A Novel Myc Target Gene, mina53, That Is Involved in Cell Proliferation*

Myc is a ubiquitous mediator of cell proliferation and can transactivate the expression of various genes through E-box sites. Here we report a novel gene, mina53(Myc-inducednuclear antigen with a molecular mass of 53 kDa). The mina53 gene encodes a protein with a molecular weight of 53 kDa, which is localized in the nucleus and with part of the protein concentrated in the nucleolus. When serum-starved cells were activated by serum, the level of c-myc mRNA was elevated, and an increase in mina53 mRNA followed the elevation of c-myc mRNA. When expression of c-myc was reduced in human promyelocytic leukemia HL60 cells by phorbol 12-myristate 13-acetate, the expression ofmina53 mRNA and protein was reduced. The expression ofmina53 mRNA and Mina53 protein was induced by ectopic introduction of wild type c-Myc but not by a mutant c-Myc lacking the transactivation domain. When c-Myc in the c-MycER chimeric protein was activated, mina53 mRNA was increased, even in the presence of an inhibitor for protein synthesis. E-box sites are present in a region proximal to the transcription initiation sites of themina53 gene. The gene expression from themina53 promoter was elevated by c-Myc through E-box sites. c-Myc protein bound to the mina53 promoter region in vivo in HL60 cells in the proliferating phase but not after treatment of cells with phorbol 12-myristate 13-acetate. Specific inhibition of mina53 expression by an RNA interference method severely suppressed cell proliferation. Taken together, these results indicate that mina53 is a direct target gene of Myc, suggesting that mina53 is involved in mammalian cell proliferation.

The myc family of proto-oncogenes consists mainly of three genes, c-myc, N-myc, and L-myc (1)(2)(3)(4)(5). Although the three genes exhibit distinct patterns of expression with respect to cell types and developmental stages of cells, they can substitute for each other in certain situations and appear to have basically the same biological activity. Deregulated expression of myc family genes, through gene amplification, viral promoter insertion, chromosomal translocation, or promoter mutation has long been known to be associated with neoplastic diseases in a wide range of vertebrates including humans. Embryonic mice with c-myc or N-myc deleted develop multi-organ hypoplasia and die during mid-embryogenesis (6 -8). These results indicate that the myc family genes are central regulators of cell growth (4,5,9).
c-myc is one of the most widely studied proto-oncogenes and is the best characterized member of the myc gene family. In general, c-myc expression is associated with cell proliferation and is down-regulated in quiescent and differentiated cells. The protein encoded by c-myc is a member of the basic helixloop-helix leucine zipper transcription factors (5,9). Dimerization of c-Myc protein with its obligate partner Max results in the formation of a heterodimer that binds to E-box sites (mainly CACGTG elements). Besides the basic helix-loop-helix leucine zipper domain (Max binding and DNA binding sites), c-Myc has another domain, the transactivation domain (TAD), 1 and the c-Myc-Max heterodimer activates transcription of various genes by activities of these domains. c-Myc has been shown to directly transactivate the expression of a number of genes (10), including ornithine decarboxylase (11), cdc25A (12), RCC1 (13), cyclin D2 (14), and Id2 (15). But the transactivation of these genes by c-Myc is generally weak, between two to severalfold. Attempts to identify genes capable of re-establishing normal proliferative rates in c-mycϪ/Ϫ cells have resulted in the repeated identification of c-myc and N-myc (16,17). These results suggest that myc controls not a gene but genes to regulate cell proliferation. Although much effort has been made to investigate c-Myc, c-myc still remains enigmatic, and information about additional genes controlled by c-myc can help elucidate the function of c-Myc.
Here we report a novel gene, mina53, whose expression is directly induced by c-Myc. The mina53 gene encodes a protein with a molecular mass of 53 kDa, which localizes in the nucleus and some of which is concentrated in the nucleolus. Specific inhibition of mina53 expression by an RNA interference method severely suppressed cell proliferation, suggesting that the mina53 contributes to cell growth induced by c-myc.

EXPERIMENTAL PROCEDURES
Cell Culture-Human glioblastoma cell line T98G cells were cultured in Eagle's medium supplemented with nonessential amino acids and 10% fetal calf serum (FCS). Human cervical carcinoma HeLa cells and rat fibroblast cell line 3Y1 and its derivatives were cultured in Dulbec-* This work was supported by Grant-in-aid from the Ministry of Education, Science, and Culture of Japan 3214107 and a grant-in-aid from the Novartis Foundation (Japan) for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
co's modified Eagle's medium supplemented with 10% FCS. Human promyelocytic leukemia HL60 cells were cultured in RPMI 1640 medium supplemented with 20% FCS. Rat colon cancer cell line RCN-9 (Riken Cell Bank, Saitama, Japan) was cultured in RPMI 1640 medium supplemented with 10% FCS. Human cancer cell lines, human erythroid leukemia (HEL), MCAS (ovarian cancer), and WiDr (colon cancer) were obtained from Health Science Research Resources Bank, Osaka, Japan. These cells were grown in appropriate media.
PCR-PCR amplifications were performed in 50 l of EX Taq buffer (Takara, Shiga, Japan) containing 10 pmol of each primer, 1.2 units of EX Taq DNA polymerase, and 200 M dNTP.
