Novel repressor of the human FMR1 gene ) identication ă of p56 human (GCC)n-binding protein as a Kruppel-like transcription factor ZF5 Sergey 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 Guliy1,2 and Andrej P Perevozchikov1,2 Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, St Petersburg, Russia Department of Embryology, St Petersburg State University, Russia Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia Department of Molecular Basis of Medicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia Keywords FMR1; fragile X syndrome; (GCC)n; triplet repeats; ZF5; zinc finger transcription factors Correspondence S V Orlov, Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376, Acad Pavlov Street 12, St Petersburg, Russia Fax: +7 812 234 0310 Tel: +7 812 346 0644 E-mail: serge@iem.sp.ru Present address *Department of Molecular Neurobiochemistry, Ruhr-University, Bochum 44801, Germany (Received May 2007, revised 20 July 2007, accepted 24 July 2007) A series of relatively short (GCC)n triplet repeats (n ¼ 3–30) located within regulatory regions of many mammalian genes may be considered as putative cis-acting transcriptional elements (GCC-elements) Fragile X-mental retardation syndrome is caused by an expansion of (GCC)n triplet repeats within the 5¢-untranslated region of the human fragile X-mental retardation (FMR1) gene The present study aimed to characterize a novel human (GCC)n-binding protein and investigate its possible role in the regulation of the FMR1 gene A novel human (GCC)n-binding protein, p56, was isolated and identified as a Kruppel-like transcription factor, ZF5, by ¨ MALDI-TOF analysis The capacity of ZF5 to specifically interact with (GCC)n triplet repeats was confirmed by the electrophoretic mobility shift assay with purified recombinant ZF5 protein In cotransfection experiments, ZF5 overexpression repressed activity of the GCC-element containing mouse ribosomal protein L32 gene promoter Moreover, RNA interference assay results showed that endogenous ZF5 acts as a repressor of the human FMR1 gene Thus, these data identify a new class of ZF5 targets, a subset of genes containing GCC-elements in their regulatory regions, and raise the question of whether transcription factor ZF5 is implicated in the pathogenesis of fragile X syndrome doi:10.1111/j.1742-4658.2007.06006.x A variety of human diseases have been associated with expansion of CGG ⁄ CCG triplet repeat tracts within the human genome [1–3] To date, five human chromosomal folate-sensitive fragile sites associated with expansion of 5¢-(CGG)n-3¢ trinucleotide repeats have been characterized at the molecular level: FRAXA, FRAXE, FRAXF, FRA16A, and FRA11B Expansion at the FRAXA locus results in fragile X-mental retardation syndrome, whereas expansion at the FRAXE locus leads to mild mental retardation, and the FRA11B locus has been implicated in Jacobsen’s syndrome [3,4] In the case of the FRAXA locus, the (GCC)n triplet repeat amplification has occurred within the 5¢-UTR of the fragile X-mental retardation (FMR1) gene [5] Expansions of n > 200 (n < 70 is normal) followed by cytosine methylation inactivate the FMR1 gene The FMR1 gene product is an RNA-binding protein that associates with Abbreviations CAST, cyclic amplification and selection of targets; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 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 works Sergey V Orlov et al polyribosomes as part of large messenger ribonucleoprotein complex, modulating the translation of its RNA ligands [6] FMR1 protein is thought to be involved in RNA interference machinery [7–9] The molecular mechanisms responsible for the instability of (GCC)n triplet repeats remain largely unknown Several models have been suggested, including DNA polymerase slippage [10–12], formation of unusual secondary structures by contiguous (GCC)n tracts [13,14], and interactions of (GCC)n triplet repeats with DNA-binding proteins [15,16] (GCC)n triplet repeats are not restricted to folate-sensitive fragile sites in mammalian genomes [4,17] There are relatively short (GCC)n triplet repeats (n ¼ 3–30) located within regulatory regions of many mammalian genes that may operate as cis-acting transcriptional elements (GCC-elements) [17] We previously characterized GCC-elements within the promoter of the gene that encodes mouse ribosomal protein L32 (rpL32) [18–20] and within the 5¢-UTR of the human very low density lipoprotein receptor gene (VLDLR) [21] Such GCCelements are transcriptionally active and can interact specifically with human and mouse nuclear proteins To date, only one (GCC)n-binding protein, p20, has been characterized [16,18,22] The p20 protein was first detected in HeLa cells as a protein interacting with the GCC-element of rpL32 [18], and then purified according to its ability to interact specifically with (GCC)n triplet repeats within the 5¢-UTR of the human FMR1 gene; the corresponding gene was cloned [16,22] Purified p20 (20 kDa) was found not to be homologous to any known DNA-binding proteins and was designated CGG triplet repeat binding protein (CGGBP1) Subsequently, in cotransfection experiments, CGGBP1 has been demonstrated to repress the activity of the FMR1 [23], VLDLR [21], and rpL32 (K B Kuteykin-Teplyakov, E B Dizhe, S V Orlov & A P Perevozchikov, unpublished results) genes Thus, DNA-binding proteins