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Dual functions of transcription factors, transforming growth factor-beta-inducible early gene (TIEG)2 and Sp3, are mediated by CACCC element and Sp1 sites of human monoamine oxidase (MAO) B gene.

Monoamine oxidases (MAO) A and B catalyze the oxidative deamination of many biogenic and dietary amines. Abnormal expression of MAO has been implicated in several psychiatric and neurodegenerative disorders. Human MAO B core promoter (-246 to -99 region) consists of CACCC element flanked by two clusters of overlapping Sp1 sites. Here, we show that cotransfection with transforming growth factor (TGF)-beta-inducible early gene (TIEG)2 increased MAO B gene expression at promoter, mRNA, protein, and catalytic activity levels in both SH-SY5Y and HepG2 cells. Mutation of the CACCC element increased the MAO B promoter activity, and cotransfection with TIEG2 further increased the promoter activity, suggesting that CACCC was a repressor element. This increase was reduced when the proximal Sp1 overlapping sites was mutated. Similar interactions were found with Sp3. These results showed that TIEG2 and Sp3 were repressors at the CACCC element but were activators at proximal Sp1 overlapping sites of MAO B. Gel-shift and chromatin immunoprecipitation assays showed that TIEG2 and Sp3 bound directly to CACCC element and the proximal Sp1 sites in both synthetic oligonucleotides and natural MAO B core promoter. TIEG2 had a higher affinity to Sp1 sites than CACCC element, whereas Sp3 had an equal affinity to both elements. Thus, TIEG2 was an activator, but Sp3 had no effect on MAO B gene expression. This study provides new insights into MAO B gene expression and illustrates the complexity of gene regulation.

Monoamine oxidase (MAO) 1 plays an important role in the metabolism of biogenic amines, including serotonin (5-hydroxytryptamine), norepinephrine, dopamine, tyramine, and phenylethylamine (PEA). There are two isoenzymes of MAO (MAO A and MAO B) that exhibit distinct substrate and inhib-itor specificity (1, 2; for review, see Ref. 3). Although both forms of MAO are expressed throughout the body, they differ in celland tissue-specific expressions. Placenta and fibroblasts predominantly express MAO A (4,5), and platelets and lymphocytes express only MAO B (6). In the brain, even though MAO A prefers 5-hydroxytryptamine and MAO B prefers PEA as substrate, MAO B but not MAO A was found in serotonergic neurons (7,8). MAO A was expressed in catecholaminergic neurons, whereas MAO B is rich in astrocytes. Moreover, MAO B but not MAO A activity increases progressively in the brain throughout adult life (7,9).
MAO A and MAO B are coded by different genes (10) and are closely linked on X chromosome, Xp11. 2-11.4 (11). Both consist of 15 exons with identical exon-intron organization (12). The promoter of MAO A has bi-directional activity (13) and consists of a functional polymorphism in a 30-bp repeat sequence (14). The 5Ј-flanking sequence of MAO B gene has been characterized. The maximal promoter activity for MAO B gene was found to be in the Ϫ246 to Ϫ99 region. This region consists of two clusters of overlapping Sp1 sites separated by a CACCC element (15). Previously, we have shown that Sp1 and Sp3 can bind to both proximal and distal clusters of Sp1 sites in the human MAO B core promoter (16). Overexpression of Sp1 activates MAO B promoter activity, and its activation can be repressed by overexpression of Sp3 (16). The mutation of CACCC element increased the basal transcriptional activity of MAO B promoter dramatically in HepG2 and MG 1242 cell lines, suggesting that CACCC element is a repressor element (16). In this study, we have studied the transcription factors interacting with the CACCC element.
It has been shown that transcription factor TIEG (TIEG1 and TIEG2) represses gene transcription and regulates cell growth, development and differentiation (17) by binding to GC-rich sequences (18,19). In contrast, we have shown that TIEG2 activates MAO B gene expression. TIEG2 served as a repressor through the CACCC element but an activator through proximal Sp1 sites. We also found that Sp3 represses the transcription of the human MAO B gene by means of interacting with CACCC box and activates MAO B through the proximal Sp1 sites, although transcription factor Sp3 had no overall effect on MAO B promoter, as shown previously (16).