Reverse Transcriptase (RT)-PCR-Synthesis of single-strand cDNA of human erythroleukemia HEL or rat colon cancer RCN-9 cells was performed on total RNA (1 g) using a Superscript First-strand Synthesis system (Invitrogen). One l (total 20 l) of resultant singlestrand cDNA was used as the template for PCR. The RT-PCR primers for the amplification were mina53 RT-F (5Ј-ACACCGGTGACGAGCG-CACGGAAAG-3Ј, a sequence in the 5Ј-untranslated region) and mina53 RT-R (5Ј-GCCTCTCATCTGTGGGGAAGTATTA-3Ј, a sequence in the 3Ј-untranslated region) for human and mina53 RT-F (5Ј-TTTCCTC-CCTACTTGTGAACAATGC-3Ј, a sequence in the 5Ј-untranslated region and the first four coding nucleotides) and rat mina53 RT-R (5Ј-AATGATGCTCTTTAAAGGAGTTATTT-3Ј, a sequence in the 3Јuntranslated region) for rat. The temperature profile was 35 cycles of denaturing at 98°C for 15 s, annealing at 65°C for 1 min, and extension at 72°C for 2.5 min.
Rapid Amplification of 5Ј cDNA Ends (5Ј-RACE) Analysis-Reverse transcriptase reaction, double-strand cDNA synthesis, and adapter ligation from poly(A) ϩ RNA (1 g) of HEL cells were performed using a Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA) as described previously (18). The first PCR was performed using primers, mina53-RACE-1 (5Ј-GCAGTACTGTGAGGACAATGTGGTCTT-3Ј, a sequence in the 5Ј-terminal region of expressed sequence tag (EST) clone W27666) and the AP1 primer provided by the supplier. The temperature profile was initial denaturing at 94°C for 1 min followed by 25 cycles of denaturing at 96°C for 15 s and annealing and extension at 68°C for 3 min. One l of the first RACE-PCR product diluted 1000 times was used as the template for nest RACE-PCR. Nest PCR was performed using mina53-RACE-2 primer (5Ј-CTCACTACACTGTCCA-GCCTCGGTAA-3Ј), which corresponds to a sequence in the upstream region of mina53-RACE-1 of EST clone W27666 or the mina53-RACE-3 primer (5Ј-CAACCAAGGAGCCAAAGTAACATTC-3Ј, a sequence in the middle part of mina53 cDNA) and the AP2 primer provided by the supplier. The temperature profile was initial denaturing at 94°C for 1 min followed by 25 cycles of denaturing at 96°C for 15 s and annealing and extension at 68°C for 3 min.
Plasmids Encoding c-myc-Pc-myc/CDM8, in which human c-myc is driven by a CMV promoter, was described previously (13). Pc-myc/ CDM8 was digested with PstI and self-ligated to produce pc-myc(d41-178)/CDM8, which encodes a mutant c-Myc protein lacking the TAD. The 2-kb BstXI-KpnI fragment, blunted with Klenow enzyme, of pTc-mycerS/BS (13) was inserted into a 5-kb EcoRI fragment, blunted with Klenow enzyme, and de-phosphorylated with Escherichia coli alkaline phosphatase of a pCAGGS mammalian expression vector (19) to produce pc-mycer/CAGGS in which c-mycer is driven by a chimeric promoter consisting of chicken actin and CMV promoters.
Plasmids Encoding mina53-cDNA for human mina53 was amplified by PCR with 5Ј-GAAGCTTATGCCAAAGAAAGCAAAGCCTACAG-G-3Ј, adding a HindIII site just before the initiation methionine, and 5Ј-TGAATTCATCCTCTCCTCGGCTCAGGTCTT-3Ј, which is located downstream after the stop codon, as primers from a library of HEL cells, and the amplified 1.4-kb fragment was cloned into a pGEM-T-vector (Promega, Madison, WI) to produce pT/hmina53 (465) and pT/hmina53 (464), encoding 465-and 464-amino acid proteins, respectively. The 1.5-kb HindIII-SalI fragment of pT/hmina53 (465) was inserted into pEGFP-C3 (CLONTECH) cut with HindIII and SalI to produce pEGFP/hmina53 (465) for green fluorescent protein-Mina53 fusion protein. The 1.5-kb HindIII-NotI fragment of pT/hmina53 (465) was inserted into pRc/CMV (Invitrogen), which was digested with HindIII and NotI, to produce phmina53/Rc/CMV. The DNA fragment encoding human mina53 was amplified by PCR with 5Ј-GCCATGCCATGGCAA-AGAAAGCAAAGCCTAC-3Ј, designed to have an artificial NcoI site at the initiation codon methionine, and 5Ј-GGCATGCCATGGCTAGACT-ACTTGAATTAAAC-3Ј, adding a NcoI site just after the stop codon, as primers from pEGFP/hmina53 (465). The amplified 1.4-kb fragment was cleaved with NcoI and ligated with an E. coli expression vector pET11d (Novagen, Madison, WI) cut with NcoI and de-phosphorylated with E. coli alkaline phosphatase to produce pET/hmina53. The 1.5-kb XhoI-SalI fragment, blunted with Klenow enzyme, of pEGFP/hmina53 (465) was inserted into pGEX-3X (Amersham Biosciences), which was digested with EcoRI, blunted with Klenow enzyme, and dephosphorylated with E. coli alkaline phosphatase to produce pGEX-hmina53 for glutathione S-transferase-Mina53 fusion protein. Rat mina53 cDNA was amplified by RT-PCR protocol as described above and cloned into a pGEM-T-vector (Promega) to produce pT/rmina53.
