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Originally published In Press as doi:10.1074/jbc.M605820200 on October 18, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39159-39168, December 22, 2006
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The Repression of Human Differentiation-related Gene NDRG2 Expression by Myc via Miz-1-dependent Interaction with the NDRG2 Core Promoter*Formula

Jian Zhang12, Fuyang Li1, Xinping Liu, Lan Shen, Junye Liu, Jin Su, Wei Zhang, Yanchun Deng, Lifeng Wang, Na Liu, Wei Han, Jing Zhang, Shaoping Ji, Angang Yang, Hua Han, and Libo Yao3

From the Institute of Molecular Biology and the State Key Laboratory of Cancer Biology, Fourth Military Medical University, 710032 Xi'an, China

Received for publication, June 19, 2006 , and in revised form, October 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The N-myc downstream-regulated gene 1 (ndrg1) is highly expressed in N-myc knock-out mice through an unknown regulatory mechanism. As one member of the human NDRG gene family, NDRG2 encodes a protein highly homologous to Ndrg1. However, it is uncertain whether the expression of human NDRG2 is regulated by Myc because mouse ndrg2 and -3 are not affected by Myc. In this study, we provide the novel evidence that the expression of human NDRG2 is down-regulated by Myc via transcriptional repression. A high level of NDRG2 was observed as Myc expression was reduced in differentiated cells, whereas a low level of NDRG2 was shown following increased Myc expression upon serum stimulation. The ectopic expression of c-Myc dramatically reduces the cellular Ndrg2 protein and mRNA level. We further identified the core promoter region of NDRG2 that is required for Myc repression on NDRG2 transcription, and we verified the interaction of Myc with the core promoter region both in vitro and in vivo. Moreover, the c-Myc-mediated repression of NDRG2 requires association with Miz-1, and possibly the recruitment of other epigenetic factors, such as histone deacetylases, to the promoter. The regulatory function of Myc on NDRG2 gene expression implicated the role of the Ndrg2 in regulating cell differentiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The myelocytomatosis viral oncogene (myc) was first identified as a cellular homolog of the viral oncogene myc (v-myc) from the retrovirus MC29 in 1978 by Bishop (1). Several research groups subsequently determined that the cellular myc (c-myc) proto-oncogene was activated in various animal and human tumors (2–4). The MYC gene family includes c-MYC (cellular MYC), B-MYC (brain-expressed), L-MYC (lung carcinoma-derived), N-MYC (neuroblastoma-derived), and s-MYC. However, only c-MYC, L-MYC, and N-MYC have neoplastic potential (5). MYC is undetectable in most normal tissues, but it is highly expressed in tumors. Uncontrolled expression of MYC causes abnormal proliferation that deregulates the cell cycle (6, 7). c-myc or N-myc knock-out mice result in embryonic lethality, suggesting that these genes are critical for development (5, 8). MYC is considered to be a strategic controller of the cell cycle and proliferation (9–11). As a transcription factor, Myc belongs to the basic helix-loop-helix and leucine zipper region protein family, which includes two N-terminal conserved transactivation domain Myc Boxes, I (MbI) and II (MbII), as well as the C-terminal basic helix-loop-helix and leucine zipper regions. The N-Myc and L-Myc proteins share several regions of at least 80% amino acids sequence homology to c-Myc. Myc functions as transcriptional activator by heterodimerizing with Max, another basic helix-loop-helix protein (12, 13). The Myc-Max heterodimers recognize consensus CACGTG E-box sequence in gene promoters (14, 15). Binding of the heterodimers to these enhancer sites regulates the transcription of the target genes. A growing number of cellular genes related to biosynthesis, metabolism, RNA structure determination, and cell cycle control are direct targets of Myc-Max transactivation (16). However, these genes may account for only a portion of the biological activation sites controlled by Myc. Indeed, Myc acts as a transcriptional repressor for a number of genes, including p15ink4b, p21Cip1, and p27Kip1. The mechanism for Myc repression has not been determined. Some genes are found repressed on the transcriptional level, and the response element is located at the transcriptional initiation region (17). Recently, a Myc-interacting protein, Myc-interacting zinc finger protein 1 (Miz-1), was demonstrated to participate in Myc-mediated repression on p15ink4b and p21Cip1 (9, 18–20).

Human NDRG (N-Myc downstream-regulated gene) family consists of the following four members: NDRG1, NDRG2, NDRG3, and NDRG4 (8, 21–26). Although NDRG members do not possess a clear functional peptide motif, they do share many well conserved residues among all members. The percentage of shared residue identities among members is roughly 60%. Phylogenetic analysis revealed that NDRG1 and -3 belong to one subfamily, whereas NDRG2 and -4 belong to another (24). The various NDRG family members are reportedly intimately involved in cellular differentiation and development. NDRG1, also known as DRG1, CAP43, RTP/rit42, and PROXY-1 (27–30), has been associated with differentiation (31), embryo development (23), and has a documented function in tumor suppression (32, 33). NDRG1 expression is up-regulated in various components of the nervous system, including injured sciatic nerves (34), and may play a key role in hereditary motor and sensory neuropathy, Lom. The ndrg1-deficient mice exhibit a progressive demyelinating disorder in the peripheral nervous system (35). The hypoxia and nickel reagent could induce human NDRG1 expression in an HIF-1-dependent manner (31, 36, 37). In human leukemia and intestinal cells, NDRG1 expression is up-regulated after induced differentiation (38, 39). Taken together, NDRG1 is induced under a number of stress and pathological conditions.