MATERIALS AND METHODS
Cell Lines and Reagents-The SH-SY5Y (human neuroblastoma) and HepG2 (human hepatocytoma) cell lines were purchased from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium supplemented with 10 mM Hepes, 2 mM L-glutamine, 100 units/ml penicillin, 10 g/ml streptomycin, and 10% fetal bovine serum (Invitrogen). Drosophila SL2 cells, which are deficient in Sp1like proteins, were maintained in Schneider's insect medium supple-mented with 10% (v/v) fetal bovine serum and antibiotics and grown at 25°C without CO 2 . Monoclonal anti-TIEG2 antibody was purchased from Transduction Laboratories (BD Biosciences). Polyclonal antisera against Sp3 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Site-directed Mutagenesis of the Human MAO B Proximal Promoter (Ϫ246 to Ϫ99 bp)-Site-directed mutagenesis was utilized to mutate FIG. 1. TIEG2 increases the human MAO B promoter activity. A series of the 5Ј-deletion constructs of human MAO B promoter fused with pGL2-basic luciferase vector (1 g) were cotransfected into SH-SY5Y or SL2 cells with TIEG2 expression vector (0.5 g) together with 20 ng of pRL-TK (Renilla luciferase expression vector, for normalization of transfection efficiencies). Control group was cotransfected with pcDNA3.1 vector. After 48 h, cells were harvested and then assayed for luciferase activity. Data were the mean Ϯ S.D. from three independent experiments, with triplicates for each experiment. Note that the TIEG2 response element was located within the core promoter region (Ϫ246 to Ϫ99 bp) of the human MAO B. The same result was obtained in HepG2 cells. The A in the initiation site ATG is defined as ϩ1.

FIG. 2. Expression of the human MAO B was increased by TIEG2 in a dose-dependent manner.
A, luciferase reporter gene assay. The core promoter region of MAO B, from Ϫ246 to Ϫ99, in pGL2 basic vector was cotransfected with different amounts of TIEG2 expression vector into SH-SY5Y and HepG2 cell lines for 48 h, as indicated in the figure. The luciferase activity was then determined. B, catalytic activity assay. TIEG2 expression vector (0, 100, and 400 ng) was transfected into SH-SY5Y or HepG2 cell lines for 72 h. The MAO B catalytic activity was determined by using 100 g of whole-cell protein extracts from SH-SY5Y or HepG2 cells. [ 14 C]PEA (10 M) was used as substrate. The assay is described under "Materials and Methods." C, Northern blot analysis. Thirty micrograms of total RNA from SH-SY5Y or HepG2 cells that were cotransfected with TIEG2 expression vector (0, 100, and 400 ng) and grown to confluence were loaded onto each gel lane and hybridized with the human MAO B probe (specific activity ϭ 2 ϫ 10 8 cpm/g). D, Western blot analysis. 40 g of whole-cell protein extracts from SH-SY5Y or HepG2 cells that were cotransfected with TIEG2 expression vector (0, 100, and 400 ng) were incubated with anti-MAO B antibody (1:500 dilution). Data were the mean Ϯ S.D. from three independent experiments. potential transcription element, CACCC box, and two clusters of Sp1 sites in both the core promoter region (Ϫ246 to Ϫ99) and MAO B 2-kb promoter (Ϫ2099 to Ϫ99). Mutant promoter constructs were generated using pGLB-(Ϫ246) construct or pGLB-(Ϫ2099) as a template. Mutagenesis was carried out using the Amersham Mutagenesis kit (Amersham Biosciences) following the manufacturer's instructions. The primers used for mutagenesis (with mutations in lower case) were 5Ј-GGCCCTGCGCTGCttCCGGCCTGCCTGG-3Ј (for CACCC element), 5Ј-GGCTGCAGAGCTGCGttCGGGttCGGCGGCCCTGCGC-3Ј (for the distal Sp1 sites), and 5Ј-GCCTGCGCGTAGGCttGCGttCTGGGCTGC-GCGTCCGG-3Ј (for the proximal Sp1 sites). The mutated nucleotide sequences of all mutant constructs were confirmed by DNA sequencing.