Reporter Plasmids Having a mina53 Genomic DNA Fragment-A genomic DNA fragment of the human mina53 gene, which extends from the promoter region to intron 1, was amplified by PCR with 5Ј-CGG-GATCCTGAACGCGGAACACCGCCGGGTAGC-3Ј, adding a BamHI site, and 5Ј-CCCAAGCTTCCTCTTCCTCCCAGTCTATCCTTC-3Ј, adding a HindIII site. The 0.8-kb amplified fragment was cleaved with BamHI and HindIII and inserted into the 4.8-kb fragment of pGL3basic vector containing firefly luciferase (Promega) cut with BglII and HindIII to produce pMina(W)luci. pMina(W)luci was cleaved with HindIII and PmaCI, blunted with Klenow enzyme, and self-ligated to produce pMina(dE)luci, in which two E-boxes were deleted. Mutation was introduced at E-box sites of pMina(W)luci using a Gene Editor TM in vitro site-directed mutagenesis system (Promega) to produce pMina(mE1)luci, pMina(mE2)luci, and pMina(mE1/2)luci, in which one or two E-boxes (CACGTG elements) were mutated to CACCTG.
Other Plasmids-pActHyg, which contains a hygromycin resistance gene under control of an actin promoter, was a kind gift from Dr. M. Nakanishi (13). pRL-CMV containing the Renilla reniformis luciferase gene under control of the CMV promoter was purchased from Promega.
Establishment of Cell Lines Expressing c-Myc, Mutant c-Myc, and c-MycER Chimeric Protein-To establish rat fibroblast 3Y1 cells highly expressing c-Myc or mutant c-Myc lacking a large part of TAD, cells were transfected with 0.4 g of pActHyg and 20 g of pc-myc/CDM8 or pc-myc(d41-178)/CDM8 by a calcium phosphate method (20). Cells were cultured for 2 weeks in the medium containing 200 g/ml hygromycin. Individual clones were isolated, and the expression of c-Myc or mutant c-Myc protein was detected by Western blot analysis using anti-c-Myc antibody. Clones 3Y1MycA and 3Y1MycB (expressing c-Myc protein) and 3Y1Myc(dTAD)A and 3Y1Myc(dTAD)B (expressing mutant c-Myc protein lacking the transactivation domain) were established. To establish human glioblastoma T98G cells expressing c-MycER chimeric protein (T98Gmycer-2 cells), cells were transfected with 20 g of pc-mycer/CAGGS and 0.4 g of pActHyg, and the expression of c-MycER in individual clones was detected as described above.
RNA Preparation-Total RNA was isolated from cells by the acid guanidinium thiocyanate-phenol/chloroform extraction method using a diethyl pyrocarbonate-treated RNA preparation solution set (Nakalai, Kyoto, Japan).
Differential Display Using DNA Chip (cDNA Micro Array)-Total RNA from untreated T98Gmycer-2 cells and from cells treated by 4-hydroxytamoxifen (OHT) for 20 h was isolated. Poly(A ϩ ) RNA was recovered and subjected to differential display using a DNA chip (Incyte Genomics, Palo Alto, CA). About 9000 kinds of cDNAs including EST clones were plated on the chip (UniGEM Human V Version. 2).
Transient Expression Assay-T98Gmycer-2 cells were grown in medium supplemented with 10% FCS. For transfection, 3 ϫ 10 4 cells were plated into a dish (12-well plate; well diameter, 22 mm) and cultured for 20 -24 h. Transfections were carried out using FuGENE 6 reagent (Roche Molecular Biochemicals) with 1 g of the reporter plasmids and 20 ng of pRL-CMV as an internal transfection marker. One day later, OHT (final concentration, 0.2 M) was added to activate MycER chimeric protein, and cells were further cultured for 17 h. Cells were then collected and analyzed for firefly and R. reniformis luciferase activities using a Dual luciferase reporter assay system (Promega). After normal-ization of R. reniformis luciferase activities, firefly luciferase activities with activated c-MycER were expressed as the ratio of activities without the MycER activation. To normalize the effect of OHT not through MycER activation, the values from T98Gmycer-2 cells were divided by the values from T98G parent cells, which were treated by exactly the same procedures as those of T98Gmycer-2 cells described above.
Chromatin Immunoprecipitation-Chromatin immunoprecipitation assay was performed basically as described by Boyd et al. (21,22). Immune complexes were recovered by adding 20 l of protein A beads blocked by DNA and BSA (50% volume/volume). The beads were washed, the DNA fragments were eluted, and the eluted solutions were phenol/chloroform-extracted and ethanol-precipitated. Then, immunoprecipitated DNA fragments were detected by PCR. PCR primers were 5Ј-GCCGGCGCTGTGGTTGCGGGACCTG-3Ј and 5Ј-TCCTCTTCCTC-CCAGTCTATCCTTC-3Ј, which amplified a 483-bp fragment containing E-boxes near the two transcription start sites of the human mina53 gene, and 5Ј-TTACAGGTAAGCCCTCCAATGACC-3Ј and 5Ј-GCAAAG-CTACCATTTAGGAACCC-3Ј, which amplified the genomic sequence of a region containing an E-box in chromosome 22. This E-box is located in a chromosomal region without any detectable genes (14).