We initially identified the human NDRG2 (GenBankTM accession number AF159092 [GenBank] ) as a candidate for tumor suppressor gene. The expression of NDRG2 in human glioblastoma tissues is significantly lower than that in the normal brain. Transfection of human glioblastoma U373 cells with NDRG2 markedly reduced proliferation of the glioblastoma cells (26). Recently, NDRG2 was shown to be up-regulated in Alzheimer disease brains (38) and could induce the differentiation of dendritic cells (40). Meanwhile, Takahashi and co-workers (39, 41) reported that NDRG2 may facilitate neurite outgrowth of nerve growth factor-differentiated PC12 cells, suggesting the involvement of Ndrg2 in differentiation. These findings implicate Ndrg2 as being associated with cell growth and differentiation.

Mouse ndrg1 expression is repressed by N-Myc and c-Myc (25) via an unknown mechanism. So far, it is uncertain whether human NDRG2 is really regulated by Myc because mouse ndrg2 or -3 is not affected. Defining the functional interaction between Myc and NDRG2 will facilitate the functional understanding of both molecules in cell differentiation and proliferation.

In this study, we first displayed an inverse regulatory relationship between human NDRG2 and MYC gene expression events in induced cell differentiation and proliferation. It was confirmed that the ectopic expression of MYC could down-regulate expression of NDRG2. Finally, through a series of in vitro and in vivo experiments, we demonstrate that Myc represses NDRG2 gene expression via Miz-1-dependent interaction with NDRG2 core promoter region.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs—The human NDRG2 promoter was amplified from BAC clone R-998D10 (The Children's Hospital of Philadelphia, Philadelphia; GenBankTM accession number AL161668.6) with the Advantage-GC Genomic polymerase (Clontech). The resulting amplicon was cloned into the pGL3-basic vector (Promega, WI) to generate the NDRG2/luciferase reporter plasmid. Various mutants of the human NDRG2 promoter were generated with PCR by using the cloned promoter as template. All of the newly constructed plasmids were confirmed by sequencing. The pcDNA3.1-c-MYC was generously provided by Dr. Michael Cole (Lewis Thomas Laboratory, Princeton University Princeton, NJ). The pBabepuro Myc-ERTM and pBabepuro {Delta}Myc106–143-ERTM were the gifts from Dr. Gerard Evan (Cancer Research Institute, University of California, San Francisco). The pcDNA3.1-MIZ-1 was kindly provided by Dr. Ben-Zion Levi (Dept. of Biotechnology and Food Engineering, Technion, Haifa, Israel).

The siRNA4 vectors of pSilencer3.1-MIZ-1 and pSilencer3.1-YY-1 were constructed as described (42, 43). The sequences of MIZ-1 and YY-1 siRNAs are 5'-gccttacctgtgtgataag-3' and 5'-ggagcagaagcaggtgcag-3', respectively.

Cell Culture and the Induction of Differentiation and Proliferation—All cell lines, including HEK293, HeLa, human intestinal cells Caco-2, HT-29, human leukemia cells HL60, U937, and mouse fibroblasts NIH3T3 cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) in a humidified 5% CO2 atmosphere at 37 °C. HT-29 cell differentiation was induced with DMEM (without glucose) supplemented with 10% dialyzed FBS and 2.5 mM inosine for four passages and then cultured for 10 days (44). Differentiation of Caco-2 cells to enterocyte-like cells was induced with high glucose DMEM with 10% FBS. Cells at 70–80% confluency were trypsinized and placed onto 12 Transwell plates (24 mm diameter, 3.0-µm pore size; Costar, Cambridge, MA), at density of 105 cells/well, and cultured for 21 days (45). Exponentially growing HL60 and U937 cells were treated with 1 µM all-trans-retinoic acid (Promega) (44) to induce cell cycle arrest and differentiation for the indicated time periods. In serum stimulation, the full culture medium was replaced with DMEM with 0.1% FBS for 48 h. Then the media were changed with DMEM containing 10% FBS, and the cells were collected at the indicated time points.

RT-PCR—The cells were collected at the indicated time points, and the total RNA was isolated from each sample by using TRIzol (Invitrogen) and then quantified. Two µg of total RNA was reverse-transcribed with reverse transcriptase (Promega, WI) according to the manufacturer's instructions. All PCR experiments were performed with Taq polymerase (Promega, WI) and the primers 5'-gcccagcgatccttacctacc-3' and 5'-ggctgcccaatccatccaacc-3' for NDRG2, 5'-gatgacatggtcacgctggctacc-3' and 5'-tgggctgtgtggattcgcacatgc-3' for MIZ-1,5'-gatgctctatcttgctctgtaatc-3' and 5'-tttgaagtatgcatgtaactgccc-3' for YY-1, and 5'-gcctcaagatcatcagcaat-3' and 5'-aggtccaccactgacacgtt-3' for GAPDH control.