Transient Transfection and Luciferase Assay-Transfections in SH-SY5Y, SL2, and HepG2 cells were performed using Superfect transfection reagent (Qiagen) following the manufacturer's instructions. Exponentially growing cells were plated at a density of 5 ϫ 10 5 cells/well in six-well plates (Costar, Cambridge, MA) with 2 ml of medium and 10% fetal bovine serum; cells were grown until 50% confluence (24 -36 h). For promoter deletion and mutagenesis studies, 1 g of MAO B promoter-luciferase construct was co-transfected into the cells with 20 ng of plasmid pRL-TK (the herpes simplex virus thymidine kinase promoter fused upstream to the Renilla luciferase gene which is used as an internal control; Promega), as described previously (16). Full-length human TIEG2 expression vector was a gift from Dr. Raul Urrutia (20), Mayo Clinic, Rochester, MN. The expression plasmid pCMV-Sp3 was kindly provided by Dr. P. Charnay. For co-transfection experiments, the total amount of DNA for each transfection was kept constant by the addition of empty expression vector pCMV3.1.
Gel-shift Assays-The DNA fragments (CACCC element, 5Ј-TGC-GCTGCACCCGGCCTGCCTGG-3Ј, and the proximal Sp1 sites, 5Ј-CG-TAGGCGGGCGGGCGGGGCTGG-3Ј) for the gel-shift assay were radiolabeled by Klenow fill-in. Labeled probes were purified by gel electrophoresis (5% polyacrylamide) and eluted in TE buffer (10 mM Tris/1 mM EDTA, pH 8.0). For DNA-protein binding, 15 g of nuclear extracts were diluted in binding buffer (40 mM HEPES, pH 8.0, 50 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 10% glycerol, 10 g/ml of poly(dI-dC); Sigma) with a total volume of 20 l. Antibody against TIEG2 or Sp3 was added (when required), and the mixture was incubated for 20 min at room temperature. Labeled probe (0.2 ng) was added to the mixture and incubated for an additional 20 min at room temperature. The binding affinity of TIEG2 or Sp3 to the CACCC element or Sp1 sites was evaluated by electrophoretic mobility-shift assay using increasing concentrations of radiolabeled CACCC element or Sp1 sites, followed by quantification of bound and free oligonucleotide (21). For super-shift assays, 1 l of antibody was included in the reaction mix along with the extract. The samples were then run on a 5% non-denaturing polyacrylamide gel in 1ϫ Tris/borate/EDTA at 150 V for 3 h. Gels were dried and visualized by autoradiography.
MAO B Catalytic Activity Assay-SH-SY5Y and HepG2 cells were grown to confluence, harvested, and washed with phosphate-buffered saline. One hundred micrograms of total proteins were incubated with 10 M 14 C-labeled PEA (Amersham Biosciences) in the assay buffer (50 mM sodium phosphate buffer, pH 7.4) at 37°C for 20 min and terminated by the addition of 100 l of 6 N HCl. The reaction products were then extracted with ethyl acetate/toluene (1:1) and centrifuged at 4°C for 10 min. The organic phase containing the reaction product was extracted, and its radioactivity was obtained by liquid scintillation spectroscopy (22).
Northern Blot Analysis of MAO B mRNA-Total RNA was purified using TRIzol reagent (Invitrogen). Thirty micrograms of total RNA from SH-SY5Y and HepG2 cells that were cotransfected with TIEG2 expression vector (0, 100, and 400 ng) and grown to confluence were loaded onto each gel lane. Electrophoresis, transfer onto BrightStar nylon membrane, and hybridization were carried out using NorthernMax according to the manufacturer's protocol (Ambion). The human MAO B probe (specific activity ϭ 2 ϫ 10 8 cpm/g) was labeled by randompriming technique using the Multiprime kit (Amersham Biosciences) following the manufacturer's instructions. Membrane hybridized with the MAO B probe was autoradiographed for 48 h.
Western Blot Analysis of MAO B Protein-Cells were harvested and washed with phosphate-buffered saline. The protein concentration was determined by the Bradford protein assay (Bio-Rad). Forty micrograms of total protein was separated by SDS-10% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After the transfer, membranes were blocked at room temperature for 2 h with 5% bovine serum albumin in TTBS (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20). The membranes were then incubated with rabbit anti-MAO B antibody (1:1000) overnight at room temperature. After incubation with goat anti-rabbit IgG conjugated with horseradish peroxidase secondary antibody at room temperature for 2 h, the bands were visualized by horseradish peroxidase reaction using diaminobenzidine as substrate (Sigma).