that interact with (GCC)n repeats may be involved in stabilization ⁄ destabilization of the triplet repeats [16,22] and ⁄ or transcriptional regulation of GCC-triplet containing genes [19–21] Hence, in our previous studies, we proposed the existence of other yet uncharacterized mammalian (GCC)n-binding proteins [19–21] In the present study, we report the isolation of a novel (GCC)n-binding protein p56 from human hepatoma HepG2 cells that interacts specifically with the GCC-elements of the rpL32 and FMR1 genes This protein was identified herein to be a Kruppel-like Zn-nger transcription factor ZF5 ă Mammalian ZF5 protein was first identified in mouse as a transcriptional repressor of the murine c-myc gene with a molecular mass of approximately A novel human (GCC)n-binding protein 52 kDa It was subsequently shown to repress the herpes simplex virus thymidine kinase gene promoter [24] ZF5 homologues have since been cloned in humans and in chicken [25–27] Vertebrate ZF5 proteins are highly conserved and contain ve Kruppel-like Zn-nă gers at the C-terminus They also have a hydrophobic BTB ⁄ POZ domain (124 N-terminal amino acid residues), which is responsible for protein–protein interactions, and a nuclear localization signal [25,26] The DNA-binding domain of ZF5 consists of the third and forth Zn-fingers [28] Cyclic amplification and selection of targets (CAST) assays performed independently by two groups confirmed the GC-rich content of ZF5 binding sites with a common core sequence 5¢-GCGCG-3¢ [28,29] Interestingly, none of the ZF5 sequences isolated by CAST assays contained (GCC)n triplet repeats Several novel ZF5 targets have been described [29,30] Transcription factor ZF5 has been reported to have both positive and negative effects on target gene transcription Moreover, in silico content analysis of the core promoter regions of human genes revealed the presence of ZF5 consensus sites within approximately 60% of human gene core promoters [31] In spite of significant over-representation of these sites in promoter regions relative to nonpromoter background data, these results must be verified experimentally, especially in terms of the relative low complexity of the ZF5 consensus sequence In the present study, we have shown for the first time that endogenous ZF5 protein acts as a repressor of the FMR1 gene in HepG2 cells Results Characterization of human nuclear proteins that interact with a composite cis-acting element of rpL32 ()24 +11) We have previously shown that the mouse rpL32 gene contains a composite cis-acting element that spans the transcription start site ()24 +11) [19]; this composite element consists of a GCC-element and a pyrimidine block (Fig 1A) Prior tissue distribution studies of mammalian nuclear proteins interacting with the rpL32 fragment ()24 +11) revealed similarities between the DNA–protein complexes formed by the rpL32 fragment ()24 +11) with nuclear proteins from human embryonic fibroblasts and HepG2 cells [19] Electrophoretic mobility shift assay (EMSA) competition experiments in the present study indicated that the complete rpl32 fragment ()24 +11) has a greater affinity to nuclear proteins from human fibroblasts FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works 4849 A novel human (GCC)n-binding protein Sergey V Orlov et al Fig Characterization of human nuclear proteins interacting with the rpL32 fragment ()24 .+11) (A) Composite cis-acting element of mouse ribosomal protein L32 gene The numbers indicate position relative to the rpL32 transcription start point The sequences corresponding to the GCC-element and pyrimidine tract are underlined (B,C) Results of EMSA competition experiments with nuclear proteins from human embryonic fibroblasts The competitors and their molar excesses are shown above the lanes: (GCC)3-element; rpL32 ()6 .+11)pyrimidine part of the rpL32 fragment ()24 .+11); K–-negative control (without nuclear proteins); K+-binding reaction with no competitors; CI and CII refer to the specific DNA-protein complexes I and II formed by the rpL32 fragment ()24 .+11); F refers to the free rpL32 fragment ()24 .+11) (D) EMSA experiments with chelators specific to bivalent cations: 1, free rpL32 fragment ()24 .+11) without human embryonic fibroblast nuclear proteins (negative control); 2, binding reaction of the rpL32 fragment ()24 .+11) with human embryonic fibroblast nuclear proteins with no chelators; 3, binding reaction with addition of mM 1,10-phenanthroline; 4, binding reaction with addition of mM 1,10-phenanthroline and mM ZnCl2; 5, binding reaction with addition of mM EGTA CI and CII refer to the specific DNA-protein complexes I and II formed by the rpL32 fragment ()24 .+11) (E) Southwestern assay of HepG2 nuclear proteins with the rpL32 fragment ()24 .