. Effect of ZF5 overexpression on activity of the rpL32 pro-moter relative to the synthetic NFjB-dependent promoter. (A)HepG2 cells were cotransfected with pL32luc (5 lg), pCMVL(2.5 lg) and different amounts of pCMVZF5: 1, without pCMVZF5;2, with 50 ng pCMVZF5; 3, with 150 ng pCMVZF5; 4, with 375 ngpCMVZF5. (B) HepG2 cells were cotransfected with pNFjBluc(5 lg), pCMVL (2.5 lg) and different amounts of pCMVZF5: 1, with-out pCMVZF5; 2, with 50 ng pCMVZF5; 3, with 150 ng pCMVZF5;4, with 375 ng pCMVZF5.Sergey V. Orlov et al. A novel human (GCC)n-binding proteinFEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4855possible novel human (GCC)n-binding transcriptionfactors. First, the DNA-binding activity of nuclearproteins that interact with the rpL32 fragment()24 +11) is Zn2+-dependent (Fig. 1D), althoughthe CGGBP1 protein does not contain any Zn-bind-ing domains [22]. Second, the affinity of purifiedCGGBP1 to (GCC)nrepeat sequences is dramaticallydecreased if there are fewer than eight repeats [16].However, a few GCC repeats are sufficient toregulate an artificial herpes viral thymidine kinasepromoter (three repeats) or the rpL32 promoter (fourrepeats) [19,20].Purification of transcription factors can be challeng-ing due to their low abundance in cells and their weakaffinity to bait sequences. Indeed, (GCC)ntripletrepeats with n > 10 are needed to bind mammaliannuclear proteins with high affinity [15,16]. However theregulatory regions of many mammalian genes containshorter (GCC)nmotifs (n ¼ 3–10) that are adjacent tobinding sites of other transcription factors (i.e. Sp1,Egr1, WT1) [17]. Thus, low affinity binding of short(GCC)nrepeats to (GCC)n-binding proteins may becompensated for by cooperative protein–protein inter-actions with transcription factors that bind to adjacentsequences.We previously characterized a fragment of mouserpL32 promoter ()24 +11) containing an adjacentGCC-element ()19 )6) and pyrimidine motif()5 +11) as a composite cis-acting element thatinteracts with nuclear proteins from mammalian cells[18–20]. The present data (Fig. 1B,C) suggest thatthere may be cooperation between DNA–protein andprotein–protein interactions within complexes formedby the rpL32 fragment ()24 +11). We proceeded todetect, by Southwestern assay, only two polypeptidesin nuclear extracts from HepG2 cells that interact withthe rpL32 fragment ()24 +11) (Fig. 1E). These find-ings indicate that the rpL32 fragment ()24 +11)would serve as an effective bait for purifying GCC-ele-ment binding proteins.The rpL32 fragment ()24 +11) formed two majorcomplexes with nuclear proteins from HepG2 cells andhuman embryonic fibroblast cells (Fig. 1B,C). In ourattempt to isolate these complexes, an exploratory pre-parative EMSA revealed that the slower migratingComplex I converted spontaneously to the fastermigrating Complex II (Fig. 2C). This observation sug-gests that the difference between the DNA-bindingcomplexes might be caused by reversible post-transla-tional modifications of rpL32-binding proteins. Alter-natively, those complexes may differ by unstableproteins that were lost during the purification process.Additional experiments are needed to distinguishbetween these possibilities.We detected two purified DNA-binding proteins spe-cifically interacting with the rpL32 fragment ()24 +11) that coincided with the rpL32-binding proteinsrevealed by Southwestern assay. The results of theMALDI-TOF analysis indicated that one of these, p56,is a Kru¨ppel-like transcription factor ZF5. Although allZF5-binding sites found previously by CAST assay areGC-rich, none are within (GCC)ntriplet repeats [28,29].Our EMSA experiments using recombinant proteinFig. 