Báo cáo khoa hoc Multiple NF-Y-binding CCAAT boxes are essential for transcriptional regulation of the human C7orf24 gene, a novel tumor-associated gene

Multiple NF-Y-binding CCAAT boxes are essential for transcriptional regulation of the human C7orf24 gene, a novel tumor-associated gene Yuji Ohno1, Akira Hattori1, Masamichi Ueda2, Susumu Kageyama3, Tatsuhiro Yoshiki4 and Hideaki Kakeya1

1 Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan 2 Institute for Virus Research, Kyoto University, Japan 3 Department of Urology, Shiga University of Medical Science, Japan 4 Department of Clinical Oncology, Kyoto Pharmaceutical University, Japan

Keywords C7orf24; CCAAT box; NF-Y; tumor biomarker

Correspondence A. Hattori & H. Kakeya, Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan Fax: +81 75 753 4591 Tel: +81 75 753 9267 & +81 75 753 4524 E-mail: ahattori@pharm.kyoto-u.ac.jp & scseigyo-hisyo@pharm.kyoto-u.ac.jp

(Received 28 June 2011, revised 12 August 2011, accepted 22 August 2011)

doi:10.1111/j.1742-4658.2011.08314.x

Human chromosome 7 ORF 24 (C7orf24) has been identified as a tumor- related protein, and shown to be a c-glutamyl cyclotransferase. In the cur- rent study, we characterized the promoter region of the human C7orf24 gene to explore the transcriptional regulation of the gene. We revealed that the human C7orf24 promoter is a TATA-less promoter, containing five CCAAT boxes aligned in a forward orientation. By performing a luciferase reporter assay with 5¢-deleted and site-directed mutated constructs in HeLa, MCF-7 and IMR-90 cells, we found that three proximal CCAAT boxes are important for basal transcription. Electrophoretic mobility gel shift assay and chromatin immunoprecipitation assay demonstrated that NF-Y specifically bound to all three CCAAT boxes. In addition, the mRNA and protein expression levels of C7orf24 were significantly reduced in HeLa cells depleted of NF-YB, a subunit of NF-Y. These results suggested that NF-Ys bound to the three proximal CCAAT boxes play a central role in the transcription of the gene. Furthermore, as in the case of other genes transcribed under the control of multiple NF-Ys, such as human E2f1 and cyclin b1, the C7orf24 gene expression profile oscillated during the cell cycle, implying that C7orf24 is a novel cell cycle-associated gene.

Introduction

Abbreviations ARE, antioxidant-responsive element; C7orf24, chromosome 7 ORF 24; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility gel shift assay; siRNA, small interfering RNA.

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By comparison of protein expression profiles between bladder urothelial carcinoma tissue and normal urothe- lium tissue obtained from patients, Kageyama et al. [1] identified 15 cancer-specific spots that could constitute a diagnostic marker for bladder cancer. They demon- strated that one of the spots was a hypothetical gene product of chromosome 7 ORF 24 (C7orf24), the function of which has not been determined. They also found that ectopic expression of C7orf24 promoted the proliferation of murine normal fibroblasts, and silenc- ing of the gene by small interfering RNA (siRNA) showed an antiproliferative effect on cancer cell lines, suggesting a role of C7orf24 in cell proliferation. In addition, it was reported that C7orf24 was expressed in a range of cancers, such as prostate and breast can- cers [2–6].

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forward orientation. Several potential regulatory cis- elements bound by transcription factors, such as Nrf-2 (antioxidant response factor) and Sp1, were also identi- fied within this region [11].

On the other hand, C7orf24 was identified as an apoptosis-induced factor by Masuda et al. [7]. By mon- itoring cytochrome c-releasing activity in a cell-free system, they purified C7orf24 from the cytosol fraction of geranylgeraniol-treated U937 cells. They also reported that ectopic expression of C7orf24 in HeLa cells resulted in the release of cytochrome c from mito- chondria, which, in turn, promoted apoptosis. These results suggested that C7orf24 plays a role in the apop- totic pathway induced by geranylgeraniol.

cysteine

To determine the regulatory elements mediating the activation of C7orf24 gene transcription, we initially performed a luciferase reporter gene assay with HeLa cells, which express large amounts of C7orf24. When the the reporter plasmid harboring the 2127 bp of C7orf24 promoter was introduced, a marked increase in luciferase activity was found (> 40-fold increase in pGL3-Basic-transfected cells) (Fig. 2A, construct a). In contrast, when the cells were transfected with con- struct k harboring the full-length 5¢-flanking region in an inverted orientation, its luciferase activity was com- parable to that of the promoter-less pGL3-Basic-trans- fected cells (construct j). These results indicated that this (cid:2) 2.1-kbp region contains a functional promoter

functional Recently, Oakley et al. [8] also identified C7orf24 as a c-glutamyl cyclotransferase. They then characterized recombinant C7orf24, and showed that it converted c-glutamyl to 5-oxoproline and cysteine, suggesting that C7orf24 may play an essential role in glutathione homeostasis. Furthermore, they resolved the crystal structure of human C7orf24 and showed a structural similarity between the protein and BtrG, a c-glutamyl cyclotransferase of Bacillus circulans, sup- porting their relationship as c-glutamyl cyclotransferases [9].

Although these findings suggested a biological signif- icance of C7orf24, its definitive role in cellular function remains unclear. In this study, we investigated the reg- ulatory mechanism of the human C7orf24 gene to gain basic insights into the physiological relevance of the gene product. By performing a luciferase reporter gene assay, we characterized the promoter region of the human C7orf24 gene. We found that three CCAAT boxes located proximally to the initiator element are functional, and that NF-Y plays an essential role in basal transcription of the gene through binding to these CCAAT boxes.

Results

Characterization of the human C7orf24 gene promoter

consensus initiator

Fig. 1. The 5¢-flanking region of the human C7orf24 gene. (A) Sche- matic structure of the human C7orf24 promoter. The thin line and a closed box denote the 5¢-flanking region and exon 1 of the gene, respectively. The CCAAT box and ARE-like motif are depicted as open and hatched boxes, respectively. Nucleotide sequences around the distal CCAAT motifs (C4 and C5) and ARE-like motif are shown below. (B) Nucleotide sequence of the proximal promoter region of the human C7orf24 gene. The exon sequence is shown in upper-case, and that of the untranscribed region is given in lower-case. The transcriptional initiation site is shown as +1. Tran- scription factor-binding sites were searched for with TRANSFAC [37], and are underlined. Arrowheads indicate the start points of the constructs used for the luciferase reporter gene assay.