+11) revealing the p56 and p68 bands (arrowheads) than does either the GCC-element or pyrimidine block alone (Fig 1B,C) Similar results were obtained with nuclear proteins from HepG2 cells (data not shown) These results suggest that this rpL32 fragment is a synergetic composite cis-acting element EMSA was conducted with 1,10-phenanthroline (a chelator that specifically removes Zn2+ from metalloproteins) [32] to examine whether Zn-finger DNA-binding human proteins are involved in DNA–protein complexes formed by the rpL32 fragment The presence of 1,10-phenanthroline in the reaction mixture inhibited 4850 formation of DNA–protein complexes (Fig 1D) and the addition of Zn2+ partially restored complex formation The relatively weak Zn-chelator EGTA produced less inhibition of DNA–protein interactions than 1,10-phenanthroline These results suggest that the DNA-binding activity of human nuclear proteins interacting with rpL32 fragment ()24 +11) depends on Zn2+, which is consistent with the supposition that those proteins contain Zn-finger DNA-binding domains Although there are similarities between the DNA– protein complexes formed by the rpL32 fragment FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works Sergey V Orlov et al ()24 +11) with human fibroblast nuclear proteins and those formed with HepG2 cell nuclear proteins, the latter were found to be much more abundant Therefore, nuclear proteins from HeG2 cells were employed in the subsequent Southwestern assays Two major putative (GCC)n-binding proteins, p56 and p68, were detected by Southwestern assays (Fig 1E) Purification of human nuclear proteins that interact with a composite cis-acting element of rpL32 ()24 +11) We designed a four-step purification procedure to isolate proteins that interact with the rpL32 fragment ()24 +11) (Fig 2A) This approach included a consequent preparative EMSA stage, two-step purification by DNA-affinity chromatography with the rpL32 fragment ()24 +11) immobilized on magnetic beads as a substrate, and DNA-affinity chromatography on a nonspecific substrate to remove the remaining nonspecific DNA-binding proteins Figure 2B shows the elution profile of the preparative EMSA of the rpL32 fragment ()24 +11) with nuclear proteins from HepG2 cells The first peak corresponds to unbound labeled DNA probe, the second and third peaks correspond to DNA–protein complexes formed by the rpL32 fragment ()24 +11) with studied proteins Fractions 21–27, corresponding to latter two peaks, were collected, concentrated by ultrafiltration and checked 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 22–25 with high DNA-binding activity were pooled, analyzed by SDS electrophoresis (Fig 2E, lane 1), and subjected to purification by DNA-affinity chromatography In the final purification step, the proteins were eluted from immobilized rpL32 fragment ()24 +11) onto immobilized human lactoferrin binding sites (nonspecific DNA substrate) The DNA-binding activity analysis results are summarized in Fig 3D The final fraction containing unbound last stage proteins was analyzed by SDS electrophoresis and compared with proteins bound to nonspecific sorbent (negative control) (Fig 2E, lanes and 2, respectively) Only two proteins, p56 (approximately 56 kDa) and p68 (approximately 68 kDa), that were not in the negative control fraction remained in a final fraction There were also two bands at 65 kDa and 72 kDa that were detected in both the negative control and final fractions The p56 and p68 proteins in the final fraction fit with the rpL32 fragment ()24 +11)binding proteins detected by Southwestern assay (see above) Together, these findings indicate that p56 and A novel human (GCC)n-binding protein p68 are DNA-binding proteins that interact specifically with composite cis-acting element of rpL32 Identification of rpL32-binding protein p56 by MALDI-TOF assay as a Kruppel-like transcription ă factor ZF5 MALDI-TOF assays were employed to identify the p56 protein After SDS electrophoresis, the corresponding bands from silver-stained gel were cut out, digested by trypsin and subjected to analysis A typical representative spectrum fragment containing several peaks corresponding to p56-derived peptides is shown in Figure 3A Identification of p56 protein was performed using the Mascot search engine Despite the presence of substantial noise, we were able to detect among the potential candidates one Zn-finger protein with a molecular weight of approximately 52 kDa: human Kruppel-like Zn-finger transcription factor ZF5 ă (Fig 3B) The cDNA gene encoding human ZF5 protein has been cloned by two-step RT-PCR and subcloned into a pBluescript vector Experiments to identify the p68 protein are currently in progress Recombinant fusion protein GST-ZF5-ZF5 purified from bacterial cells specifically interacts with (GCC)n repeats EMSA experiments with a recombinant fusion protein containing ZF5 Zn-finger domains linked to bacterial glutathione transferase (GST-ZF5) were performed to test the capacity of ZF5 to interact with the rpL32 fragment ()24 +11) and to examine the possible (GCC)n-binding activity of ZF5 The recombinant protein was isolated from bacterial cells by glutathione affinity chromatography (Fig 4) Recombinant GSTZF5 protein efficiently recognized the ZF5 consensus sequence (Fig 4A, lanes and 6) Molar excesses of unlabeled ZF5 binding site depleted the corresponding bands in a dose-dependent manner (Fig 4A, lanes 3–5), indicating a good specificity of the DNA–protein interactions Interestingly, the unlabeled rpL32 fragment ()24 +11) depleted the corresponding band even more efficiently (Fig 4A, lanes 7–9) Similar results were obtained in reciprocal EMSA experiments (Fig 4B) Recombinant GST-ZF5 protein specifically interacted with the rpL32 fragment ()24 +11) Moreover, the rpL32 fragment ()24 +11) was a more effective competitor than the ZF5 binding site (Fig 4B, lanes 2–4 and 6–8, respectively) The irrelevant lactoferrin binding site (negative control) did not disrupt the corresponding DNA–protein complex (Fig 4B, lanes 10–12) Therefore, these FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works 4851 A novel human (GCC)n-binding protein Sergey V Orlov et al Fig Purification of HepG2 nuclear proteins interacting with the rpL32 fragment ()24 .