Cell Lysis and Western Blot—The cells were collected and lysed in 100 µl of buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride. Fifty µg of cell lysate (quantificationally measured by BCA protein assay; Pierce) was resolved by SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences). The blots were probed with various antibodies, such as anti-c-Myc rabbit polyclonal (sc-764; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Ndrg2 goat polyclonal (Santa Cruz Biotechnology), anti-CD18 mouse monoclonal antibody (Santa Cruz Biotechnology), anti-Miz-1 rabbit polyclonal (sc-22837; Santa Cruz Biotechnology), and anti-beta-actin rabbit polyclonal (Boster, Wu Han, China). To visualize primary antibody-bound protein, the secondary antibodies conjugated to horseradish peroxidase (1:4000 dilution; Santa Cruz Biotechnology) and ECL detection solutions (Pierce) were applied. The scanned images were quantified using Kodak Digital Science one-dimensional software (Eastman Kodak Co., New Haven, CT).

Transfection and Reporter Gene Assay—The cells used for transfection (HEK293 and HeLa) were seeded in 96-well plates at a density of 2 x 104 cells/well and transfected the next day using Lipofectamine 2000 (Invitrogen) at a confluency of about 80%. Briefly, 0.1 µg of reporter DNA was co-transfected with 50 ng of pcDNA3.1-c-MYC (designated as Myc) or its mutant counterparts (the total amount was normalized with empty vector). Five ng fo pRL-CMV and 0.1 µg of green fluorescent protein were co-transfected to monitor the transfection efficiency. Forty eight hours after transfection, the luciferase activity was measured by using the dual luciferase reporter assay system (Promega).

Inducible Activation of Myc—HeLa cells were transfected with MycER or {Delta}MycER expression plasmid, the latter deleted the N-terminal 106–143 residues of Myc. After 24 h, 4-hydroxytamoxifen (4-OHT) (Sigma) was added into cells to a final concentration of 300 nM. The activation (nuclear translocation) of Myc was confirmed by immunofluorescence assay with anti-Myc rabbit polyclonal (sc-764, Santa Cruz Biotechnology) and fluorescein isothiocyanate-conjugated anti-rabbit antibody. Cells were harvested at indicated time points and applied to Western blotting with antibody against Ndrg2.

DNA Pulldown Assay—DNA pulldown assay was performed as described (49). DNA covering the core promoter region of NDRG2(–79 to +57 bp) was amplified by PCR with primers, one of which was labeled with biotin. Two µg of purified PCR product was attached to streptavidin-Sepharose beads according to the manufacturer's guidelines (Pierce). The beads were washed twice with buffer A (20 mM HEPES, pH 7.9, 1 mM MgCl2, 40 mM KCl, 0.1 mM EGTA, pH 8.0, 1 mM dithiothreitol, and 10% glycerol (v/v)) and blocked in the same buffer supplemented with 0.5% bovine serum albumin at room temperature for 30 min. Before use, the beads were washed three times with buffer A and then incubated with 50 µg of nuclear extract protein in 350 µl of buffer A containing 5 µg of poly(dI·dC) at room temperature for 30 min. Sepharose beads with adsorbed proteins were pelleted and washed, and then the bound proteins were analyzed by SDS-PAGE and Western blotting analysis as described, using antibodies against c-Myc, Miz-1 as indicated. The double strand DNA covering putative NF-{kappa}B recognizing sequence (–1455/–1160) upstream of the NDRG2 promoter and T7 primer sequence were used as control.

Chromatin Immunoprecipitation—The cells were cross-linked with 1% formaldehyde and lysed as described (49). The chromatin DNA was sonicated to an average size of 300–500 bp. The immunoprecipitations were carried out in a ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl, pH 8.1, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A) with the following antibodies: anti-Myc rabbit polyclonal (sc-764; Santa Cruz Biotechnology), anti-FLAG mouse M2 monoclonal (F3165; Sigma), anti-acetylhistone H3 rabbit polyclonal antibody (06-599; Upstate), antiacetylhistone H4 rabbit polyclonal antibody (06-866; Upstate), and normal rabbit serum as control. The precipitated DNA was amplified by PCR using primers 5'-ggcattgaccccagagtccctg-3' (–75/–54) and 5'-gaagttggacaacaaggcgggg-3' (+29/+50) for NDRG2 core promoter region.