[10].

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To characterize the 5¢-flanking region of the human C7orf24 gene, we referred to the nucleotide sequence deposited in GenBank (accession no. NW_001839003). We obtained the 2127-bp fragment of the 5¢-flanking region of the gene by genomic PCR, cloned it into the pGL3-Basic reporter plasmid, and then confirmed the sequence by DNA sequencing. This region contains a sequence (CCAGTCC) that perfectly matches the pyrimidine-rich sequence, in which Py represents a PyPy(A+1)N(T ⁄ A)PyPy, pyrimidine residue (Fig. 1) In addition, five CCAAT boxes were located in this flanking region, whereas no canonical TATA box was found. It is notable that all of the CCAAT boxes are aligned in a

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containing 5¢-flanking

that regulates C7orf24 gene expression. Subsequently, we generated sequential 5¢-deletion constructs and examined their promoter activities to identify the prox- imal promoter region (constructs b–i). Although sub- stantial promoter activity was detected when chimeric constructs sequence the upstream from position )135 (constructs b–d) were transfected, it gradually decreased in proportion to the insert size (constructs e–h). In cells transfected with construct i, no significant increase in luciferase activity was observed. These results suggested that a minimal promoter region is located (cid:2) 58 bp distal from the ini- tiator. We next examined the promoter activity of

5¢-flanking region resulted in a gradual reduction in C7orf24 promoter activity, and the activity was com- pletely abolished in construct h. A similar promoter activity profile was obtained in IMR-90 cells, although the maximum promoter activity was apparently lower than that in HeLa and MCF-7 cells. It was noted that constructs a and b showed comparable promoter activ- ity in all cells tested, and similar promoter activity pro- files were obtained for all cells, despite different expression levels of C7orf24 protein. Collectively, these results indicated that the minimal promoter essential for basal transcription of the C7orf24 gene is located between positions )58 and +14 from the initiation site, and that the proximal upstream region may be involved in enhancement of human C7orf24 gene expression.

Fig. 2. Significance of the CCAAT boxes in the human C7orf24 promoter. (A) Reporter plasmids harboring sequentially deleted fragments of the C7orf24 promoter were transfected into HeLa, MCF-7 and IMR-90 cells. Firefly luciferase activity was measured as described in Experi- mental procedures, and normalized to the Renilla luciferase activity of a cotransfected internal control plasmid, pCMV-Renilla. (B) The lucifer- ase reporter assay was conducted with the plasmids harboring a mutation at the CCAAT motif of the C7orf24 promoter in HeLa, MCF-7 and IMR-90 cells. A schematic representation of the reporter constructs is shown on the left. Open boxes indicate intact CCAAT boxes, and crossed boxes indicate mutated CCAAT boxes (CCAAA sequence). Relative promoter activity is expressed as fold increase as compared with that of the promoter-less pGL3-Basic vector. Error bars are the standard errors of quadruplicate results. Similar results were obtained in five separate experiments. LUC, luciferase.

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the C7orf24 gene 5¢-flanking region in human breast carci- noma MCF-7 cells (which express large amounts of C7orf24) and human diploid fibroblast IMR-90 cells (which barely express C7orf24) (Figs 2A and S1). When construct a or construct b was transfected into MCF-7 cells, a significant increase in luciferase activity was observed. Elimination of the distal part of the As shown in Fig. 1A, the human C7orf24 promoter contains a putative antioxidant-responsive element (ARE) [12]. In addition, it has been suggested that C7orf24 is involved in glutathione homeostasis though c-Glu-Cys hydrolysis [8]. We thus investigated the role

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(Fig. 3B). Taken together,

its band was observed in each probe. Subsequently, binding specificity was examined by employing an excess amount of unlabeled oligoprobe. A 100-fold molar excess amount of unlabeled probe completely inhibited the formation of complexes between nuclear extracts and radiolabeled probes. When EMSA was carried out with mutant oligonucleotide probes, replacing the CCAAT motifs with the CCAAA sequences, no retarded band was detected in any mutant probes these results indicated the sequence-specific formation of a CCAAT motif-protein complex.

of the element in C7orf24 gene expression. As it is well known that a transcription factor, Nrf-2, binds to the ARE and plays an essential role in the antioxidant response [13,14], we explored the effect of Nrf-2 activa- tion on C7orf24 promoter activity. When HeLa cells transfected with construct a were exposed to t-butyl hydroquinone, an Nrf-2 activator [15], no difference was observed in luciferase activity between Nrf-2-acti- vated cells and control cells (Fig. S2A). Furthermore, although the mRNA level of a well-known antioxidant response gene, heme oxygenase-1 (HMOX1), was mark- edly elevated by t-butyl hydroquinone treatment [16], no change was observed in C7orf24 mRNA levels, indi- cating that oxidative stress did not affect C7orf24 gene expression (Fig. S2B).

Significance of the three proximal CCAAT boxes in the human C7orf24 gene promoter

NF-Y is one of the major CCAAT-binding proteins, and is also referred as CCAAT-binding factor [17]. It is also known that NF-Y is not able to bind to the CCAAA sequence, because the protein is the only CCAAT-binding factor that absolutely requires the pentanucleotide sequence CCAAT for DNA sequence recognition [18,19]. Hence, we examined the binding of NF-Y to the three CCAAT boxes (C1–C3) in the proximal promoter of the gene. When a mAb against subunit A of NF-Y (NF-YA) was employed, a super- shift of the probe–protein complex was detected in each probe (Fig. 3A), suggesting the binding of NF-Y to each CCAAT box. Specific binding of NF-Y to each CCAAT box of the C7orf24 proximal promoter was also observed in IMR-90 cells (Fig. 3A,B). We further evaluated direct

these findings suggest

interactions between NF-Y and CCAAT boxes of the C7orf24 gene pro- moter in vivo with a chromatin immunoprecipitation (ChIP) assay. As shown in Fig. 3C, the proximal C7orf24 promoter fragment containing three CCAAT boxes (C1–C3) ()179 to )19) was specifically amplified from the immunoprecipitate with antibody against NF-YB, but not control IgG. Furthermore, no signifi- cant amplification of the distal promoter of the gene either with C4 and C5 CCAAT boxes ()1218 to )1088) or without any CCAAT box ()1577 to )1467) was observed in either immunoprecipitate. These data strongly indicated the in vivo recruitment of NF-Y to the proximal promoter region of the gene.