+11) (A) Summary of purification scheme (B) Preparative EMSA elution profile (C) Testing of the rpL32 fragment ()24 .+11)-binding activity of 21st to 27th fractions obtained on a preparative EMSA purification stage (D) EMSA testing of the rpL32 fragment ()24 .+11)-binding activity in the samples after DNA affinity chromatography: 1, negative control (without proteins); 2, positive control (EMSA with HepG2 crude nuclear extracts); 3, the proteins eluted from the immobilized rpL32 ()24 .+11) fragment (first round of DNA affinity chromatography); 4, the proteins eluted after second round of DNA affinity chromatography; 5, proteins not bound to immobilized lactoferrin binding site (final fraction) (E) Analysis of fractions obtained from the preparative EMSA and DNA affinity chromatography stages by SDS electrophoresis: 1, pooled fractions 22–25 obtained from the preparative EMSA stage; 2, the proteins bound by the lactofferin binding site in the last purification stage; 3, unbound proteins obtained from the 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) 4852 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works Sergey V Orlov et al A novel human (GCC)n-binding protein Fig Identification of p56 polypeptide as Kruppel-like transcription factor ZF5 by MALDI-TOF assay (A) Fragment of MALDI-TOF spectrum of ă p56-derived peptides (B) Mascot search results Peptides with matched mass values are listed with their locations in the total ZF5 protein FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works 4853 A novel human (GCC)n-binding protein Sergey V Orlov et al Fig 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) EMSA using 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 increasing amounts of competitor applied C, Specific DNA–protein complex; F, free DNA probe data provide a robust demonstration of the capacity of ZF5 to interact specifically with rpL32 composite cisacting element Furthermore, the affinity of recombinant GST-ZF5 for the rpL32 fragment ()24 +11) is higher than its affinity for a classic ZF5 binding site EMSA experiments with a labeled GCC-element probe or pyrimidine motif probe were conducted to determine whether the ZF5 binds to the GCC-element or pyrimidine motif of rpL32 composite cis-acting element The pyrimidine part of the rpL32 fragment 4854 bound nuclear proteins from HepG2 cells, but did not interact with recombinant GST-ZF5 (Fig 4C) Meanwhile, a (GCC)9 sequence corresponding to a 5¢-UTR fragment of the human FMR1 gene (normal allele) bound specifically to GST-ZF5 Molar excesses of unlabeled rpL32 fragment ()24 +11), (GCC)9 sequence, or ZF5 binding site, but not lactoferrin binding site, efficiently disrupted (GCC)9 ⁄ ZF5 complexes (Fig 4D,E) These data demonstrate unequivocally that ZF5 transcription factor interacts specifically with FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works A novel human (GCC)n-binding protein Sergey V Orlov et al (GCC)n triplet repeats and, hence, may be considered a novel (GCC)n-binding protein ZF5 overexpression leads to repression of rpL32 promoter that contains GCC-element The capacity of ZF5 transcription factor to interact with GCC-elements suggests that it may regulate rpL32 gene expression To investigate this possibility, we constructed a eukaryotic expression vector containing the ZF5 coding region under the control of the human cytomegalovirus early gene promoter (pCMVZF5) and performed cotransfection experiments When HepG2 cells were transiently cotransfected with a pCMVZF5 expression vector and pL32luc plasmid containing a firefly luciferase reporter gene under the control of rpL32 promoter, ZF5 overexpression strongly downregulated rpL32 promoter activity in dose-dependent manner (Fig 5A) relative to its impact on the activity of a synthetic minimal promoter containing five tandem cis-acting elements for transcription factor nuclear factor kappa B NFeB (negative control) (Fig 5B) These ˆ results demonstrate that human ZF5 can regulate transcription of genes that contain (GCC)n triplet repeats within their regulatory regions Endogenous ZF5 down-regulates expression of the FMR1 gene in HepG2 cells RNA interference was used to test whether ZF5 is involved in regulation of the human FMR1 gene Three small interfering RNAs (siRNAs) matching different regions of ZF5 mRNA were selected based on general rules for siRNA design [43] The efficiency of ZF5 down-regulation in HepG2 cells by siRNA transfection was estimated by semiquantitative RT-PCR HepG2 cells were transfected with the most active siRNA (matched bases 945–963 of human ZF5 mRNA; accession number D89859) The amount of FMR1 mRNA in siRNAZF5 transfected cells harvested after 48 h was accessed by semiquantitative RT-PCR and compared with the mRNA from nontransfected cells and cells transfected with control siRNA that was not homologous to any human mRNAs (Fig 6A,B) ZF5 down-regulation increased FMR1 mRNA levels in HepG2 cells; transfection of HepG2 cells with control siRNA did not influence FMR1 mRNA levels It was