Statistics Analysis—We performed t tests and analysis of variance on independent samples using the statistical software SPSS (version 10.0; SPSS, Chicago, IL) to evaluate the differences. Statistical significance was set at p < 0.01 and p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NDRG2 Expression Was Enhanced in Differentiated Cells Along with the Reduced Myc Content—Reportedly, the differentiation programs of intestinal cells and leukemia cells can induce the down-regulation of Myc (16). To explore the possible relationship between Myc and NDRG2 expression regulation, we induced the differentiation of the intestinal cell lines Caco-2 and HT-29 and the leukemia cell lines U937 and HL60, respectively, and we analyzed the resulting changes of gene and protein expression levels. The differentiation status of Caco-2 and HT-29 cells was confirmed by examining the presence of regular brush borders and tight junctions, which are structural markers of differentiation (supplemental Fig. S1), using transmission electron microscopy. The Myc protein level significantly declined after the Caco-2 and HT-29 cells differentiated, meanwhile the protein level of Ndrg2 increased markedly (Fig. 1A). A similar phenomenon also occurred in the differentiated leukemia cells U937 and HL60. The expression of CD18 was used as the marker of differentiation here (48). During the retinoic acid-induced differentiation, the c-Myc protein concentration declined distinctly over time in the U937 and HL60 cells and was almost undetectable at day 9 (Fig. 1B). In contrast, the Ndrg2 protein amount showed simultaneous augmentation.

NDRG2 Was Down-regulated following the Induction of c-Myc in Growth-stimulated HeLa Cells—To further confirm the functional interactions between NDRG2 and c-MYC, we examined their gene expression behaviors in serum-stimulated HeLa cells. Serum deprivation followed by serum stimulation results in a transient induction of c-Myc (45). In this experiment, c-Myc expression was induced rapidly 2 h after serum stimulation and reached the highest level after 4 h. Following 10 h of stimulation, the c-Myc level declined gradually, until it reached basal level after 24 h (Fig. 1C). In contrast, Ndrg2 displayed the opposite kinetics. As shown in Fig. 1C, the amount of the Ndrg2 started to decline following 4 h of serum stimulation, an event that lagged ~2 h behind the decline of c-Myc expression. Ndrg2 maintained its lowest level from 6 to 12 h post-stimulation. After 24 h of stimulation, the Ndrg2 level recovered partially. This staggered induction time line between c-Myc and Ndrg2 suggests a regulatory role for c-Myc on the NDRG2 gene.

Ectopic Expression of c-Myc Resulted in Down-regulation of Ndrg2—To assess directly whether c-Myc can down-regulate the NDRG2 expression, we transfected a construct harboring a c-Myc fused with the ligand-binding domain of the estrogen receptor (ER) into HeLa cells. This system permits an inducible nuclear translocation of MycER by 4-OHT, which was confirmed here by immunofluorescence analysis (supplemental Fig. S2). The expression of MycER and {Delta}MycER relative to the endogenous Myc protein is shown in Fig. 2A. The Ndrg2 level declined following the MycER activation and reached the lowest level at 24 h post-activation in HeLa cells (Fig. 2B). However, the Ndrg2 level was not changed in the cells transfected with the mutant {Delta}MycER, in which the sequences responsible for transcriptional repression (9, 17) were deleted (Fig. 2B). The data from the semi-quantitative RT-PCR assay were consistent with those of the Western blotting analysis (Fig. 2, C and D), suggesting the repression of Myc on NDRG2 occurred at the transcriptional level.


Figure 1
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  		FIGURE 1.
Inverse regulation of Myc and human Ndrg2 in differentiated cells and in HeLa cells under proliferation stimuli. A, protein levels of Ndrg2 and c-Myc in induced differentiation of Caco-2 and HT-29 cells. For the differentiation of Caco-2, HT-29 was induced as described under "Materials and Methods." U, undifferentiation; D, differentiation. B, protein levels of Ndrg2, c-Myc, and the CD18 along the process of all-trans-retinoic acid-induced differentiation of U937 and HL60 cells. C, expression kinetics of c-Myc and Ndrg2 in serum-stimulated HeLa cells. The protein levels was detected by Western blotting.

 
Myc Represses the Promoter Activity of NDRG2—To address the mechanism of Myc-induced transcriptional repression, we amplified the putative promoter region flanking 5' of the first exon of the NDRG2 gene (–1455/+274) (supplemental Fig. S3). Reporter gene assay confirmed the promoter activity, which is partly consistent with the prediction based with an on-line promoter scan service. The core promoter region was mapped within –79/+57 with series deletion assay (see Fig. 3A). Co-transfection of c-MYC or N-MYC with the NDRG2/luciferase reporter gene in HEK293 cells, respectively, resulted in repression of the NDRG2 promoter (Fig. 3B). To map the domains of Myc required for repression, we deleted the N-terminal Myc box II (MbII) and the C-terminal helix-loop-helix domain of c-Myc and N-Myc, and we designated them as {Delta}N and {Delta}C, respectively. As shown in Fig. 3B, the N-terminal deletion reduced the Myc-induced repression of NDRG2 partially, whereas the C-terminal deletion abolished Myc-induced repression completely. Similar results were obtained in HeLa cells (data not shown). The relative equivalent expression of wild type and mutant c-Myc and N-Myc proteins was confirmed by Western blotting (Fig. 3B).

For determining whether Myc could repress NDRG2 at the physiological level, endogenous c-Myc was knocked down by antisense RNA in HEK293 cells. The endogenous c-Myc protein decreased to undetectable levels upon antisense RNA treatment, which led to an enhanced activity of NDRG2 promoter. In contrast, overexpression of Myc resulted in reduced activity of the NDRG2 promoter (Fig. 3C).