The proximal promoter sequence from )205 to )35 contains three CCAAT sequences; therefore, we next explored the significance of these motifs in C7orf24 gene transcription. We made a mutation in the proxi- mal CCAAT boxes (C1, C2, and C3) to generate a CCAAA sequence on the reporter plasmid a, and mea- sured promoter activity in HeLa and MCF-7 cells (Fig. 2B). Although a mutation at a single CCAAT site did not show any effect on luciferase activity in either type of cell, when mutations were introduced into two CCAAT sequences, C7orf24 promoter activity was significantly decreased, except for the combination of C1 and C3 (construct a-mcm). Strikingly, mutations in both C1 and C2 (construct a-cmm) caused a marked decrease in C7orf24 promoter activity in HeLa cells. When all three CCAAT motifs were replaced with CCAAA sequences, a further decrease in promoter activity was observed in both types of cell. Collec- the significance of all tively, three CCAAT boxes in C7orf24 gene expression. The importance of all three proximal CCAAT boxes was further demonstrated in IMR-90 cells. It was noted that construct a-cmc, harboring a mutation only in C2, showed reduced promoter activity in the cells. Involvement of NF-Y in C7orf24 gene expression

Binding of NF-Y to CCAAT boxes in the proximal promoter of the human C7orf24 gene

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For further evaluation of the involvement of NF-Y in C7orf24 gene transcription, we examined promoter activity in cells depleted of NF-YB. By gene silencing through an RNA interference mechanism, the NF-yb mRNA level was decreased to nearly 15% (48 h after transfection) in NF-yb-specific siRNA (NF-YB-1 and NF-YB-2)-transfected HeLa cells (Fig. 4A). In NF-yb- eliminated cells, the mRNA level of C7orf24 declined to nearly 61% of that of mock-transfected cells. With We next investigated the interaction of trans-acting factor(s) on proximal CCAAT boxes in the C7orf24 gene promoter by electrophoretic mobility gel shift assay (EMSA). As shown in Fig. 3A, when the oli- goduplex probe for each CCAAT box was incubated with nuclear extracts of HeLa cells, a single retarded

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Fig. 3. NF-Y binds to all proximal CCAAT boxes of the human C7orf24 promoter. Nuclear extracts prepared from HeLa and IMR-90 cells were incubated with radiolabeled probes. Sequence-specific interactions between nuclear extracts and radiolabeled probes were analyzed in a 4% native polyacrylamide gel. (A) An excess amount (100-fold) of the unlabeled probe was employed for competition. For the supershift assay, antibody against NF-YA was added to the reaction mixture and incubated for 2 h at 4 (cid:2)C before addition of labeled probes. (B) EMSA was carried out with mutated oligonucleotide probes. W, probe harboring an intact CCAAT sequence; M, invalid probe harboring a CCAAA sequence. (C) Cross-linked chromatin complexes were immunoprecipitated with antibody against NF-YB or normal rabbit IgG. The amounts of precipitated DNA fragments derived from the C7orf24 promoter, i.e. )179 to )19 containing C1–C3 (closed bar), )1218 to )1088 contain- ing C4 and C5 (gray bar), and )1577 to )1467 lacking a CCAAT box (open bar), were measured by quantitative PCR. Data are expressed as a percentage of the input DNA with the standard errors of quadruplicate results. Similar results were obtained in three separate experi- ments. Ab, antibody.

regard to the E2f1 gene, which is known to be tran- scribed in an NF-Y-dependent manner [20], elimina- tion of NF-YB reduced E2f1 mRNA expression to a similar degree as observed for the C7orf24 mRNA level ((cid:2) 68% of control siRNA-transfected cells). In accordance with the parallel reduction in Nf-yb and C7orf24 mRNAs, C7orf24 protein expression was shown to be significantly decreased in Nf-yb-eliminated HeLa cells (Fig. 4B). These results strongly suggest that NF-Y plays an important role in transcriptional regulation of the human C7orf24 gene.

Oscillation of C7orf24 gene expression during cell cycle progression

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(CCNB1), is mediated through the promoter harboring multiple CCAAT boxes that are bound by NF-Y [20– 22]. Therefore, we analyzed the gene expression profile of human C7orf24 with M phase-synchronized HeLa cells. After release from thymidine ⁄ nocodazole block- ade, the C7orf24 mRNA level gradually increased from the late stage of the G1 phase and peaked in the mid- S phase (9–12 h after release) (Figs 5A and S3A). Although the expression level of each gene varied, this cell cycle-dependent profile was similar to that of the E2f1 gene, a typical S phase gene, but not of cyclin b1, a G2 ⁄ M phase gene [20,21]. Moreover, dynamic recruitment of NF-Y to the C7orf24 promoter was observed at late G1 ⁄ early S phase, followed by an ele- vation of the C7orf24 mRNA level (Fig. 5B). Fig- ures 5C and S3B also show the cell cycle-dependent oscillation of C7orf24 mRNA expression in IMR-90 It has been reported that the transcription of some cell cycle-associated genes, such as E2f1 and cyclin b1

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the human transcriptional regulatory mechanism of C7orf24 gene. In this study, we identified and charac- terized the promoter region of the human C7orf24 gene for the first time. Analysis of the human C7orf24 promoter sequence showed that it is a TATA-less pro- moter, but that it contains five CCAAT boxes aligned in a forward orientation within an (cid:2) 2.1-kb 5¢-flanking region of the gene. We found that the three proximal CCAAT boxes function in the regulation of C7orf24 gene expression.