6. Down-regulation of endogenous ZF5 in HepG2 cells by RNAinterference. (A) Effects of HepG2 cell transfection by siRNAs onZF5 expression (semiquantitative RT-PCR): lanes 1–6, RT-PCR ofGAPDH mRNA (18 cycles, control); lane 7, marker; lanes 8–13,RT-PCR of ZF5 mRNA (27 cycles); 1, 8, negative control; 2, 9, trans-fection of HepG2 cells by siRNAZF5with oligofectamine; 3, 10,transfection of HepG2 cells by control siRNA with oligofectamine;4, 11, transfection of HepG2 cells by siRNAZF5with RNAFect; 5,12, transfection of HepG2 cells by control siRNA with RNAFect; 6,13, untreated cells. (B) Effects of HepG2 cell transfection bysiRNAZF5on FMR1 expression (semiquantitative RT-PCR, 34 cycles):1, marker; 2, negative control; 3, transfection by siRNAZF5with oli-gofectamine; 4, transfection by control siRNA with oligofectamine;5, transfection by siRNAZF5with RNAFect; 6, transfection by controlsiRNA with RNAFect; 7, untreated cells. (C) Effects of HepG2 celltransfection by siRNAgrp58on FMR1 expression (semiquantitativeRT-PCR): 1–3, RT-PCR of GAPDH mRNA (18 cycles, control); 4, mar-ker; lanes 5–7, RT-PCR of grp58 mRNA (27 cycles); 8–10, RT-PCR ofFMR1mRNA (34 cycles); 1, 5, 8, negative control; 3, 7, 10, untreatedcells; 2, 6, 9, transfection by siRNAgrp58with RNAFect.A novel human (GCC)n-binding protein Sergey V. Orlov et al.4856 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government workscontaining the DNA-binding domain of ZF5 fused withGST confirmed that recombinant GST-ZF5 specificallyinteracts with its canonical binding site, the rpL32fragment ()24 +11), and the GCC-element alone(Fig. 4). Moreover, competition experiments showedthat the affinity of ZF5 for the rpL32 fragment()24 +11) is greater than its affinity for its ownconsensus sequence.Although the rpL32 fragment ()24 +11) containsa core part of the ZF5 consensus sequence GCGCimmediately before the GCC-element (Fig. 1A), a(GCC)9sequence that does not contain any ZF5consensus core was recognized specifically by ZF5(Fig. 4C). Thus, the high affinity of ZF5 for the rpL32fragment may be due to cooperation interactions ofZF5 with both its consensus core sequence and theGCC-element. Hence, ZF5 appears to bind GCC-ele-ments with an affinity that is similar to that for itsconsensus sequence. This finding may explain why pre-vious CAST assays did not reveal GCC-elements to beamong ZF5-binding sites. The affinity of ZF5–DNAinteractions may depend on the length of the bindingsite. Random sequences used for CAST assays areapproximately 20 bp in length [28], and the selectionprocedure is based on single site affinities to theprotein of interest. In the case of the rpL32 fragmentor the (GCC)9sequence, there are several repeatedZF5-binding sites within a single oligonucleotide.Therefore, the affinity of those sequences for ZF5revealed in the present study may be the result ofcooperative interactions between several ZF5 moleculesand adjacent ZF5-binding sites. Such a cooperativemechanism is especially interesting with respect to therole of (GCC)ntriplet repeat amplification in inactiva-tion of the FMR1 gene. That is, ZF5 affinity for(GCC)nrepeat tracts in the FMR1 gene would beexpected to be greatly enhanced in alleles with veryextended (GCC)nrepeats.Mammalian ZF5 protein was first identified as atranscriptional repressor of the murine c-myc gene.Although ZF5-mediated transcriptional activation hasbeen reported [29,30], ZF5 is most often observed todown-regulate target genes. Here, we show that ZF5overexpression led to down-regulation of the rpL32promoter. Moreover, down-regulation of ZF5 byRNA interference caused up-regulation of the FMR1gene. These data clearly support the assertion thatendogenous ZF5 acts as a transcriptional repressor ofthe human FMR1 gene. Given that FMR1 inactivationin FMR patients has been attributed to long (GCC)ntriplet