Core Promoter Region of NDRG2 Is Responsible for the Myc-specific Repression—To further map the elements on the NDRG2 promoter that respond to Myc repression, a series of promoter deletion mutants were produced and used in the reporter assays (Fig. 3A). Deletions from the nucleotide positions –1455 to –79 bp, relative to the transcription start site of NDRG2, did not abolish the relative repression by c-Myc. This finding suggested that the c-Myc repressor response region is located within –79 to +57 bp of the NDRG2 promoter (Fig. 3D). Then the core promoter region (–79/+57) was replaced with the one from the thymidine kinase promoter (–206/+10). As shown in Fig. 3E, the replacement abolished the repression of Myc on the chimera promoter, indicating that the core region of human NDRG2 is responsible for the transcriptional repression of Myc.

Myc Associates with the Core Promoter of NDRG2—To examine whether Myc associates with the NDRG2 core promoter region, nuclear extract from HeLa cells expressing ectopic c-Myc was applied to a DNA pulldown assay with the double strand DNA covering the core promoter region (–79 bp to +57 bp) of NDRG2. As shown in Fig. 4A, c-Myc bound to the core promoter region of NDRG2, but not to the region covering a putative NF-{kappa}B recognizing sequence located upstream of NDRG2 core promoter. To confirm the Myc/NDRG2 promoter association in vivo, ChIP assay was performed in HeLa cells expressing ectopic or endogenous c-Myc, respectively. The core NDRG2 promoter fragment was co-precipitated by anti-c-Myc antibody specifically. On the other hand, DNA sequence 1 kb away from the core promoter of NDRG2 (–1455/–1304, and +1093/+1215) failed to be enriched by anti-Myc antibody (supplemental Fig. S4A), revealing the association of Myc with the core promoter region of NDRG2 in vivo (see Fig. 4B). Additionally, we failed to co-precipitate the core promoter fragments of NDRG3 and NDRG4 with the same antibody (supplemental Fig. S5), suggesting that c-Myc was not recruited to the core promoter fragments of NDRG3 and NDRG4.


Figure 2
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  		FIGURE 2.
Ectopic expression of Myc represses Ndrg2 expression. A, ectopic expressions of MycER and {Delta}MycER detected by Western blotting. B, level of Ndrg2 detected by Western blotting. C, transcription of NDRG2 assayed by RT-PCR following the time course of MycER and {Delta}MycER activation induced by 4-OHT. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, relative quantification of the expression of NDRG2 mRNA. The result of the RT-PCR product is analyzed with Kodak Digital Science one-dimensional software. The figure shows the tendency of each group as indicated. All the assays were duplicated for at least three independent experiments. The results are shown as the mean ± S.D.

 
Miz-1 Participates in the Transcriptional Repression of Myc on NDRG2—As a Myc-interacting zinc finger protein, Miz-1 is presently one of the major molecules mediating the transcriptional repression of Myc on its target genes (20, 49). To investigate the possible involvement of Miz-1 in Myc-mediated repression on the NDRG2 gene, we first checked the binding of Miz-1 with NDRG2 core promoter. The FLAG-MIZ-1 cDNA was transfected into HeLa cells, and ChIP assay was conducted using anti-FLAG antibody. The core NDRG2 promoter fragment was co-precipitated with anti-FLAG antibody (Fig. 5A). The sequences located 1 kb away from the core promoter region of the NDRG2 (both upstream and downstream) were not enriched in DNA from anti-FLAG-Miz-1 ChIP (supplemental Fig. S4B), suggesting the binding of Miz-1 with the core promoter region of NDRG2 particularly in vivo. To confirm the binding, we performed DNA pulldown assay using a double strand DNA oligonucleotide covering the core promoter region (–79 bp to +57 bp) of NDRG2. The initiator element of p15ink4b promoter served as a positive control. As shown in Fig. 5B, Miz-1 displayed equivalent binding to core region of both NDRG2 and p15ink4b promoter.

To evaluate the possible contribution of Miz-1 to Myc-mediated repression on the NDRG2 gene, we transfected MIZ-1 vector together with c-MYC and NDRG2/luciferase reporter gene into HEK293 cells. The lower dose of ectopic expression of Miz-1 in HEK293 cells enforced the repression of c-Myc on the NDRG2 promoter (Fig. 5C). However, increasing the amount of Miz-1 reduced the repression ability of Myc on the NDRG2 promoter. It is possible that the excessive Miz-1 redistributes Myc in cytoplasm by specific interaction, which results in the reduction of Myc in nucleus for repression on target genes (50). To determine whether the Myc/Miz-1 association is required for Myc-mediated repression of NDRG2, we deleted the C-terminal 637–803 amino acid residues of Miz-1, a region contributing mostly to the interaction with Myc (20). The expression of the mutant Miz-1 abolished the repression of Myc on NDRG2. Surprisingly, the repression was reversed with an excessive amount of Miz-1 mutant. The maximal enhancement of NDRG2 promoter activity by the Miz-1 mutant reached about 3.5-fold. Similar results were observed in the cells without the ectopic expression of c-Myc (supplemental Fig. S6). Because the Miz-1 mutant used here still holds its DNA binding ability, its up-regulation effect on NDRG2 promoter activity may attribute to the recruitment of other co-activators instead of c-Myc.