Western Blot Analysis and Indirect Immunofluorescence Staining-Cells were trypsinized and extracted in 3% SDS solution containing 100 mM Tris, pH 6.8, 0.1 M dithiothreitol, and 20% glycerol. Cell extracts were separated on 4 -20% polyacrylamide gels and transferred to a polyvinylidene difluoride microporous membrane (Millipore, Bedford, MA). After treatment with antibodies, bands were detected using an enhanced chemiluminescence technique (Amersham Biosciences).
For indirect immunofluoresence staining, HeLa cells grown on glass coverslips in a 6-well plate were fixed in methanol for 10 min at Ϫ20°C. Anti-Mina53 rabbit antibody and mouse anti-nucleolin monoclonal antibody were added and incubated for 120 min at 37°C. After washing in 0.1% skim milk in phosphate-buffered saline three times, Alexa 488conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG were added, incubated for 120 min at 37°C, and washed with 0.1% skim milk three times. Finally, cells were embedded in Immunon (Thermo Shandon, Pittsburgh, PA) and observed via fluorescence microscopy.
Introduction of Small Interference RNA (siRNA) into Cells-RNA with 21 nucleotides was chemically synthesized (Hokkaido System Science, Sapporo, Japan). The siRNA sequences targeting human Mina53 and rat Mina53 are in positions 45-63 relative to the first nucleotide of the start codon and the 2-nucleotide 3Ј overhang of deoxythymidine. A control siRNA sequence was an inverted sequence of the siRNA directed to human mina53. siRNAs were annealed and transfected essentially as described previously by Elbashir et al. (24). Twenty-four hours before transfection, cells in an exponentially growing phase were trypsinized and transferred to a 12-well plate. Transfection was carried out with 100 pmol of siRNA per well using OligofectAMINE (Invitrogen) according to their instructions, except that HeLa cells were cultured for 20 h for transfection without serum, and 3Y1MycB cells were cultured for 10 h.

A Method Used to Identify Genes Whose Expression Was
Induced by c-Myc-To conditionally activate c-Myc activity, the estrogen-inducible Myc system (25) was used. The chimeric protein c-MycER consists of human c-Myc and the estrogen binding domain of the human estrogen receptor. c-MycER anchors to cytoskeletal components of cells in the absence of estrogen. When estrogen or its derivative, OHT, binds to the chimeric protein, it becomes free to function as c-Myc. The human glioblastoma cell line T98G was used as the parental cells into which ectopic c-Myc activity was introduced. A T98G cell line expressing c-MycER protein (T98Gmycer-2 cells) was established. Total RNAs from T98Gmycer-2 cells in an exponentially growing phase in the presence or absence of OHT for 20 h were processed by a cDNA micro array analysis. Specific signals for Myc target genes, ornithine decarboxylase (11) and nucleolin (26), were increased by 2.6-and 1.6-fold, respectively, with c-MycER activation. These results suggest that Myc target genes could be detected in this experimental system.
Identification of Human mina53-To shed light on a new facet of c-Myc functions, we focused on human EST sequences among genes stimulated by c-MycER activation. The signal for EST clone W27666 was stimulated by 1.9-fold with c-Myc activation. This stimulation rate was similar to those of the Myc-targeted genes, ornithine decarboxylase and nucleolin, measured in this study.
cDNA encoding the 5Ј upstream part of EST clone W27666 was isolated using the 5Ј-RACE protocol from a library of HEL cells. We isolated two kinds of cDNAs with different sequences at the 5Ј terminus. From the results of the nucleotide sequences from 5Ј-RACE experiments and ESTs, we predicted the whole sequence of the mRNA molecule. Using primers consistent with 5Ј-part and 3Ј-part sequences of the predicted molecule, a 2.3-kb-length cDNA was amplified from total RNA of HEL cells by RT-PCR protocol. The amplified cDNA was cloned into a vector and sequenced. The majority of cDNA clones encode a protein of 465 amino acids with a predicted molecular mass of 52,800.28 Da (Fig. 1A). The subcellular localization of this protein was visualized by indirect immunofluorescence staining with the specific antibody. As shown in Fig. 1B, this protein is localized in the nucleus. For these reasons, we refer to this gene and protein as mina53 and Mina53 (Myc-induced nuclear antigen with a molecular mass of 53 kDa). In addition to the diffuse localization in the nucleus, strong dotted staining was observed within the nucleus and supposed to be nucleoli. Double staining with anti-nucleolin antibody indicated that these dots were consistent with nucleoli. Nuclear and nucleolar localization of Mina53 was also observed when green fluorescent protein-Mina53 fusion protein was expressed (data not shown). These results suggest that Mina53 is a nuclear protein, some of which is concentrated in nucleoli.
The cDNA encoding a 464-amino acid protein with a predicted molecular mass of 52,672.15 Da was also cloned. This protein lacks Gln-297 of the major protein. We also detected a cDNA that has 101 bp inserted in the middle part of the mina53 cDNA. There is a stop codon in the inserted sequence, and the cDNA encodes 262 amino acids of Mina53 protein from the initiation codon and 18 additional novel amino acids at the 3Ј end. Although the amount of this mRNA was small, we detected this mRNA molecule in cDNA libraries made from a human ovary mucinous cystadenocarcinoma cell line (MCAS) and a human colon adenocarcinoma cell line (WiDr) (data not shown).