repeats within the 5¢-UTR of the FMR1 gene,further investigations to clarify the role of ZF5 inFMR pathogenesis are warranted.In conclusion, we report that p56 protein purifiedfrom HepG2 cells interacts with GCC-elements of therpL32 promoter and the 5¢-UTR of the humanFMR1 gene. We further identify p56 as the Kru¨ppel-like transcription factor ZF5. We show, for the firsttime, that recombinant mammalian ZF5 proteininteracts specifically with (GCC)ntriplet repeats, andfurther show that endogenous ZF5 acts to down-reg-ulate the FMR1 gene. The present study has revealeda novel class of ZF5 target genes that have GCC-ele-ments within their regulatory regions and implicatesZF5 in the pathogenesis of fragile X-mental retarda-tion syndrome.Experimental proceduresMaterialsChemicals were purchased from Sigma (St Louis, MO,USA), Amersham Biosciences (Piscataway, NJ, USA),Roche Applied Science (Mannheim, Germany), Invitrogen(Carlsbad, CA, USA), Promega (San Luis Obispo, CA,USA) and from local Russian manufacturers (analytical orhigh purity grade). Enzymes used in gene engineering werepurchased from Fermentas (Vilnius, Lithuania) and the Sci-entific Industrial Corp. SibEnzyme (Novosibirsk, Russia).Genetic constructionspL32luc plasmid containing the reporter gene encodingfirefly luciferase under the control of rpL32 promoter()155 .+195 relative to the transcription start point) wasconstructed as follows. First, rpL32 promoter was cut outfrom pL3A plasmid containing the genomic rpL32 gene (agift from Dr N. V. Tomilin, Institute of Cytology, RussianAcademy of Sciences) by AccI and SmaI and inserted intopUC19 vector. Next, it was digested from pUC19 byEcoRI, blunt ended by Klenow fragment of Escherichia coliDNA polymerase I, digested by HindIII and inserted into apGL3basic plasmid containing the luciferase gene (Pro-mega). The pCMVL plasmid containing the reporter genelacZ and encoding bacterial b-galactosidase driven by apromoter of early human CMV genes have been describedpreviously [19]. pNFjBluc plasmid contains luciferase genedriven by synthetic minimal promoter carrying five bindingsites for transcription factor NFjB was purchased fromStratagene (La Jolla, CA, USA).Cell cultureHepG2 cells were obtained from the Cell Culture Bank ofthe Institute of Cytology, Russian Academy of Sciences.Human embryonic fibroblast cells were obtained from theInstitute of Influenza, Russian Academy of MedicalSergey V. Orlov et al. A novel human (GCC)n-binding proteinFEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS. No claim to original Russian government works 4857[...]... assay RNA interference assay Three siRNAs for human ZF5 mRNA were selected based on general rules for siRNA selection [43] The first siRNAZF5 5¢-GGUUGAGGAUGUGAAAUUCUU-3¢ and 5¢-GAAUUUCACAUCCUCAACCUU-3 ) matched bases 192–210; the second siRNAZF5 (5¢-GAGGAAGCAUGA GAAACUCUU-3¢ and 5¢-GAGUUUCUCAUGCUUCCU CUU-3 ) matched bases 945–963; and the third siRNA ZF5 (5¢-GGUCCUGAACUACAUGUACUU-3¢ and 5¢-GUAC AUGUAGUUCAGGACCUU-3 ). .. which amplify overlapping 5¢- and 3¢-parts of ZF5 cDNA, were designed using Primer software developed by V Prutkovsky and O Sokur of the Institute of Influenza, Ministry of Health of the Russian Federation The first pair of primers (5¢-GGCCTTCAAGGCATTAAG-3¢, 5¢-AAACAAATGG CCTGTCCG-3 ) spanned a 958 bp 5¢-part of the ZF5 coding region; the second pair (5¢-CCCCTCAAGCCTT AACAT-3¢, 5¢-TCTCCACTTTCCAGGCAA-3 ). .. AUGUAGUUCAGGACCUU-3 ) matched bases 333–351 of the ZF5 mRNA sequence (accession number D8985 9) siRNAgrp58 sequence (5¢-UAGUCCCAUUAGCAAAGG UUU-3¢ and 5¢-ACCUUUGCUAAUGGGACUAUU-3 ) was published previously [44] and used as a control The control siRNA sequence did not match any human mRNAs FEBS Journal 274 (200 7) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works 