To confirm the requirement of interaction between Miz-1 and Myc for the repression of Myc on NDRG2, we then introduced a single mutation (V394D) into Myc, which was reported to disrupt the interaction between Myc and Miz-1 specifically (20). As shown in Fig. 5C, the mutation compromised the Myc-mediated repression of NDRG2.


Figure 3
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  		FIGURE 3.
Myc represses human NDRG2 promoter activity on the core region. A, promoter activity of NDRG2 and its deletion mutants. The upper panel illustrates schematically the structure of NDRG2 promoter. Numbers on the left side of the bars indicate the relative position of the deletion. HEK293 cells were transfected with these different fragments of the NDRG2 promoter, respectively. The relative luciferase activity (firefly luciferase/Renilla luciferase) was analyzed 48 h later. B, co-transfection of N-MYC or c-MYC or their mutants with NDRG2(–1455/+274)/luciferase. {Delta}C represents the deletion of helix-loop-helix-ZIP region of Myc. {Delta}N represents the deletion of MbII region. The Western blot (WB) results show the relative expression of wild type and mutants of c-Myc and N-Myc. The left and right panels represent c-Myc and N-Myc, respectively. C, NDRG2/luciferase reporter gene activity in HEK293 cells under different levels of Myc. The left panel shows the expression of endogenous Myc (control), enforced expression of Myc by transfection with c-MYC, and the elimination of endogenous Myc by antisense RNA, respectively. D, co-transfection of c-MYC with series deletions of the NDRG2 reporter gene. The numbers at the left side of the bars are the relative position of each deletion. E, core promoter region of NDRG2 was replaced by the thymidine kinase core promoter (–206/+10) and named as NDRG2(–1455/–300)-TK. The luciferase activity was analyzed. All the values of relative luciferase activity assay show as the treated group standardized by their control, respectively. The value of luciferase activity of the reporter gene co-transfected with pcDNA-3.1(+) vector (control) was set to 1.0. The results are the mean ± S.D. All the assays were duplicated for at least three independent experiments. In each experiment, the individual data were calculated as the means of triplicates. *, p < 0.01; **, p < 0.05 versus control.

 
To investigate whether Miz-1 is required for Myc-mediated repression of NDRG2 in particular, we constructed the siRNA vectors of pSilencer3.1-MIZ-1 and pSilencer3.1-YY-1, respectively. The knockdown efficiency of MIZ-1 and YY-1 was verified by RT-PCR. As shown in Fig. 5D, knockdown of MIZ-1 by siRNA reduced the repression of c-Myc on the NDRG2 promoter significantly. Neither pSilencer3.1 vector nor pSilencer3.1-YY-1 interfered with the repression of Myc on the NDRG2 promoter. The endogenous expression of NDRG2 was detected and showed a similar tendency as the changes of the promoter activity. This result indicated the specificity of Miz-1 required for Myc-mediated repression of NDRG2. This result shows the specific requirement of Miz-1 required for Myc-mediated repression of NDRG2.


Figure 4
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  		FIGURE 4.
Myc was associated with core region of NDRG2 promoter. A, DNA pulldown assays of HeLa cells express ectopic c-Myc with double strand DNA covering the core promoter region of NDRG2. Double strand DNA from upstream of the NDRG2 promoter (–1455/–1160) containing a putative NF-{kappa}B-binding site was set as control. B, ChIP assay of HeLa cells using antibody against Myc and primers specific to core promoter region of NDRG2. The left panel is the amplified products of co-precipitated DNA from HeLa cells expressing ectopic c-Myc. The right panel is from HeLa cell expression of endogenous c-Myc. An arrow indicates the amplified fragment.

 
The Transcriptional Repression of Myc on NDRG2 May Require HDAC Activity—Many transcriptional repressors associate with HDACs by bridging proteins that function as co-repressors. We therefore tested whether transcriptional repression of the NDRG2 promoter by Myc requires HDACs activity. We first detected the expression kinetics of Myc and Ndrg2 under serum stimulation in the presence of the HDAC inhibitor TSA. In the presence of TSA, c-Myc expression was still up-regulated rapidly upon serum stimulation as usual, but the NDRG2 gene expression level was not repressed as expected (Fig. 6A). The abolishment of the transcription repression of NDRG2 is not because of the direct effect of TSA on the NDRG2 gene because TSA treatment itself had no effect on the expression of NDRG2 (supplemental Fig. S7). Reporter gene assays were also performed as described above using NDRG2(–1455/+274)/luciferase in the presence or absence of TSA. As shown in Fig. 6B, TSA treatment compromised the repression of Myc on NDRG2 in a dose-dependent manner, suggesting that HDACs activity is required for Myc-mediated repression on the NDRG2 promoter.