Fig. 4. Involvement of NF-Y in the regulation of C7orf24 gene expression. HeLa cells were transfected with siRNA (50 nM) spe- cific for Nf-yb (NF-YB-1 and NF-YB-2) or green fluorescent protein (GFP) (control). (A) After 48 h of siRNA transfection, mRNA levels of Nf-yb (open bar), C7orf24 (closed bar) and E2f1 (gray bar) were measured by quantitative RT-PCR, and normalized to the level of 18S rRNA, which is transcribed by RNA polymerase I. Relative mRNA levels are shown as ratios against those of the control siRNA-transfected cells. Error bars are the standard errors of qua- druplicate results. Similar results were obtained in three separate experiments. (B) C7orf24 protein levels (at 72 h after siRNA trans- fection) were examined by western blot analysis with antibodies against NF-YB, C7orf24, and a-tubulin. The values below represent expression levels of the proteins in Nf-yb-depleted cells relative to those in control siRNA-transfected cells.

three contains

cells. These results imply that C7orf24 is an S phase gene and that NF-Y plays a central role in transcrip- tional regulation of the gene.

Discussion

It was estimated that the CCAAT box is present in (cid:2) 30% of eukaryote genes [23]. It was recently pro- posed that the CCAAT box is not coupled with the TATA box in various promoters, and is frequently found in close proximity to ((cid:2) 100 bp) the RNA poly- merase II initiation site [24]. The CCAAT box is typi- cally found as a single-copy element; however, multiple NF-Y-binding CCAAT motifs are found in a few TATA-less promoters, especially in cell cycle-regulated gene promoters such as E2f1, cyclin b1, and Cdc25 [20–22,24,25]. Given our finding that the TATA-less C7orf24 gene promoter functional CCAAT boxes in close proximity to the initiation site, it is tempting to speculate that C7orf24 is a cell cycle- associated gene. Consistent with this hypothesis, we demonstrated that the mRNA expression of the human C7orf24 gene did indeed oscillate during the cell cycle, and that its profile was similar to that of E2f1 mRNA in synchronized HeLa cells (Fig. 5) [22]. In addition, on the basis of ectopic expression and gene-silencing experiments, it was proposed that C7orf24 might be involved in cancer cell proliferation [1]. Thus, the func- tion and relevance of C7orf24 in cell cycle progression are important issues to be addressed in a future study. NF-Y is one of the major transcriptional factors binding to the CCAAT box, and is involved in the transcription of many types of genes, i.e. inducible or constitutive genes, tissue-specific or ubiquitous genes, and housekeeping genes [26,27]. NF-Y is a heterotri- metric protein composed of NF-YA, NF-YB, and NF-YC. Because NF-YB and NF-YC contain a his- tone-like motif (a histone 2B-like or histone 2A-like motif, respectively), this NF-Y complex is able to inter- act with the core octamer of histones in the nucleosome, resulting in distortion of the chromatin structure fol- lowed by transcriptional initiation [28,29]. Otherwise, NF-Y binding to the CCAAT box may interact with other trans-acting factors, such as Sp1, E2F1, and so on, to regulate gene transcription [20,30–33].

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Recent studies have revealed that the expression of human C7orf24 is highly upregulated in several differ- ent types of cancer, but not in normal tissues [2–4]. Currently, C7orf24 is thought to be a promising tumor to clarify the biomarker; is important therefore, it By EMSA, we showed that NF-Y bound to all three functional CCAAT boxes (C1–C3). The results were also supported by considerable matches of CCAAT- centered sequences in the promoter to an NF-Y-binding

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Fig. 5. Cell cycle-dependent expression of C7orf24 mRNA. (A) HeLa cells were synchronized in metaphase with a single thymidine block (2.5 mM, 20 h) followed by a nocodazole block (100 ngÆmL)1, 12 h). Total RNA was recovered from the cells at the indicated times after release from nocodazole arrest. mRNA levels of C7orf24, E2f1 and cyclin b1 were quantified by quantitative PCR, and normalized to the 18S rRNA level. The mRNA level at each period after arrest release is shown as a ratio against that of metaphase-synchronized cells (0 h). Error bars are the standard errors of quadruplicate results. Similar results were obtained in three separate experiments. (B) Crosslinked chromatin complexes were prepared from HeLa cells at the indicated times after release from nocodazole arrest. The ChIP assay was carried out with antibody against NF-YB or normal rabbit IgG. The precipitated C7orf24 proximal promoter (between )179 and )19) was measured by quanti- tative PCR. Data are expressed as a percentage of the input DNA for each sample, with the standard errors of quadruplicate results. Similar results were obtained in three separate experiments. (C) IMR-90 cells were synchronized into G0 phase by serum deprivation for 48 h. mRNA levels of C7orf24 at the indicated times after release from serum starvation were quantified by quantitative PCR, and normalized to 18S rRNA levels. The mRNA level at each period after arrest release is shown as a ratio against that of G0-synchronized cells (0 h). Error bars are the standard errors of quadruplicate results. Similar results were obtained in three separate experiments.

sequence, 5¢-YYRRCCAATCAG-3¢ [17,28]. It

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(C1, consensus 91.7%; C2, 83.3%; C3, 75.0%) is well known that the action of NF-Y-mediated transcription is influenced by the position of the CCAAT box on a it has also been shown promoter [17]. Furthermore, that TFIID is recruited onto a TATA-less promoter through the interaction with TATA-binding protein(s) associated with NF-YB and ⁄ or NF-YC [34]. Given that the most proximal CCAAT box of the human C7orf24 gene promoter (C1) is only 50 bp from the initiator, it is plausible that C1 acts as a ‘landmark’ for recruitment of the general transcriptional machin- ery to the initiator of the gene. Indeed, in the current study, substantial promoter activities of reporter plas- mids, construct g and construct a-mmc were found in HeLa and MCF-7 cells (Fig. 2), indicating the signifi- cance of the NF-Y-binding C1 for C7orf24 gene tran- scription. Likewise, NF-Y bound to C2, which is located within 90 bp upstream from the initiator, is able to activate the general transcriptional machinery. On the other hand, the spatial relationship between the NF-Y-binding CCAAT and other cis-elements has also been shown by parallel bioinformatic analysis and bio- chemical studies [24]. For example, by computer analy- sis, consecutive NF-Y-binding sites separated by three to five DNA helix turns have generally been found in promoters with multiple CCAAT boxes, and the func- tional interaction between the neighboring NF-Ys has been experimentally elucidated in a subset of the genes, such as cyclin b1. Accordingly, as the distance between