Chromatin Immunoprecipitation (ChIP) Assays and Quantitative Real-time PCR-SH-SY5Y or HepG2 cells (7 ϫ 10 6 cells/150-mm dish) were plated and grown in Dulbecco's modified Eagle's medium as described above for 2 days. Cells were cross-linked by incubating with formaldehyde (1% final concentration) at room temperature for 10 min. The dishes were rinsed twice with ice-cold PBS. Cells were then scraped into PBS containing protease inhibitors (Sigma) and centrifuged for 3 min at 2000 rpm. Cells were resuspended in 350 l of lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1) containing protease inhibitors before sonication. Cell debris was removed by centrifugation. The resulting cell lysates were prepared for immunoprecipitation after the methods described by Jia et al. (23). 35 l of the supernatant was saved as input DNA. The nuclear protein-DNA complex was immunoprecipitated by incubating with anti-TIEG2 (with BioMag goat antimouse) or anti-Sp3 antibody (with BioMag anti-rabbit) overnight at 4°C with rotation. They were recovered from the beads with an elution buffer (1% SDS and 0.1 M NaHCO 3 ) and were analyzed by real-time PCR using an iCycler optical system (Bio-Rad). The PCR product was determined by SyBr Green reagent (2ϫ SyBr Green Supermix, Bio-Rad) following the manufacturer's instructions. The primers for MAO B core promoter (153 bp, from Ϫ287 bp to Ϫ134 bp) and irrelevant locus (160 bp, from Ϫ1940 to Ϫ1780 bp for negative control) were, for D5 forward, 5Ј-TCTCCGCCCAGGCACCCGCCCTCC-3Ј (bases Ϫ287 to Ϫ263); for D5 reverse, 5Ј-TCGGCGAGCCGCTATATTAGCCCC-3Ј (bases Ϫ157 to Ϫ134); for 5Ј irrelevant locus forward, 5Ј-TTTGCTGTCTCAGGCCCTT-TATA-3Ј (bases Ϫ1940 to Ϫ1917); and for 5Ј irrelevant locus reverse, 5Ј-ATGAATGGAGAGGATCTGCTACG-3Ј (bases Ϫ1802 to Ϫ1780).
The PCR reactions for input, TIEG2, Sp3-associated DNA, or 5Ј irrelevant locus were done in triplicate under the following conditions: 95°C for 3 min, followed by 35 cycles of PCR consisting of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The average threshold cycle (C T ) for the triplicate was used in all subsequent calculations. A ⌬C T value was calculated for each sample using the C T value for the input DNA samples to normalize the ChIP assay results, as outlined in the iCycler optical systems protocol. The ⌬C T were converted to the fold induction required to reach the threshold amount of PCR by raising 2 to the ⌬C T power. The relative differences between input sample and TIEG2 or Sp3 ChIP assay or negative control were determined using the ⌬C T method and presented as the percent of input (taken as 100%). Data were the means Ϯ S.D. from triplicate samples of three independent experiments.

TIEG2 Was a Transcriptional Activator of the Human MAO B
Promoter-To understand the function of TIEG2 in the human MAO B promoter and to identify the promoter regions which were responsible for TIEG2 function, the 5Ј-flanking sequence of MAO B promoter was systematically deleted, and each one was ligated to the luciferase reporter gene (pGL2basic) and transfected into SH-SY5Y, SL2, and HepG2 cells. Full-length human TIEG2 expression vector was then cotransfected with each MAO B promoter-luciferase construct. As shown in Fig. 1 in SH-SY5Y cells, co-expression of TIEG2 increased the MAO B promoter activity up to 4-fold, when the deletion was up to Ϫ246 bp. However, the TIEG2 activation of MAO B promoter activity was abolished (Fig. 1, construct 5) when the Ϫ261 to Ϫ99 bp region was deleted. Thus, the TIEG2 response element was located in Ϫ246 to Ϫ99 bp, the core promoter region of MAO B (Fig. 1, constructs 1-4). Similar results were found in HepG2 cells (data not shown) and SL2 cells, which lack the Sp1 family (Fig. 1). Because the SL2 cell line does not have Sp1-like proteins, we found that the TIEG2 activation of MAO B promoter in SL2 cells (ϳ3-fold) was weaker than in SH-SY5Y cells (ϳ4-fold), which suggested that the interaction of TIEG2 with other Sp1-like proteins were important for the activation of MAO B promoter activity.