Mina53 Is Conserved in Human, Rat, and Mouse-A mouse cDNA that appeared to be an orthologue of human mina53 was found by a Blast search (GenBank TM NM_025910). A rat EST (GenBank TM H32933) that is 300 bp in length and is homologous to the 3Ј-untranslated region of mouse and human mina53 was also found. Using primers that were predicted to amplify rat mina53 mRNA, we isolated rat mina53 cDNA from RCN-9 cells by RT-PCR protocol. The predicted amino acid sequences for Mina53 from the three species were aligned (Fig.  1A). The open reading frames of rat and mouse mina53 encode 465 amino acid proteins, as did human mina53. Human and mouse amino acid sequences of Mina53 proteins were 72% identical to each other, those of human and rat were 75% identical, and those of rat and mouse were 86% identical, suggesting that the mina53 is conserved in mammals. A nucleotide 3 bp upstream of the methionine initiation codon is A in mina53 of all the three species, conforming to a Kozak consensus sequence (27).
Expression of mina53 mRNA-The expression profile of mina53 mRNA was examined by Northern blot analysis after T98G cells were stimulated by serum ( Fig. 2A). Serum stimulation of serum-starved T98G cells resulted in a 5-fold increase in the expression of mina53 mRNA. The increase was detecta- ble at 6 h after serum addition and peaked at 12 h. The increase of c-myc mRNA level was detected at 3 h after serum addition and peaked at 12 h. These results suggest that up-regulation of mina53 mRNA followed the increase of c-myc mRNA.
Human promyelocytic leukemia HL60 cells are terminally differentiated by phorbol 12-myristate 13-acetate (TPA) in which the c-myc expression level reduces (28,29). This experimental system was used to investigate whether Myc-targeted genes are affected during the shut-off of Myc that accompanies hematopoietic differentiation (30). When HL60 cells were cultured with 10 nM TPA, the expression of a differentiation marker CD18 mRNA (31) increased, and a 3-fold-induction was observed at 24 h, confirming the differentiation in this system (Fig. 2B). At 3 h after the addition of TPA, the level of c-myc mRNA started to decline and reached 1 ⁄5 at 7 h. Down-regulation of mina53 mRNA followed the decrease in c-myc mRNA, and the level of mina53 mRNA decreased to 1 ⁄5 at 12 h. These experiments demonstrate that the expression pattern of mina53 correlated with c-myc expression.
Next, the effect of c-Myc activation on mina53 mRNA was investigated (Fig. 2C). The mina53 mRNA level rose steadily for 9 h in OHT-treated T98Gmycer-2 cells, showing nearly 3-fold induction. The OHT treatment of T98G parent cells did not stimulate the mina53 mRNA level. Induction of mina53 mRNA by OHT in T98Gmycer-2 cells was maintained in the presence of the protein synthesis inhibitor cycloheximide. Treatment with cycloheximide had little effect on the mina53 mRNA level in T98G parent cells. These results indicate that the mina53 gene is a direct target of Myc.
Induction of Mina53 Protein by Myc Activation-To examine the correlation between expression of Mina53 and c-Myc proteins, a specific antibody against Mina53 protein was used in Western blotting. Bands with similar mobility to that predicted from the amino acid sequence of Mina53 were detected in T98G and HL60 cells (Fig. 3A). When c-MycER was activated by OHT in T98Gmycer-2 cells, the intensity of the band for Mina53 was increased, whereas that for ␤-actin was not. When HL60 cells were cultured with 10 nM TPA for 24 h, the expression of c-Myc and Mina53 proteins was decreased, whereas the expression of ␤-actin was not.
The control of Mina53 expression by c-Myc was also verified in rat fibroblast cell line 3Y1. Human c-Myc or mutant c-Myc that lacks TAD was stably expressed in 3Y1 cells. The expression of Mina53 protein was increased in the dose-dependent manner of wild type c-Myc expression (Fig. 3B, lanes for  3Y1MycA and 3Y1MycB). When the mutant Myc was expressed at a level comparable with that of wild type c-Myc (lanes for 3Y1MycA and 3Y1Myc(dTAD)A) or even at a much higher level than that of wild type Myc (lanes for 3Y1MycB and 3Y1Myc-(dTAD)B), expression of Mina53 protein was not increased.
To confirm the results, mRNA for mina53 was detected by Northern blotting in these 3Y1 cell lines (Fig. 3C). The mina53 mRNA level was stimulated by 1.5-and 3-fold in 3Y1MycA and 3Y1MycB, respectively. On the other hand, mina53 mRNA level was not stimulated in 3Y1Myc(dTAD)A and 3Y1Myc-(dTAD)B. These results suggest that c-Myc transactivates the expression of mina53.