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the human C7orf24 gene and examined its role in tran- scription of the gene; however, t-butyl hydroquinone, an activator of Nrf-2, did not show any effects on C7orf24 gene expression, indicating that C7orf24 is not an antioxidant response gene. Further study is required for better understanding of the multifunctionality of C7orf24.

this is one of

In conclusion, we have revealed the gene regulatory mechanism of the human C7orf24 gene for the first time. We have demonstrated that NF-Ys bound to the three CCAAT boxes in the proximal region of the C7orf24 promoter and plays a central role in transcrip- tion of the gene. The promoter structure and regulatory mechanism of the gene support the cell cycle-dependent oscillation of C7orf24 gene expression. Thus, the results obtained this study may provide basic and valuable insights into the physiological and pathological signifi- cance of C7orf24. C1 and C2 of the C7orf24 gene is 40 bp, correspond- ing to four turns of the double nucleotide helix, it is possible to speculate that there is an interaction between NF-Y binding to both CCAAT boxes, result- ing in enhanced transcription of the gene; however, we were not able to rule out the possibility of other NF-Y–NF-Y interactions among the three proximal CCAAT boxes of the C7orf24 gene promoter. It is also well known that NF-Y cooperates with another tran- scriptional factor, Sp1, which binds to the GC box, resulting in activation of gene transcription. In the human C7orf24 gene promoter, the GC box is located the typical 15 bp upstream of C3; distances between the two elements [24], suggesting an interplay between NF-Y binding to C3 and to Sp1 to accelerate C7orf24 gene transcription. It should be noted that, even in other multiple CCAAT promoters, such as G2 ⁄ M gene promoters, NF-Y plays a central role in transcriptional regulation [21,22].

Experimental procedures

Mouse monoclonal anti-NF-YA IgG (sc-17753) and rab- bit polyclonal anti-NF-YB IgG (sc-13045) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti- a-tubulin IgG (T5168) was from Sigma-Aldrich (St Louis, MO, USA). Mouse monoclonal anti-C7orf24 IgG was prepared by immunizing mice with recombinant human C7orf24 [1].

Materials

Cell culture

All human cells were purchased from the American Tissue Culture Collection (Manassas, VA, USA). Normal lung fibroblasts, IMR-90 cells, cervical carcinoma HeLa cells and breast adenocarcinoma MCF-7 cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum. These cells were cultured in a humidified 5% CO2 ⁄ 95% air incubator at 37 (cid:2)C.

C7orf24 was originally identified as a bladder cancer- specific protein, and is now recognized as a good candidate for a diagnostic marker [1,4]. Similarly, we also found that C7orf24 gene expression (in mRNA and protein levels, respectively) is of higher amplitude in cancer cell lines but not in normal diploid fibro- blasts (Fig. S1). Despite the variable expression level of C7orf24, similar promoter activity profiles were obtained for all cells tested in the reporter assay with 5¢-deletion constructs (Fig. 2A). In the luciferase assay with CCAAT-mutated constructs, however, a cell-type- specific difference in NF-Y-mediated transcription of the C7orf24 gene was observed (Fig. 2B, constructs a-cmc and a-mcm). Hence, more comprehensive under- standing of the transcriptional regulatory mechanism of the C7orf24 gene, focusing on NF-Y-interacting fac- tor(s) or the epigenetic state of the C7orf24 promoter, may reveal the physiological and pathological signifi- cance of the gene and ⁄ or its usefulness as a diagnostic marker.

A series of fragments of the 5¢-flanking region of the human C7orf24 gene containing a putative transcriptional initiation site was amplified by PCR from genomic DNA of MCF-7 cells, and cloned into the HindIII site of a promoter-less reporter plasmid, pGL3-Basic vector (Promega, Madison, WI, USA). Mutagenesis of the three proximal CCAAT sequences was performed with a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Oligonu- cleotide sequences of PCR primers used for the creation of each mutant were as follows: for C1 ()50 to )46), 5¢-GG

Construction of reporter plasmids

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C7orf24 has been shown to be one of the enzymes involved in glutathione metabolism, and hydrolyzes a glutathione metabolite, c-Glu-Cys dipeptide [8]. The expression of a large proportion of glutathione metab- olism enzymes is governed by the Nrf-2 ⁄ Keap-1 pathway [35]. Under basal conditions, Nrf-2 is ubiqui- tinylated by Keap-1, an E3 ligase, and degraded via the ubiquitin–proteasome pathway. Once cells are exposed to oxidative stress, e.g. t-butyl hydroquinone treatment, a conformational change of Keap-1 is induced, resulting in a loss of E3 ligase activity. Nrf-2, translocates which escapes proteolytic degradation, into the nucleus and binds to ARE with c-Maf. We found an ARE-like sequence in the distal promoter of

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Transcriptional regulation of human C7orf24

(sense) ACCGGCAGCCAAACAGGAGAGCGCTCGC-3¢ and 5¢-GCGAGCGCTCTCCTGTTTGGCTGCCGGTCC-3¢ (antisense); for C2 ()90 to )86), 5¢-GTGGGGATGGCC (sense), and 5¢-GAGT AAACAAAGGGGGCGACTC-3¢ (antisense); CGCCCCCTTTGTTTGGCCATCCCCAC-3¢ and for C3 ()174 to )170), 5¢-TAGGTCGTGTCCAAAG (sense) and 5¢-CGGCGCTG AGGAGCCAGCGCCG-3¢ GCTCCTCTTTGGACACGACCTA-3¢ (antisense). The mutated nucleotides are underlined.