suggested previously that FMR1 protein may be involved in the RNA interference machinery [7–9] Thus, accumulation of FMR1 mRNA may be due to stimulation of FMR1 gene expression in response to activation of RNA interference machinery in the cells trasfected by siRNAZF5 Fig Effect of ZF5 overexpression on activity of the rpL32 promoter 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 ng pCMVZF5 (B) HepG2 cells were cotransfected with pNFjBluc (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 ng pCMVZF5 but not to down-regulation of ZF5 To test this assumption, we transfected HepG2 cells by siRNA for human grp58 gene [44] siRNAgrp58 down-regulated expression of grp58 gene but did not affect FMR1 mRNA (Fig 6C) This, these data indicate that endogenous transcription factor ZF5 is a repressor of the human FMR1 gene Discussion CGGBP1 is the only DNA-binding protein described to date that specifically interacts with (GCC)n repeats [16,22] Two observations led us to hypothesize that other (GCC)n-binding proteins exist and to search for FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works 4855 A novel human (GCC)n-binding protein Fig Down-regulation of endogenous ZF5 in HepG2 cells by RNA interference (A) Effects of HepG2 cell transfection by siRNAs on ZF5 expression (semiquantitative RT-PCR): lanes 1–6, RT-PCR of GAPDH mRNA (18 cycles, control); lane 7, marker; lanes 8–13, RT-PCR of ZF5 mRNA (27 cycles); 1, 8, negative control; 2, 9, transfection of HepG2 cells by siRNAZF5 with oligofectamine; 3, 10, transfection of HepG2 cells by control siRNA with oligofectamine; 4, 11, transfection of HepG2 cells by siRNAZF5 with RNAFect; 5, 12, transfection of HepG2 cells by control siRNA with RNAFect; 6, 13, untreated cells (B) Effects of HepG2 cell transfection by siRNAZF5 on FMR1 expression (semiquantitative RT-PCR, 34 cycles): 1, marker; 2, negative control; 3, transfection by siRNAZF5 with oligofectamine; 4, transfection by control siRNA with oligofectamine; 5, transfection by siRNAZF5 with RNAFect; 6, transfection by control siRNA with RNAFect; 7, untreated cells (C) Effects of HepG2 cell transfection by siRNAgrp58 on FMR1 expression (semiquantitative RT-PCR): 1–3, RT-PCR of GAPDH mRNA (18 cycles, control); 4, marker; lanes 5–7, RT-PCR of grp58 mRNA (27 cycles); 8–10, RT-PCR of FMR1mRNA (34 cycles); 1, 5, 8, negative control; 3, 7, 10, untreated cells; 2, 6, 9, transfection by siRNAgrp58 with RNAFect possible novel human (GCC)n-binding transcription factors First, the DNA-binding activity of nuclear proteins that interact with the rpL32 fragment ()24 +11) is Zn2+-dependent (Fig 1D), although the CGGBP1 protein does not contain any Zn-binding domains [22] Second, the affinity of purified CGGBP1 to (GCC)n repeat sequences is dramatically decreased if there are fewer than eight repeats [16] However, a few GCC repeats are sufficient to regulate an artificial herpes viral thymidine kinase 4856 Sergey V Orlov et al promoter (three repeats) or the rpL32 promoter (four repeats) [19,20] Purification of transcription factors can be challenging due to their low abundance in cells and their weak affinity to bait sequences Indeed, (GCC)n triplet repeats with n > 10 are needed to bind mammalian nuclear proteins with high affinity [15,16] However the regulatory regions of many mammalian genes contain shorter (GCC)n motifs (n ¼ 3–10) that are adjacent to binding sites of other transcription factors (i.e Sp1, Egr1, WT1) [17] Thus, low affinity binding of short (GCC)n repeats to (GCC)n-binding proteins may be compensated for by cooperative protein–protein interactions with transcription factors that bind to adjacent sequences We previously characterized a fragment of mouse rpL32 promoter ()24 +11) containing an adjacent GCC-element ()19 )6) and pyrimidine motif ()5 +11) as a composite cis-acting element that interacts with nuclear proteins from mammalian cells [18–20] The present data (Fig 1B,C) suggest that there may be cooperation between DNA–protein and protein–protein interactions within complexes formed by the rpL32 fragment ()24 +11) We proceeded to detect, by Southwestern assay, only two polypeptides in nuclear extracts from HepG2 cells that interact with the rpL32 fragment ()24 +11) (Fig 1E) These findings indicate that the rpL32 fragment ()24 +11) would serve as an effective bait for purifying GCC-element binding proteins The rpL32 fragment ()24 +11) formed two major complexes with nuclear proteins from HepG2 cells and human embryonic fibroblast cells (Fig 1B,C) In our attempt to isolate these complexes, an exploratory preparative EMSA revealed that the slower migrating Complex I converted spontaneously to the faster migrating Complex II (Fig 2C) This observation suggests that the difference between the DNA-binding complexes might be caused by reversible post-translational modifications of rpL32-binding proteins Alternatively, those complexes may differ by unstable proteins that were lost during the purification process Additional experiments are needed to distinguish between these possibilities We detected two purified DNA-binding proteins specifically interacting with the rpL32 fragment ()24 + 11) that coincided with the rpL32-binding proteins revealed by Southwestern assay The results of the MALDI-TOF analysis indicated that one of these, p56, is a Kruppel-like transcription factor ZF5 