Structure of the Human mina53 Gene-Human genomic DNA sequences, which include mina53 cDNA sequences, were found in the High Throughput Genomic Sequence (HTGS) data base (GenBank TM AC026100, AC073245, and AC024892), and our cDNA sequences for mina53 were aligned with these genomic sequences from the GenBank TM data (Fig. 4A). The length of intron 2 was controversial between the GenBank TM data. To know the length of intron 2, the genomic DNA fragment was amplified by PCR using oligonucleotides correspond-ing to the sequences in exon 2 and exon 3 as primers, and the length of intron 2 was determined to be 5.2 kb (Fig. 4A). The human mina53 gene consists of 12 exons. The translation start site locates in exon 2, which follows two distinct exons, exon 1a and exon 1b. Two exon 1s are consistent with the two cDNAs with two kinds of 5Ј-terminal sequences that we had isolated in the present study. Thus, there are two transcription initiation sites in the mina53 gene. The exon 1b exists 0.25 kb downstream of the exon 1a. The stop codon (TAG) exists in the last exon, exon 10. cDNA encoding 464 amino acids (lacking Gln-297) was generated by alternative splicing due to the lack of the first 3 bp of exon 7. The minor form of cDNA contained the insertion of a 101-bp sequence, which was found between exon 5 and exon 6. We designated this exon as exon 5Ј. Mapping data from the University of California Santa Cruz Genome Browser showed that the mina53 gene maps to chromosome 3 (3q12.1).
c-Myc Stimulates the Gene Expression through a mina53 Genomic DNA Fragment-To investigate promoter activity of the mina53 gene, a human mina53 genomic DNA fragment including a 0.8-kb region from upstream of exon 1a to part of intron 1 was joined to firefly luciferase cDNA to construct a reporter plasmid, pMina(W)luci (Fig. 4B). Transient expression assays in T98Gmycer-2 cells indicate that the DNA fragment has promoter activity (Fig. 4C). After c-MycER activation by OHT, the luciferase activity was increased up to 3-fold. This stimulation is comparable with that observed for an increase in mina53 mRNA level by OHT in T98Gmycer-2 cells (Fig. 2C).
Exon 1b has two E-box sites (CACGTG elements). We designated the first and second CACGTG elements as E-box 1 and E-box 2, respectively. When the two E-box sites were deleted from pMina(W)luci, luciferase activity was not increased by the activation of c-MycER chimeric protein (Fig. 4C, a lane for  pMina(dE)luci). Expression from pMina(mE1/2)luci, whose two CACGTGs were mutated to CACCTG, was not increased by c-Myc activation. These results indicate that c-Myc stimulates the expression of pMina(W)luci through the CACGTG elements. To determine which CACGTG elements are functional, reporter plasmids pMina(mE1)luci and pMina(mE2)luci were constructed with each CACGTG element mutated to CACCTG (Fig. 4B). As shown in Fig. 4C, mutation of E-box 1 had little effect. On the other hand, mutation of E-box 2 severely decreased the stimulation of luciferase activity by c-Myc. These results suggest that c-Myc functions through E-box 2.
c-Myc Binds to the mina53 Gene in Vivo-To examine c-Myc protein binding to the endogenous mina53 gene in vivo during proliferation of HL60 cells, chromatin immunoprecipitation was performed as described previously (21,22). After immunoprecipitation, enrichment of the endogenous mina53 gene fragment in each sample was monitored by PCR using primers that specifically amplify part of exon 1b and intron 1 in the mina53 gene. As shown in Fig. 5, two different anti-c-Myc antibodies immunoprecipitated the mina53 DNA fragment from HL60 cells in the proliferating phase, whereas the same antibodies did not immunoprecipitate detectable levels of DNA fragment from cells treated with TPA. Enrichment for mina53 genomic DNA fragments is dependent on c-Myc binding to the mina53 gene, because nonspecific antibody did not immunoprecipitate mina53 DNA fragments. Additionally, binding of c-Myc detected in the mina53 gene is specific, since antibodies against c-Myc did not enrich the genomic DNA fragment containing an E-box that is located in a chromosomal region without any detectable gene in chromosome 22. These results demonstrate that the mina53 gene is bound by c-Myc specifically at the proliferating phase of HL60 cells.