20 mm KCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dith- iothreitol, 0.2 mm phenylmethanesulfonyl fluoride, 25% glycerol, pH 7.9). Then, a half-volume of high-salt buffer (20 mm Hepes, 1.4 m KCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 0.2 mm phenylmethanesulfonyl fluo- ride, 25% glycerol, pH 7.9) was added stepwise; the mixture was then incubated at 4 (cid:2)C for 30 min and centrifuged at 21 500 g for 45 min. The supernatant was then collected. Nuclear extracts were incubated with 10 ng of radiolabeled [100 mm KCl, 1 mm oligonucleotide in binding buffer MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 4% glycerol, 0.2 lgÆlL)1 poly(dI-dC) (Roche Diagnostics, Basel, Switzer- land), pH 7.9] for 30 min at 4 (cid:2)C. For the competition assay, a 100-fold excess amount of unlabeled oligonucleo- tide was added to the binding reaction prior to the addition of the radiolabeled probe. For supershift analysis, 1.7 lg of anti-body against NF-YA was added, following the binding reaction, for 2 h at 4 (cid:2)C. These incubation mixtures were then electrophoresed in a 4% native polyacrylamide gel with Tris ⁄ borate ⁄ EDTA buffer. The gel was dried and ana- lyzed with a BAS-5000 image analyzer (Fuji Film, Tokyo, Japan).

Luciferase reporter gene assay

For transfection of reporter plasmids, cells were plated on a 24-well plate and grown for 24 h. Plasmid DNA (500 ng) was mixed with a transfection reagent and transfected into the cells, following the manufacturer’s protocol [HeLa cells, (Invitrogen); MCF-7 cells, LipofectAmine 2000 Reagent cells, LipofectA- IMR-90 FuGENE HD (Promega); mine LTX (Invitrogen)]. A pCMV-Renilla (50 ng) plasmid was employed as an internal control of transfection effi- ciency. After 24 h of transfection, cells were washed three times with NaCl ⁄ Pi and then lysed in reporter lysis buffer (Promega). Firefly and Renilla luciferase activities were then measured with a dual-luciferase reporter assay system (Pro- mega), according to the manufacturer’s instructions. Rela- tive luciferase activity was measured in quadruplicate, averaged, and then normalized to Renilla luciferase activity to correct for transfection efficiency.

ChIP assay

containing

EMSA was conducted in the same manner as described pre- viously [36]. Complementary double-stranded oligonucleo- tides (C1–C3) were each CCAAT box radiolabeled with [32P]ATP[cP] at the 5¢-end by T4 polynu- cleotide kinase (Toyobo, Osaka, Japan) and then purified with a Sigma Spin postreaction purification column (Sigma- Aldrich). Sense sequences of the synthesized oligonucleo- tides used were as follows: for native C1 ()50 to )46), 5¢-GGACCGGCAGCCAATCAGGAGAGCGCTCGC-3¢; for native C2 ()90 to )86), 5¢-GTGGGGATGGCCAAT CAAAGGGGGCGACTC-3¢; for native C3 ()174 to )170), 5¢-TAGGTCGTGTCCAATGAGGAGCCAGCGCCG-3¢; for mutated C1, 5¢-GGACCGGCAGCCAAACAGGAG AGCGCTCGC-3¢; for mutated C2, 5¢-GTGGGGATGGC CAAACAAAGGGGGCGACTC-3¢; and for mutated C3, 5¢-TAGGTCGTGTCCAAAGAGGAGCCAGCGCCG-3¢. The mutated nucleotides are underlined.

1.5 mm MgCl2,

)1088,

Cells were crosslinked with 1% formaldehyde (37 (cid:2)C, 10 min), and this was followed by addition of glycine to 200 mm final concentration for quenching. Chromatin was sheared into fragments in a range between 500 bp and 800 bp by sonication (Bioruptor UCD-250HSA; Cosmo bio, Tokyo, Japan) in SDS lysis buffer (50 mm Tris ⁄ HCl, 10 mm EDTA, 1% SDS, pH 8.0) supplemented with prote- ase inhibitors. Precleared chromatin supernatants were immunoprecipitated with 2 lg of antibody against NF-YB coupled to 20 lL of salmon sperm DNA-blocked protein G sepharose for 2 h at 4 (cid:2)C. Normal rabbit IgG (Vector Lab- oratories, Burlingame, CA, USA) was employed as a control. After immunoprecipitates had been extensively washed with wash buffer 1 (twice) (50 mm Tris ⁄ HCl, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxych- olate, pH 8.0), wash buffer 2 (10 mm Tris ⁄ HCl, 0.5% NP-40, 0.5% sodium deoxycholate, 250 mm LiCl, pH 8.0), and finally twice with TE buffer (10 mm Tris ⁄ HCl, 1 mm EDTA, pH 8.0), the crosslinks were reversed by incubating the beads in 200 mm NaCl at 65 (cid:2)C for 5 h. The immuno- precipitated DNA was purified and subjected to quantita- tive PCR with FastStart Universal SYBR Green Master (Roche Diagnostics) on an ABI StepOne Plus (Applied Bio- systems, Foster City, CA, USA). Oligonucleotide sequences specific for the human C7orf24 promoter were as follows: )179 to )19, 5¢-CGTGTCCAATGAGGAGCCA-3¢ (sense) and 5¢-GTCGAGTCAGGAGCGAGC-3¢ (antisense); )1218 5¢-GAGTAAAGTTGGTGTAATAGTC-3¢ to (sense) and 5¢-CAAACTGAAAAATGATTGGAA-3¢ (anti- sense); and )1577 to )1467, 5¢-GAGCCGACATCAC

Nuclear extracts were prepared as follows. Cells were sus- ice-cold buffer A (10 mm Hepes, pended in 500 lL of 0.5 mm dithiothreitol, 10 mm KCl, 0.2 mm phenylmethanesulfonyl fluoride, pH 7.9) and lysed in a Dounce homogenizer. The nuclei were pelleted by cen- trifugation at 3300 g for 15 min, and then suspended in (20 mm Hepes, an equal volume of

low-salt buffer

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EMSA

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Transcriptional regulation of human C7orf24

and 5¢-TACTCCCTGGGGATTA

(sense) ACCACT-3¢ CTGC-3¢ (antisense).

washed three times with NaCl ⁄ Tris ⁄ Tween, and incubated for 1 h with horseradish peroxidase-conjugated goat anti- body against mouse IgG (Promega), diluted 1 : 10 000 in blocking buffer. After washing of the filter three times with NaCl ⁄ Tris ⁄ Tween, the blot was detected by an enhanced chemiluminescence method with an ECL plus western blot- ting kit obtained from GE Healthcare. The results were visualized with a Fuji LAS-3000.