To examine the affects of c-Myc on histone acetylation status in the core promoter region/proximal regulatory region of the NDRG2 gene, ChIP assays with anti-acetylhistone H3 and anti-acetylhistone H4 antibody were performed. HeLa cells were transfected with wild type or the mutants of c-Myc. However, the amounts of acetylated histones bound to the NDRG2 core promoter were not different among each of the transfected groups (Fig. 6C), suggesting the recruitment of c-Myc to the NDRG2 core promoter does not necessarily influence the amount of acetylated histones at this region (51).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Myc, as an essential oncoprotein family member, participates in the transcriptional program driving cell proliferation (45, 46) and inhibiting cell differentiation (52). As a master switch for cell proliferation and differentiation, Myc exerts its biological function mainly by transcriptional regulation of its target genes (47). Mouse ndrg1 expression was found to be repressed by N-Myc and c-Myc (23), suggesting ndrg1 to be a downstream target gene of Myc. However, mouse ndrg2 seems not to be regulated by N-Myc, because the expression of ndrg2 was not up-regulated in tissues of N-myc knock-out mice (22). By using reporter gene assay, we did not detect repression of Myc on the mouse ndrg2 promoter in NIH 3T3 cells (supplemental Fig. S8), supporting the concept that mouse ndrg2isnot regulated by Myc.

Human NDRG family members have been found intimately involved in cell proliferation and differentiation. Human NDRG2 was named as N-Myc downstream-regulated gene 2 mainly because of its sequence similarity to NDRG1. Nonetheless, it is uncertain whether NDRG2 is regulated by Myc or not. In this study, we provide the first evidence to indicate that human NDRG2 is a real Myc downstream-regulated gene.

First, we found the inverse regulation between Myc and human Ndrg2 under induced differentiation and serum stimuli, respectively, which suggested the functional consequence between Myc and Ndrg2 in cell differentiation and proliferation. We further show that enforced expression of Myc represses NDRG2 at least in part at a transcriptional level. This is further supported by reporter gene assay, in which enforced expression of c-Myc represses the NDRG2 promoter. On the other hand, knockdown of the endogenous c-Myc in HEK293 cells enhances the expression of the transfected NDRG2 reporter gene, suggesting that the repression of c-Myc on the NDRG2 promoter acts on physiological level. Combining the observation above, it is reasonable to propose that NDRG2isa Myc downstream-regulated gene, as it is named. Regarding the expression divergence of the NDRG2/ndrg2 gene in human and mouse, the different response of the genes to Myc suggests the differential regulation and function of the two genes in mouse and human (53).

Myc represses several target genes on the core promoter region in which the initiator is responsive to the repression (17). In the case of NDRG2, the region responsive to Myc-mediated repression is also located in the core promoter region in which no E-box is found. It suggests that the mechanism of repression of Myc on NDRG2 is different from that of activation, which is dependent on the direct binding of the Myc/Max heterodimer to the E-box in the target genes. In agreement to this proposition, the N-terminal MbII is required for the repression but not for activation of Myc on target genes (17).


Figure 5
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  		FIGURE 5.
Miz-1 is required in the transcriptional repression of Myc on NDRG2. A, ChIP assay. ChIP was performed using the anti-FLAG antibody. The co-precipitated DNA was amplified using primers targeting the NDRG2 core promoter. An arrow indicates the amplified fragment. The data are representative of three experiments. B, DNA pulldown assay of HeLa cell transfected ectopic MIZ-1 with double strand DNA covering the core promoter region of NDRG2. T7 and p15ink4b transcriptional initiator served as negative and positive control, respectively. C, HEK293 was transfected with c-MYC (0.05 µg) or MYCV394D (0.05 µg), respectively, or co-transfected c-MYC (0.05 µg) with increasing amounts (0.01, 0.05, and 0.1 µg) of Miz-1 and its mutant. The NDRG2(–1455/+274)/luciferase reporter was transfected in all of the above groups. The total DNA amount of each well was balanced with pcDNA3.1(+). The luciferase activity was assayed and analyzed as above. The results are the mean ± S.D. All the assays were duplicated for at least three independent experiments. In each experiment, the individual data were calculated as the means of triplicates. WB, Western blot. **, p < 0.01; *, p < 0.05 versus control. D, transfected the NDRG2(–1455/+274)/luciferase plasmid with c-MYC (0.05 µg) or with pSilencer3.1-MIZ-1 (0.05 µg), pSilencer3.1 control vector (0.05 µg), and pSilencer3.1-YY-1 (0.05 µg), respectively, in HEK293 cells. The total DNA amount of each well was balanced with pcDNA3.1(+). The luciferase activity was assayed and analyzed as above. The results are the mean ± S.D. All the assays were duplicated for at least three independent experiments. In each experiment, the individual data were calculated as the means of triplicates. *, p < 0.05 among each compared group. The endogenous expression of NDRG2 and the knockdown of MIZ-1 and YY-1 were detected by RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the control.