Mina53  2. Expression of mina53 mRNA. A, correlation of mina53 mRNA levels with c-myc expression levels in T98G cells. Serum-starved T98G cells were stimulated by the addition of serum final to 10%. RNA was isolated at the indicated time points and analyzed by Northern blotting to detect mina53 and c-myc mRNAs (left). 28 S and 18 S ribosomal RNAs are shown to indicate the amount of total RNA electrophoresed. The results were quantified and plotted (right). B, decrease in mina53 mRNA levels after c-myc mRNA disappearance in human promyelocytic leukemia HL60 cells. RNA was isolated at the indicated time points after exposure to 10 nM TPA and analyzed by Northern blotting to detect mina53, c-myc, and CD18 mRNAs (left). 28 S and 18 S ribosomal RNAs are shown. The results were quantified and plotted (right). C, increase of mina53 mRNA levels in T98G cells by activation of MycER protein (T98Gmycer-2 cells). After T98Gmycer-2 cells (mycer) and parental T98G cells (cont) were cultured in the medium supplemented with 0.25% serum for 40 h, cells were treated with 200 nM OHT, and RNA was isolated at the indicated time points. When indicated as ϩ, 20 g/ml cycloheximide (CHX) was added 20 min before the addition of OHT. RNA was analyzed by Northern blotting to detect mina53 mRNA (left). 28 S and 18 S ribosomal RNAs are also shown. The results were quantified and expressed as a bar graph (right). that a 21-nucleotide siRNA duplex specifically suppresses gene expression in mammalian cell lines including human cervical carcinoma HeLa cells (24). To gain insight into the biological function of mina53, this new technique was applied to specifically inhibit the expression of Mina53 protein. As shown in Fig.   6A, the expression of Mina53 protein was specifically reduced by the cognate siRNA duplex in HeLa cells at 40 h after siRNA transfection but by neither a control siRNA (a duplex with inverted mina53 sequence) nor a duplex directed against rat mina53 in which 5 nucleotides of a 19-nucleotide sequence were different from that for human mina53. The expression of rat Mina53 protein was specifically reduced by the cognate siRNA duplex in rat 3Y1 cells highly expressing c-Myc (3Y1MycB) but by neither the control siRNA nor the duplex directed against human mina53. When Mina53 expression was reduced by siRNA transfection, cell proliferation was severely suppressed (Fig. 6B), i.e. proliferation of HeLa and 3Y1MycB cells were almost completely stopped by transfection of siRNA duplex directed against human and rat mina53, respectively. DISCUSSION We identified a novel gene, mina53, that is controlled by c-Myc and investigated the regulation of mina53 expression and the nature of Mina53 protein. We found evidence that c-Myc directly induces the expression of mina53. When serumstarved T98G cells were activated by serum, the level of c-myc mRNA was elevated, and the mina53 mRNA followed the elevation of c-myc mRNA. In HL60 cells, reduction of c-myc mRNA expression by TPA preceded the down-regulated expression of mina53 mRNA. The TPA treatment of HL60 cells also decreased the expression of both c-Myc protein and Mina53 protein. Ectopic expression of wild type c-Myc, but not the mutant c-Myc in which TAD was deleted, stimulated mina53 expression in rat fibroblast cell line 3Y1. Stimulation of the expression of mina53 mRNA and Mina53 protein was observed when c-Myc was activated in the c-MycER chimeric protein. Induction of mina53 mRNA by c-MycER activation was maintained in the presence of a protein synthesis inhibitor, cycloheximide, indicating that the mina53 is a direct Myc target gene. Luciferase activity that was expressed from a reporter plasmid constructed with a human mina53 genomic DNA fragment and luciferase cDNA was elevated by the activation of c-Myc in transient expression experiments. When the putative Myc binding sites (CACGTG elements) in the reporter plasmid were destroyed, stimulation of reporter gene expression by the activation of c-Myc was abolished. Although a mutation of one of the CACGTG elements (E-box 1) had little effect, the mutation of the other CACGTG element (E-box 2) severely decreased the stimulation of promoter activity by c-Myc, suggesting that c-Myc transactivates the mina53 expression through a specific CACGTG element. c-Myc protein binds to the mina53 genomic DNA that contains the E-box 2 in vivo in proliferating HL60 cells but not HL60 cells treated with TPA. Together, these results demonstrate that c-Myc directly induces the expression of mina53.
The high conservation of amino acid sequences of Mina53 protein between human and rodents suggest that the protein plays an important role in mammals. The expression of mina53 correlated with cell proliferation, suggesting a role of mina53 in cell proliferation. To gain insight into the biological function of Mina53, expression of mina53 was specifically inhibited in human cervical carcinoma HeLa cells and rat 3Y1 cells highly expressing c-myc. In HeLa cells, c-myc is highly expressed and plays an essential role in cell proliferation (32). A new technique, siRNA technique (24), was applied for the specific inhibition. RNA interference (RNAi) is the process of sequencespecific, post-transcriptional gene silencing, which is initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene due to sequence-specific dsRNAmediated mRNA degradation. Recently it has been shown that 21-nucleotide siRNA duplexes specifically suppress the expression of endogenous genes in mammalian cells (24). Specific inhibition of Mina53 expression by siRNA duplex was clearly shown here because the expression of Mina53 protein in human and rat cells was specifically reduced by each cognate siRNA duplex for human and rat mina53, respectively, but not by a control siRNA (inverted sequence for human mina53) or a duplex directed against mina53 of different species. Five nucleotides of the 19-nucleotide sequence were different between human and rat mina53 siRNA duplexes. When the expression of Mina53 protein was reduced by siRNA, severe inhibition of cell proliferation was observed in both human and rat cells.
These results indicate the importance of mina53 in proliferation of mammalian cells.
Recently, the role of c-myc in vivo has been extensively studied in mice in which c-myc expression is incrementally reduced to zero (33). These studies showed that reduction of c-Myc levels resulted in reduced body mass owing to multiorgan hypoplasia (reducing cell numbers). They stated that c-Myc activity determines the ratio of activated T cells that re-enter the cell cycle and the rate of cell division of fibroblasts. A recent in vitro study using time-lapse microscopy also suggested that c-myc regulates the decision of cells to enter or exit the cell cycle in rat cell culture systems (34). Therefore, it is possible that Mina53 may be one of factors directly involved in the cell cycle machinery, as other Myc target genes, including cyclin D2 (35,36) and Id2 (15), are. It was shown that loss of Drosophila Myc (dMyc) retards cellular growth (accumulation of cell mass) and reduces cell size, whereas dMyc overproduction increases growth rate and cell size, suggesting that dMyc regulates the primary targets involved in cellular growth (37). Deregulated expression of Myc sometimes induces an increase of cell size in mammals also (38). For example, constitutive expression of a c-myc transgene under control of the Ig heavychain enhancer resulted in an increase in cell size of pretransformed B lymphocytes at all stages of B cell development independently of cell cycle phase. This increase correlated with an increase in protein synthesis (39). Recent reports also suggest that myc enhanced the expression of a large set of genes functioning in protein synthesis, including ribosome biogenesis (40,41). Thus, mina53 may be one of those genes.