Silencing of the human Nf-yb gene through RNA interference

Acknowledgements

siRNAs used for the silencing of human Nf-yb gene were obtained from Invitrogen [NF-YB-1:HSS-107170, 5¢-AUC UGUUGUAGAACUGUCACCAUCC-3¢ (sense) and 5¢-GG AUGGUGACAGUUCUACAACAGAU-3¢ (antisense); NF-YB-2:HSS181522, 5¢-UUCUCUUGAUGGCACCUUU CACUUG-3¢ (sense) and 5¢-CAAGUGAAAGGUGCCAU CAAGAGAA-3¢ (antisense)]. Stealth RNAi Reporter Con- trol Duplexes for green fluorescent protein (Invitrogen) were used as a control. Transfection of siRNA into HeLa cells was carried out with TransIT-TKO transfection reagent, according to the manufacturer’s instructions (Mirus Bio, Madison, WI, USA). The silencing effect was evalu- ated by monitoring Nf-yb gene expression at both the mRNA (48 h after transfection) and protein (72 h after transfection) levels.

We are grateful to E. Moriyoshi for technical assis- tance with the quantitative PCR study. This work was supported in part by research grants from the Japan Society for the Promotion of Science, the Ministry of Education, Culture, Sports, Science and Technology of Japan and the 3rd Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor and Welfare of Japan.

References

1 Kageyama S, Iwaki H, Inoue H, Isono T, Yuasa T,

(antisense);

for human C7orf24,

Nogawa M, Maekawa T, Ueda M, Kajita Y, Ogawa O et al. (2007) A novel tumor-related protein, C7orf24, identified by proteome differential display of bladder urothelial carcinoma. Proteomics Clin Appl 1, 192–199. 2 Zhang C, Li HR, Fan JB, Wang-Rodriguez J, Downs T, Fu XD & Zhang MQ (2006) Profiling alternatively spliced mRNA isoforms for prostate cancer classifica- tion. BMC Bioinformatics 7, 202, doi:10.1186/1471- 2105-7-202.

3 Xu L, Geman D & Winslow RL (2007) Large-scale

integration of cancer microarray data identifies a robust common cancer signature. BMC Bioinformatics 8, 275, doi:10.1186/1471-2105-8-275.

4 Gromov P, Gromova I, Friis E, Timmermans-Wielenga V, Rank F, Simon R, Sauter G & Moreira JM (2010) Proteomic profiling of mammary carcinomas identifies C7orf24, a gamma-glutamyl cyclotransferase, as a poten- tial cancer biomarker. J Proteome Res 9, 3941–3953. 5 Uejima D, Nishijo K, Kajita Y, Ishibe T, Aoyama T,

Total RNA was extracted with an RNAspin Mini RNA isolation kit (GE Healthcare, Chalfont St Giles, UK), according to the manufacturer’s instructions. The RNA was reverse transcribed to cDNA with ReverTra Ace a (Toyobo) and PCR-amplified with the following primer pairs: for human Nf-yb, 5¢-TCTCTGCAGACTATATTG GAGGAA-3¢ (sense) and 5¢-TCATGATCATTCATGCTG TCC-3¢ 5¢-GAGAG TTTTCTGTACTTTGCCTACG-3¢ (sense) and 5¢-TGG GAATTGCCAAAGTCAA-3¢ (antisense); for human E2f1, 5¢-TCCAAGAACCACATCCAGTG-3¢ (sense) and 5¢-CTG GGTCAACCCCTCAAG-3¢ (antisense); for human cyclin b1, (sense) and 5¢-AG 5¢-CATGGTGCACTTTCCTCCTT-3¢ GTAATGTTGTAGAGTTGGTGTCC-3¢ (antisense); and for human 18S rRNA, 5¢-GCAATTATTCCCCATGAACG-3¢ (anti- (sense) and 5¢-GGGACTTAATCTACGCAAGC-3¢ sense). Quantitative RT-PCR analysis was carried out with FastStart Universal SYBR Green Master on an ABI StepOne Plus.

RT-PCR analysis

Kageyama S, Iwaki H, Nakamura T, Iida H, Yoshiki T et al. (2011) Involvement of cancer biomarker C7orf24 in the growth of human osteosarcoma. Anticancer Res 31, 1297–1305.

6 Azumi K, Ikeda Y, Takeuchi T, Nomura T, Sabau SV, Hamada J, Okada F, Hosokawa M & Yokosawa H (2009) Localization and characterization of gamma- glutamyl cyclotransferase in cancer cells. Mol Med Report 2, 385–391.

7 Masuda Y, Maeda S, Watanabe A, Sano Y, Aiuchi T,

Test samples were separated by SDS ⁄ PAGE on a 10% sep- arating gel and transferred to poly(vinylidene difluoride) membranes (Pall, East Hills, NY, USA). The membranes were blocked with NaCl ⁄ Tris (pH 7.4) containing 0.1% Tween-20 (NaCl ⁄ Tris ⁄ Tween) with 5% skimmed milk for 1 h at room temperature, and then incubated with mouse monoclonal anti-human C7orf24 IgG (0.2 lgÆmL)1) in blocking buffer for 2 h at room temperature. The filter was

Nakajo S, Itabe H & Nakaya K (2006) A novel 21-kDa cytochrome c-releasing factor is generated upon

FEBS Journal 278 (2011) 4088–4099 ª 2011 The Authors Journal compilation ª 2011 FEBS

4097

Western blot analysis

Y. Ohno et al.

Transcriptional regulation of human C7orf24

21 Farina A, Manni I, Fontemaggi G, Tiainen M, Cenciar- elli C, Bellorini M, Mantovani R, Sacchi A & Piaggio G (1999) Down-regulation of cyclin B1 gene transcrip- tion in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex. Oncogene 18, 2818–2827.

treatment of human leukemia U937 cells with geranylg- eraniol. Biochem Biophys Res Commun 346, 454–460. 8 Oakley AJ, Yamada T, Liu D, Coggan M, Clark AG & Board PG (2008) The identification and structural char- acterization of C7orf24 as gamma-glutamyl cyclotrans- ferase. An essential enzyme in the gamma-glutamyl cycle. J Biol Chem 283, 22031–22042.