 
Mouse ndrg1, as the first identified N-Myc downstream-regulated gene, is repressed by N-Myc. The region responsive to the repression of N-Myc is located in the core promoter region. However, it has not been determined whether ndrg1 is a direct target gene of Myc because no direct binding of N-Myc to this region was detected (23). Although DNA pulldown and ChIP assay revealed the association of Myc with the human NDRG2 core promoter region both in vitro and in vivo, we failed to show the direct binding of Myc to this region by using gel shift assay (data not shown). It is implied that the association of Myc to NDRG2 core promoter maybe mediated by some other proteins. In agreement with this speculation, the deletion of the helix-loop-helix-ZIP region of Myc, a domain responsible for interaction with other proteins, abolished the repression of Myc completely. There are various candidate proteins that have been proposed to target Myc to the core promoters, such as TFII-I, YY-1, Sp-1, Miz-1, Smad-2 and -3, and NF-Y proteins (54). In the case of NDRG2, we provide several lines of evidence for indicating the involvement of Miz-1 in the repression of Myc as follows: 1) Miz-1 binds to NDRG2 core promoter; 2) co-transfection of MIZ-1 enforced the repression of Myc; 3) the deletion of the interacting domain in Miz-1, functioning as a dominant negative mutant, abolishes the repression of Myc on NDRG2; 4) excessive Miz-1, especially the mutant, reverses the repression of Myc; and 5) the single residue mutation (V394D) introduced into Myc, which is the critical residue for specific interaction with Miz-1, compromises the repression on NDRG2 promoter. The specific requirement of Miz-1 for Myc-mediated repression of NDRG2 was further confirmed by the knockdown of MIZ-1 siRNA.

The requirement of the MbII domain for the repression of Myc suggests a possible alternative mechanism by which Myc represses NDRG2. The function of MbII in the repression of Myc is controversial. MbII has been indicated to interact with TATA box-binding protein both in vitro and in vivo (55, 56). This interaction might contribute to the recruitment of Myc to the core promoter region. However, in the case of NDRG2, there is no canonical TATA box found in the core promoter region, thus not supporting the possibility of this interaction on the promoter. Moreover, MbII is regarded as a transactivation domain that interacts with TRRAP and TIP48/TIP49, which are components of huge complexes containing histone acetyltransferase activity involved in the regulation of chromatin structure (47). Although there is no report that Myc interacts with repressor or co-repressor, it is tempting to speculate that Myc may recruit repressors or co-repressors to the core promoter regions by an indirect way. In the case of NDRG2, we show that in the presence of TSA, a specific HDAC inhibitor, the repression of Myc on NDRG2 can be dramatically reduced, suggesting the requirement of HDAC activity for the repression. However, the repression of Myc on NDRG2 did not influence the acetylation level of histones at the core promoter. It is possible that Myc did not alter the histone acetylation status directly (51). HDACs were just recruited to the huge repression complex of Myc on human NDRG2. The mechanism whereby Myc recruits HDACs to the core promoter region remains to be explored.


Figure 6
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  		FIGURE 6.
Repression of Myc on NDRG2 is reduced in the presence of HDAC inhibitor TSA. A, kinetics of protein amount of c-Myc and Ndrg2 following serum stimulation of HeLa cells in the presence of HDAC inhibitor TSA. After 48 h of serum starvation, cells were treated with 100 ng/ml TSA and 10% FBS. B, c-Myc was co-transfected with NDRG2 (–1455/+274)/luciferase reporter gene into HeLa cells. Transfected cells were cultured for 40 h, followed by treatment with or without TSA for 8 h. Cells were collected, and luciferase activity was examined. Final TSA concentration is 100, 200, and 400 nM, respectively (represented as +, ++, and +++). pRL-CMV was co-transfected for normalization. C, effects of c-Myc on histone acetylation status in core promoter region of the NDRG2 gene. For the transfected wild type and the mutants of c-Myc for 48 h, HeLa cells were performed to ChIP assays with anti-acetylhistone H3 and anti-acetylhistone H4 antibody, respectively. The data are the representative of three experiments.

 
In summary, we indicate the first time that human NDRG2is a real Myc downstream-regulated gene repressed by Myc on its core promoter region. The repression of the NDRG2 mediated by c-Myc requires an association with Miz-1 and possibly the recruitment of other epigenetic factors, such as HDACs, to the promoter.


   FOOTNOTES
 
* This work was supported in part by Chinese National Key Basic Research and Development Program 2002CB513007, Program PCSIRT0459 for Changjiang Scholars and Innovative Research Team in University in China, and National Natural Science Foundation of China Grants 30370315, 30228012, and 30670452. 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S8. Back

1 The first two authors should be regarded as joint first authors. Back

2 Recipient of a University grant for Ph.D. thesis. Back

3 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, The Fourth Military Medical University, 17 Changle Western Rd., 710032, Xi'an, China. Tel.: 86-29-84774513; Fax: 29-84774513; E-mail: bioyao{at}fmmu.edu.cn.

4 The abbreviations used are: siRNA, short interfering RNA; DMEM, Dulbecco's modified Eagle's medium; 4-OHT, 4-hydroxytamoxifen; MycER, Myc-estrogen receptor; ChIP, chromatin immunoprecipitation; FITC, fluorescein isothiocyanate; HDAC, histone deacetylase; FBS, fetal bovine serum; RT, reverse transcription; TSA, trichostatin. Back



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