Although all ă ZF5-binding sites found previously by CAST assay are GC-rich, none are within (GCC)n triplet repeats [28,29] Our EMSA experiments using recombinant protein FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works Sergey V Orlov et al containing the DNA-binding domain of ZF5 fused with GST confirmed that recombinant GST-ZF5 specifically interacts with its canonical binding site, the rpL32 fragment ()24 +11), and the GCC-element alone (Fig 4) Moreover, competition experiments showed that the affinity of ZF5 for the rpL32 fragment ()24 +11) is greater than its affinity for its own consensus sequence Although the rpL32 fragment ()24 +11) contains a core part of the ZF5 consensus sequence GCGC immediately before the GCC-element (Fig 1A), a (GCC)9 sequence that does not contain any ZF5 consensus core was recognized specifically by ZF5 (Fig 4C) Thus, the high affinity of ZF5 for the rpL32 fragment may be due to cooperation interactions of ZF5 with both its consensus core sequence and the GCC-element Hence, ZF5 appears to bind GCC-elements with an affinity that is similar to that for its consensus sequence This finding may explain why previous CAST assays did not reveal GCC-elements to be among ZF5-binding sites The affinity of ZF5–DNA interactions may depend on the length of the binding site Random sequences used for CAST assays are approximately 20 bp in length [28], and the selection procedure is based on single site affinities to the protein of interest In the case of the rpL32 fragment or the (GCC)9 sequence, there are several repeated ZF5-binding sites within a single oligonucleotide Therefore, the affinity of those sequences for ZF5 revealed in the present study may be the result of cooperative interactions between several ZF5 molecules and adjacent ZF5-binding sites Such a cooperative mechanism is especially interesting with respect to the role of (GCC)n triplet repeat amplification in inactivation of the FMR1 gene That is, ZF5 affinity for (GCC)n repeat tracts in the FMR1 gene would be expected to be greatly enhanced in alleles with very extended (GCC)n repeats Mammalian ZF5 protein was first identified as a transcriptional repressor of the murine c-myc gene Although ZF5-mediated transcriptional activation has been reported [29,30], ZF5 is most often observed to down-regulate target genes Here, we show that ZF5 overexpression led to down-regulation of the rpL32 promoter Moreover, down-regulation of ZF5 by RNA interference caused up-regulation of the FMR1 gene These data clearly support the assertion that endogenous ZF5 acts as a transcriptional repressor of the human FMR1 gene Given that FMR1 inactivation in FMR patients has been attributed to long (GCC)n triplet repeats within the 5¢-UTR of the FMR1 gene, further investigations to clarify the role of ZF5 in FMR pathogenesis are warranted A novel human (GCC)n-binding protein In conclusion, we report that p56 protein purified from HepG2 cells interacts with GCC-elements of the rpL32 promoter and the 5¢-UTR of the human FMR1 gene We further identify p56 as the Kruppelă like transcription factor ZF5 We show, for the first time, that recombinant mammalian ZF5 protein interacts specifically with (GCC)n triplet repeats, and further show that endogenous ZF5 acts to down-regulate the FMR1 gene The present study has revealed a novel class of ZF5 target genes that have GCC-elements within their regulatory regions and implicates ZF5 in the pathogenesis of fragile X-mental retardation syndrome Experimental procedures Materials Chemicals 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 or high purity grade) Enzymes used in gene engineering were purchased from Fermentas (Vilnius, Lithuania) and the Scientific Industrial Corp SibEnzyme (Novosibirsk, Russia) Genetic constructions pL32luc plasmid containing the reporter gene encoding firefly luciferase under the control of rpL32 promoter ()155 +195 relative to the transcription start point) was constructed as follows First, rpL32 promoter was cut out from pL3A plasmid containing the genomic rpL32 gene (a gift from Dr N V Tomilin, Institute of Cytology, Russian Academy of Sciences) by AccI and SmaI and inserted into pUC19 vector Next, it was digested from pUC19 by EcoRI, blunt ended by Klenow fragment of Escherichia coli DNA polymerase I, digested by HindIII and inserted into a pGL3basic plasmid containing the luciferase gene (Promega) The pCMVL plasmid containing the reporter gene lacZ and encoding bacterial b-galactosidase driven by a promoter of early human CMV genes have been described previously [19] pNFjBluc plasmid contains luciferase gene driven by synthetic minimal promoter carrying five binding sites for transcription factor NFjB was purchased from Stratagene (La Jolla, CA, USA) Cell culture HepG2 cells were obtained from the Cell Culture Bank of the Institute of Cytology, Russian Academy of Sciences Human embryonic fibroblast cells were obtained from the Institute of Influenza, Russian Academy of Medical FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works 4857 A novel human (GCC)n-binding protein Sciences HepG2 cells and human embryonic cell lines were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD, USA) All cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere Nuclear extract preparation and EMSA Nuclear extracts were prepared from cultured cells as