9 Llewellyn NM, Li Y & Spencer JB (2007) Biosynthesis of butirosin: transfer and deprotection of the unique amino acid side chain. Chem Biol 14, 379–386. 10 Javahery R, Khachi A, Lo K, Zenzie-Gregory B &

22 Manni I, Mazzaro G, Gurtner A, Mantovani R, Haug- witz U, Krause K, Engeland K, Sacchi A, Soddu S & Piaggio G (2001) NF-Y mediates the transcriptional inhibition of the cyclin B1, cyclin B2, and cdc25C promoters upon induced G2 arrest. J Biol Chem 276, 5570–5576.

Smale ST (1994) DNA sequence requirements for tran- scriptional initiator activity in mammalian cells. Mol Cell Biol 14, 116–127.

23 Bucher P (1990) Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. J Mol Biol 212, 563–578.

11 Faisst S & Meyer S (1992) Compilation of vertebrate- encoded transcription factors. Nucleic Acids Res 20, 3–26.

24 Dolfini D, Zambelli F, Pavesi G & Mantovani R (2009)

12 Rushmore TH, Morton MR & Pickett CB (1991) The

A perspective of promoter architecture from the CCAAT box. Cell Cycle 8, 4127–4137.

antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. J Biol Chem 266, 11632–11639.

13 Venugopal R & Jaiswal AK (1996) Nrf1 and Nrf2 posi-

25 Zwicker J, Gross C, Lucibello FC, Truss M, Ehlert F, Engeland K & Muller R (1995) Cell cycle regulation of cdc25C transcription is mediated by the periodic repres- sion of the glutamine-rich activators NF-Y and Sp1. Nucleic Acids Res 23, 3822–3830.

tively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expres- sion of NAD(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci USA 93, 14960–14965.

26 Sun F, Xie Q, Ma J, Yang S, Chen Q & Hong A (2009) Nuclear factor Y is required for basal activation and chromatin accessibility of fibroblast growth factor receptor 2 promoter in osteoblast-like cells. J Biol Chem 284, 3136–3147.

14 Venugopal R & Jaiswal AK (1998) Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 17, 3145–3156.

15 Wang XJ, Hayes JD, Higgins LG, Wolf CR & Dink-

27 Kabe Y, Yamada J, Uga H, Yamaguchi Y, Wada T & Handa H (2005) NF-Y is essential for the recruitment of RNA polymerase II and inducible transcription of several CCAAT box-containing genes. Mol Cell Biol 25, 512–522.

28 Mantovani R (1999) The molecular biology of the CCAAT-binding factor NF-Y. Gene 239, 15–27. 29 Ceribelli M, Dolfini D, Merico D, Gatta R, Vigano

ova-Kostova AT (2010) Activation of the NRF2 signal- ing pathway by copper-mediated redox cycling of para- and ortho-hydroquinones. Chem Biol 17, 75–85. 16 Prestera T, Talalay P, Alam J, Ahn YI, Lee PJ &

AM, Pavesi G & Mantovani R (2008) The histone-like NF-Y is a bifunctional transcription factor. Mol Cell Biol 28, 2047–2058.

Choi AM (1995) Parallel induction of heme oxygen- ase-1 and chemoprotective phase 2 enzymes by elec- trophiles and antioxidants: regulation by upstream antioxidant-responsive elements (ARE). Mol Med 1, 827–837.

17 Mantovani R (1998) A survey of 178 NF-Y binding CCAAT boxes. Nucleic Acids Res 26, 1135–1143.

30 Bolognese F, Pitarque-Marti M, Lo Cicero V, Manto- vani R & Maier JA (2006) Characterization of the human EDF-1 minimal promoter: involvement of NFY and Sp1 in the regulation of basal transcription. Gene 374, 87–95.

31 Nicolas M, Noe V & Ciudad CJ (2003) Transcriptional regulation of the human Sp1 gene promoter by the specificity protein (Sp) family members nuclear factor Y (NF-Y) and E2F. Biochem J 371, 265–275.

18 Maity SN, Golumbek PT, Karsenty G & de Crombrug- ghe B (1988) Selective activation of transcription by a novel CCAAT binding factor. Science 241, 582–585. 19 Dorn A, Bollekens J, Staub A, Benoist C & Mathis D (1987) A multiplicity of CCAAT box-binding proteins. Cell 50, 863–872.

32 Zhou D, Masri S, Ye JJ & Chen S (2005) Transcrip- tional regulation of the mouse PNRC2 promoter by the nuclear factor Y (NFY) and E2F1. Gene 361, 89– 100.

33 Sugiura N & Takishima K (2000) Regulation of the

gene promoter for extracellular signal-regulated protein

20 Wang W, Dong L, Saville B & Safe S (1999) Transcrip- tional activation of E2F1 gene expression by 17beta- estradiol in MCF-7 cells is regulated by NF-Y- Sp1 ⁄ estrogen receptor interactions. Mol Endocrinol 13, 1373–1387.

FEBS Journal 278 (2011) 4088–4099 ª 2011 The Authors Journal compilation ª 2011 FEBS

4098

Y. Ohno et al.

Transcriptional regulation of human C7orf24

Supporting information

kinase 2 by transcription factors NF-Y and Sp3. Biochem J 347 (Pt 1), 155–161.

34 Bellorini M, Lee DK, Dantonel JC, Zemzoumi K, Roe- der RG, Tora L & Mantovani R (1997) CCAAT bind- ing NF-Y–TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues. Nucleic Acids Res 25, 2174–2181.

response of the human

35 Taguchi K, Motohashi H & Yamamoto M (2011)

Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution. Genes Cells 16, 123–140.

36 Tanioka T, Hattori A, Mizutani S & Tsujimoto M (2005) Regulation of the human leukocyte-derived arginine aminopeptidase ⁄ endoplasmic reticulum-aminopepti- dase 2 gene by interferon-gamma. FEBS J 272, 916–928.

The following supplementary material is available: Fig. S1. Aberrant expression of C7orf24 in cancer cell lines. Fig. S2. Transcriptional C7orf24 gene to the oxidant stimulus. Fig. S3. Cell cycle progression of HeLa and IMR-90 cells. This supplementary material can be found in the online version of this article.

37 Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Pod- kolodnaya OA, Kolpakov FA et al. (1998) Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26, 362–367.

FEBS Journal 278 (2011) 4088–4099 ª 2011 The Authors Journal compilation ª 2011 FEBS

4099

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