4859 A novel. .. described above The optimal numbers of PCR cycles for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene (primers 5¢-TCAC CATCTTCCAGGAGCGA-3¢ and 5¢-TACTCCTTGGAG GCCATGT-3 ) (18 cycles), ZF5 (primers 5¢-CCCCTC AAGCCTTAACAT-3¢ and 5¢-TCTCCACTTTCCAGGC AA-3 ) (27 cycles), FMR1 (primers 5¢-GGGTGAGGATT GAGGCTGA-3¢ and 5¢-GCCGTGCCCCCTATTTCT-3 ) (34 cycles), and grp58 (primers 5¢-ATCTCCGACACG... fetal bovine serum, 5% CO2 at 37 °C Ca-phosphate tranfection was performed as described previously [42] Ten micrograms of DNA per dish was used in all experiments b-galactosidase assays were performed following standard protocols, using O-nitrophenyl-b-d-galactopyranoside as a substrate Relative b-galactosidase activity was calculated as D420 nm optical density per mg of total protein in cell lysates... cDNA containing the coding region of human ZF5 transcription factor (1425 bp) was cloned by two step RTPCR Total cellular RNA was isolated from HepG2 cells using the guanidine isothiocyanate method [41] After digestion with RNase-free DNase I (Roche Applied Science), RNA was reverse transcribed using an oligo(dT) primer and reverse transcriptase (Invitrogen) to make the first cDNA strand Two pairs of. .. Oligofectamine reagent (Invitrogen Corp., Carlsbad, CA, USA) in accordance with the manufacturer’s instructions Thirty nanomolar concentrations of siRNAs were used in all experiments Cells were harvested 48 h after transfection and total RNA was isolated as described above Semiquantitative RT-PCR was used to compare ZF5 and FMR1 mRNA levels in transfected cells Reverse transcription was performed as described... Luciferase activity was measured in a scintillation counter (Beckman Coulter, Fullerton, CA, USA) employing a Luciferase Assay System (Promega, catalog number E403 0) in accordance with manufacturer’s guidelines Luciferase activity is indexed in relative light units that correspond to counts per min per mg of total protein in cell lysates Protein concentration in cell lysates was measured by the Bradford assay... (199 6) Genomic and cDNA structures of the gene encoding the chicken ZF5 DNA binding protein Biochim Biophys Acta 1308, 114–118 A novel human (GCC)n-binding protein 28 Obata T, Yanagidani A, Yokoro K, Numoto M & Yamamoto S (199 9) Analysis of the consensus binding sequence and the DNA-binding domain of ZF5 Biochem Biophys Res Commun 255, 528–534 29 Kaplan J & Calame K (199 7) The ZiN ⁄ POZ domain of ZF5. .. novel human (GCC)n-binding protein (5¢-GACGCGGGAAAAAUUAAGCUU-3¢ and 5¢-GCUU AAUUUUUCCCGCGUCUU-3 ) All RNAs were ordered from Syntol Corp (Moscow, Russia) as single stranded oligonucleotides and annealed in 300 mm NaCl by heating at 85 °C for 15 min followed by gradual cooling to room temperature Transfection of HepG2 cells by siRNAs was performed by RNAiFect reagent (Qiagen, Hilden, Germany) or Oligofectamine . and5¢-GAAUUUCACAUCCUCAACCUU-3 ) matched bases192–210; the second siRNA ZF5 (5¢-GAGGAAGCAUGAGAAACUCUU-3¢ and 5¢-GAGUUUCUCAUGCUUCCUCUU-3 ) matched bases. regu-lation of the FMR1 gene. A novel human (GCC)n-binding protein, p56, was isolated and identified as a Kru ¨ ppel-like transcription factor, ZF5, byMALDI-TOF
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Xem thêm: Báo cáo khoa học: Novel repressor of the human FMR1 gene ) identification ¨ of p56 human (GCC)n-binding protein as a Kruppel-like transcription factor ZF5 ppt, Báo cáo khoa học: Novel repressor of the human FMR1 gene ) identification ¨ of p56 human (GCC)n-binding protein as a Kruppel-like transcription factor ZF5 ppt, Báo cáo khoa học: Novel repressor of the human FMR1 gene ) identification ¨ of p56 human (GCC)n-binding protein as a Kruppel-like transcription factor ZF5 ppt