described previously [33] with slight modifications [19] The following synthetic oligonucleotides were purchased from MWG Biotech (Ebersberg, Germany) (upper strands only): rpL32 ()24 +11) fragment, 5¢-CTTGCGCGCCGCC GCCGCCTCTTCCTTCTTCCTCG-3¢; ZF5 binding site, 5¢-CAGGTACCGCGCCTTCGCTGCCA-3¢; (GCC)3 element, 5¢-AGCTTGCCGCCGCCTTCGA-3¢; (GCC)9 element, 5¢-AGCTTGCCGCCGCCGCCGCCGCCGCCGCC GCCTTCGA-3¢; rpL32 pyrimidine motif ()6 +11), 5¢-CTCTTCCTTCTTCCTCG-3¢; lactoferrin binding site, 5¢-CTAGTGCAAGTGCCA-3¢ The synthetic oligonucleotides were end-labeled with [c32P]ATP using T4 polynucleotide kinase (Scientific Industrial Corp SibEnzyme) and annealed to form double stranded probes EMSA was performed as described previously [34] Reaction mixtures contained 100 mm NaCl, 10.5 mm Hepes, pH 7.9, 0.8 mm Na2-EDTA, 0.15 mm Na2-EGTA, 0.8 mm dithiothreitol, 0.15 mm polymethanesulfonyl fluoride, mm MgCl2, 0.25 mm ZnCl2, 150 lgỈmL)1 of sonicated salmon sperm DNA, 8000–80000 cpm of labeled probe (1–10 ng), and 2–20 lg of nuclear extracts In the competition experiments, unlabeled competitors were added to reaction mixture together with labeled probe as indicated in the figure legends Southwestern assay The procedure was performed using a previously described protocol [35] Briefly, crude nuclear protein extracts were separated by electrophoresis on 10% SDS polyacrylamide gels [36] and transferred onto nitrocellulose membrane (0.4 lm; Amersham) via a semidry transfer unit The proteins on the membrane were denatured with m guanidine hydrochloride and then renatured by incubation in decreasing concentrations of guanidine hydrochloride The nitrocellulose membranes were then soaked in a Southwestern buffer (100 mm NaCl; 10 mm Hepes, pH 7.9; mm Na2EDTA; mm dithiothreitol; mm MgCl2; 0.25 mm ZnCl2; 15 lgỈmL)1 of sonicated salmon sperm DNA; 5% nonfat dry milk) for h at room temperature Labeled probe (105 cpmỈmL)1) was added and incubated for h at room temperature The membrane was washed to remove nonspecifically bound DNA and subjected to autoradiography The relative molecular sizes of the proteins bound by the probe were determined by comparison with KaleidoscopeTM prestained protein standards (Bio-Rad, Hercules, CA, USA) 4858 Sergey V Orlov et al Purification of the rpL32 fragment ()24 +11)binding proteins Protein isolation was performed using a four-step purification procedure consisting of preparative EMSA followed by three-step DNA affinity chromatography Crude nuclear extract obtained from · 1012 HepG2 cells was subjected to preparative EMSA The binding reactions were performed under the same conditions as an analytical EMSA (see above), but contained 1–3 · 106 cpm of 32 P-labeled rpL32 fragment ()24 +11) as a probe Electrophoretic separation of DNA-protein complexes was carried out in 5% nondenaturating polyacrylamide gels using V P Kalinovski’s device for preparative gel electrophoresis [37] Two-milliliter fractions were eluted in flowing Tris-borate buffer (with a flow rate of approximately 12 mLỈh)1) Fractions containing DNA–protein complexes were detected by measuring their radioactivity on a scintillation counter (Beckman Coulter Inc., Fullerton, CA, USA), concentrated and equilibrated in the EMSA binding buffer by ultrafiltration in Centricon YM-10 centrifugal filter units (Millipore, Bedford, MA, USA; catalog number 4205) Sorbent for DNA affinity chromatography was prepared as follows The rpL32 fragment ()24 .+11) was extended by oligo(T), biotin end-labeled with terminal transferase (Roche Applied Science) and immobilized on streptavidin-coated magnetic beads (Roche Applied Science, catalog number 1641778) in accordance with the manufacturer’s guidelines Partially purified proteins obtained in the previous stage were incubated with the rpL32 fragment-coated beads in binding buffer (100 mm NaCl, 10.5 mm Hepes, pH 7.9, 0.8 mm Na2-EDTA, 0.15 mm Na2-EGTA, 0.8 mm dithiothreitol, 0.15 mm polymethanesulfonyl fluoride, mm MgCl2, 0.25 mm ZnCl2, 150 lgỈmL)1 of sonicated salmon sperm DNA) for h at °C, washed three times in binding buffer, and the rpL32 binding proteins were eluted from the beads in m NaCl binding buffer Next, eluted fractions were equilibrated in binding buffer by ultrafiltration, as described above, and a second round of DNA affinity chromatography was performed Finally, the double stranded oligonucleotides containing the lactoferrin binding site were immobilized on magnetic beads as described for the rpL32 fragment and used as a nonspecific substrate to remove contaminant nonspecific DNA-binding proteins The unbound proteins were considered to be the final fraction enriched with the rpL32 fragment ()24 .