Recent studies suggest that the nucleolus might function as a "prison" for certain proteins involved in eukaryotic cell cycle regulation. Sequestration of proteins into nucleoli prevents them from reaching their targets in other regions of the cell (42). Thus, Mina53 in nucleoli may be sequestered from the place where Mina53 functions, by which an activity of Mina53 FIG. 5. Chromatin immunoprecipitation experiments using HL60 cells. HL60 cells in the proliferating phase (ϪTPA) and those treated with 10 nM TPA for 24 h (ϩTPA) were fixed with 1% formaldehyde, nuclear extracts were collected, and chromatins were immunoprecipitated using antibodies against c-Myc (anti-c-Myc antibody-1 reacting with the amino-terminal half of c-Myc and anti-c-Myc antibody-2 reacting with the carboxyl-terminal half of c-Myc), control antibody or no antibody (Ϫantibody). Mock immunoprecipitation, in which no nuclear extracts were added, was also performed (nuclear extract Ϫ). After DNA purification, samples were subjected to PCR with primers designed to amplify a DNA fragment containing exon 1b of the mina53 gene (mina53 primers) or primers to specifically amplify a DNA fragment containing a chromosome 22 E-box (control primers) (14). is controlled. The nucleolus has been considered to be a "ribosome factory" since the 1960s, and ribosome biogenesis is essential for cell proliferation. Thus, Mina53 in nucleoli may play a necessary role in ribosome biogenesis, which is consistent with the possibility we described above. Recent studies also suggest that the nucleolus participates in many other activities, including the processing or nuclear export of certain mRNAs, biogenesis of signal recognition particle RNA and telomerase RNA, and processing one of the spliceosomal small nuclear RNAs (43). These functions also appear to be essential for cell proliferation. Thus, it is possible that Mina53 plays a role in one of these functions.
It had been suggested that proliferation and differentiation represent alternative and mutually exclusive pathways for cells, and there is compelling evidence that Myc may function at a pivotal control point in the decision-making process (4). It had been reported that down-regulation of c-myc expression is correlated with differentiation in many cellular systems. It was also shown that constitutive expression of c-myc prevents cells from leaving the cell cycle and inhibits the differentiation, and suppression of c-myc alone is sufficient to leave the cell cycle and induce differentiation. However, down-regulation of c-myc is not always correlated with differentiation. Expression of c-myc is detectable in terminally differentiated cells such as keratinocytes. U937 cells can be differentiated without downregulation of c-myc in certain conditions. Furthermore, transforming function of c-myc can be distinguished from the Mycinduced block of differentiation using a Myc mutant in primary quail myoblast. Therefore, relationship between c-myc, proliferation, and cellular differentiation still include controversial problems. One of the Myc target genes might be specifically involved in cell proliferation, differentiation, or both. Thus, studies of each Myc target gene on proliferation and differentiation will help elucidate the problems, and it is a next, important issue to determine whether mina53 is involved in cellular differentiation.
Although no domains whose functions have been experimentally demonstrated are found in Mina53, the amino acid sequence 128 -271 of Mina53 represents a JmjC domain, a domain that was recently identified on the basis of significant sequence similarity among many genes (44). It was suggested that JmjC domains are present in some metalloenzymes that adopt the cupin fold, which is a flattened ␤-barrel structure containing two sheets of the five anti-parallel ␤ strands that form the walls of a zinc binding cleft. This domain has been often found together with DNA or chromatin binding domains, and JmjC domain-containing proteins are candidates for enzymes that regulate chromatin remodeling (44). Human and mouse Mina53 amino acid sequences in JmjC domains are 83.3% identical, whereas the sequences of the other portions are 66.6% identical between the two species, suggesting the functional importance of this domain. Therefore, Mina53 may regulate chromatin remodeling, which appears to largely affect patterns of gene expression.
Because of the clear relationship of mina53 with cell proliferation, mina53 appears to be an important target of c-myc.
FIG. 6. Reduction of Mina53 protein expression by RNA interference and its effect on cell proliferation. A, reduction of Mina53 protein expression by RNA interference. siRNA duplexes specific for human mina53 and rat mina53 were synthesized. As a nonspecific control, a duplex with the inverted sequence of human mina53 siRNA was also synthesized. HeLa cells (left panel) and 3Y1MycB cells, which are rat 3Y1 cells that highly express human c-myc (right panel), were transfected with siRNA duplexes. Forty hours later, cell extracts were processed by Western blot analysis using anti-Mina53 antibody. B, cell proliferation of transfected cells. 2 ϫ 10 4 HeLa cells (left panel) and 1 ϫ 10 4 3Y1MycB cells (right panel) were transfected with siRNA duplexes specific for human mina53 (q), rat mina53 (OE), and control siRNA duplex (E). At various days after transfection, cell numbers were counted and expressed on the y axis.
Further studies will clarify the function of Mina53 protein and may contribute to solving the puzzle of Myc functions.