+11) binding proteins That fraction was analyzed by SDS electrophoresis and compared with the fraction captured by the lactofferin binding site containing substrate (negative control) MALDI-TOF assay Proteins separated by one-dimensional gel electrophoresis were in-gel digested as described elsewhere [38] Samples FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works Sergey V Orlov et al were prepared using the dried-droplet method Peptide samples (0.2–1 mL) were mixed with an equal volume of 2,5-dihydroxybenzoic acid solution (20 mgỈmL)1, Sigma) containing 20% acetonitrile and 0.1% trifluoroacetic acid, and the resulting droplets were air dried Mass spectra of trypsin-digested proteins were obtained for the mass range of 500–2000 Da using a Reflex III MALDI-TOF mass spectrometer (Bruker, Ettlingen, Germany) The mass spectrometer was operated in the reflector mode using previously described settings [39] Mass spectra were calibrated using known masses of internal standards [40] Peptide peak lists were composed using bruker data analysis software (Bruker Daltonik, Bremen, Germany) Proteins were identified by the sets of proteolytic peptide masses using the Peptide Fingerprint option in Mascot software (http://www.matrixscience.com/home html) The accuracy of MH+ mass determination was 0.02% and the possible modification of cysteine residues by acrylamide and methionine oxidation was taken into consideration Isolation of human ZF5 cDNA and construction of ZF5 expression vectors 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 primers, 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¢) spanned a 686 bp 3¢-part of the ZF5 coding region Those two overlapping parts of ZF5 cDNA were amplified separately, digested by BglI, ligated and reamplified by outer primers (5¢-primer from first pair and 3¢-primer from second pair) The resulting fragment was subcloned into pBluescript vector, and the ZF5 sequence was verified by sequencing to exclude mutations that could have occurred during amplification The ZF5 eukaryotic expression vector with a full coding region of ZF5 driven by a CMV promoter (pCMVZF5) was then constructed ZF5 cDNA was digested from pBluescript by XhoI, NotI, and inserted into a pDSRed-N1 plasmid (ClonTech, Palo Alto, CA, USA) digested by SalI and NotI in place of the red fluorescent protein gene A novel human (GCC)n-binding protein Purification of recombinant GST-ZF5 fusion protein Escherichia coli strain BL21 bearing the prokaryotic expression vector pGEXZF5 encoding recombinant fusion protein, which consists of glutathione S-transferase (N-terminal part) and the DNA-binding domain of ZF5 (C-terminal part), was kindly provided by Dr S Yamamoto, Hiroshima University Bacteria were grown at 37 °C to the late logarithmic phase (D600 nm ¼ 1) and GST-ZF5 expression was induced by addition of mm isopropyl-bd-galactothiopyranoside The cells were harvested after additional incubation for h, and the recombinant protein was purified by standard glutathione-agarose bead binding procedures Cell transfection, b-galactosidase and luciferase assays HepG2 cells were seeded on 35 mm culture dishes at a density of 104 cellsỈcm)2 and grown to a subconfluent layer in Dulbecco’s modified Eagle’s medium containing 10% 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 per hour Luciferase activity was measured in a scintillation counter (Beckman Coulter, Fullerton, CA, USA) employing a Luciferase Assay System (Promega, catalog number E4030) in accordance with manufacturer’s guidelines Luciferase activity is indexed in relative light units that correspond to counts per per mg of total protein in cell lysates Protein concentration in cell lysates was measured by the Bradford 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¢) matched bases 333–351 of the ZF5 mRNA sequence (accession number D89859) 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 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works 4859 A 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 followed by gradual cooling to room temperature Transfection of HepG2 cells by siRNAs was performed by RNAiFect reagent (Qiagen, Hilden, Germany) or 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 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 GGCTCT-3¢ and 5¢-TGCTGGCTGCTTTTAGGAA-3¢ (27 cycles) were determined by preliminary PCR experiments All probes were normalized relative to GAPDH Statistical analysis The results are presented as the mean ± SEM The statistical analyses of group differences were performed using a nonpaired t-test P < 0.05 was considered statistically significant All statistical analyses were performed using the program statistica 5.0 (StatSoft, Inc., Tulsa, OK, USA) Acknowledgements We thank D Issakov for helpful discussion and proofreading, and S Yamamoto for supplying bacteria expressing the recombinant GST-ZF5 protein This work was supported by the Russian Fund for Basic Research (grants 00-04-49426, 04-04-48691, 06-0448714) and by the Higher Education Ministry of the Russian Federation (grant PD02-1.4-385) References Caskey CT, Pizzuti A, Fu YH, 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CD & Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression Nat Rev Mol Cell Biol 4, 457–467 4862 Sergey V Orlov et al 44 Hetz C, Russelakis-Carneiro M, Walchli S, Carboni S, ă Vial-Knecht E, Maundrell K, Castilla J & Soto C (2005) The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity J Neurosci 25, 2793–2802 FEBS Journal 274 (2007) 4848–4862 Journal compilation ª 2007 FEBS No claim to original Russian government works ... 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... human (GCC)n-binding protein Fig Identification of p56 polypeptide as Kruppel-like transcription factor ZF5 by MALDI-TOF assay (A) Fragment of MALDI-TOF spectrum of ă p56- derived peptides (B) Mascot... FMR1 gene These data clearly support the assertion that endogenous ZF5 acts as a transcriptional repressor of the human FMR1 gene Given that FMR1 inactivation in FMR patients has been attributed