MAO B Activity and Protein Levels Were Increased by TIEG2 in a Dose-dependent Manner-When different concentrations of TIEG2 expression vector were transfected into SH-SY5Y or
HepG2 cell lines, the MAO B promoter activity ( Fig. 2A), catalytic activity (Fig. 2B), mRNA (Fig. 2C) and protein levels (Fig. 2D) were increased in a dose-dependent manner. These results indicate that the MAO B gene expression was increased by TIEG2 in a dose-dependent manner.
TIEG2 and Sp3 Were Repressors of MAO B through CACCC Element-The CACCC element and Sp1 sites in MAO B core promoter were mutated. The wild type or mutated constructs were cotransfected with TIEG2 into SH-SY5Y (Fig. 3A), HepG2 (data not shown), or SL2 cells, which lack Sp1 family (Fig. 3B). The result showed that TIEG2 activated the MAO B promoter (Fig. 3, A and B, control 1, TIEG2 versus basal) in both cell lines. Because Sp3 was found to repress the Sp1 activation on MAO B gene (16), we investigated whether Sp3 regulated MAO B gene expression by means of the CACCC element or Sp1 sites. Sp3 itself did not have an effect upon the MAO B promoter activity (Fig. 3, A and B, construct 1, Sp3 versus basal), which is consistent with our previous finding (16).
When the CACCC element was mutated into CttCC in the MAO B core promoter (Ϫ246 to Ϫ99 bp; Fig. 3, A and B,  construct 2), the basal promoter activity was increased by 8-fold compared with the wild type in SH-SY5Y cell line (Fig.  3A, compare basal activity in construct 2 versus construct 1). This result suggested that the CACCC element was a repressor element. The mutation of the CACCC element destroyed the binding of TIEG2 and Sp3 to CACCC element by gel-shift assay (data not shown); thus, the increased basal MAO B promoter activity may be mediated by another cis-element, such as Sp1 element. In contrast to SH-SY5Y cell line, when CACCC was mutated, the basal promoter activity in the SL2 cell line (no Sp1 family) was not increased, compared with the wild type (Fig. 3B, construct 2 basal versus construct 1 basal). This result suggested that the Sp1 family and Sp1 sites were responsible for the increased promoter activity seen in CACCC mutated promoter construct.
Cotransfecting TIEG2 or Sp3 with CACCC-mutated MAO B core promoter further activated the luciferase activity in SH-SY5Y (Fig. 3A, construct 2, compare TIEG2 or Sp3 to basal promoter activity), suggesting that TIEG2 and Sp3 were repressors at CACCC element, and the activation function of these factors might occur by means of Sp1 sites. When the CACCC-mutated construct was cotransfected with TIEG2 or FIG. 5. TIEG2 and Sp3 bound to natural core promoter of MAO B directly. The occupation of TIEG2 and Sp3 on the MAO B core promoter or 5Ј-irrelevant region of MAO B was determined by ChIP assay combined with quantitative real-time PCR using SH-SY5Y or HepG2 cells. A, a schematic representation of the MAO B promoter. Real-time PCR-targeted regions containing core promoter (from Ϫ287 bp to Ϫ134 bp) and 5Ј-irrelevant locus (from Ϫ1940 to Ϫ1780 bp for negative control) were indicated. B, representative TIEG2 ChIP/quantitative real-time PCR amplification plots (triplicates). The ChIP/quantitative real-time PCR amplification was performed for cross-linked inputs, TIEG2-associated MAO B core promoter (TIEG2), or 5Ј-irrelevant locus in SH-SY5Y cells. Nuclear protein-DNA complex was immunoprecipitated by anti-TIEG2 antibody and was quantitatively analyzed by real-time PCR. Cell lysates without immunoprecipitation were used as input sample (positive control). The y-axis represents the relative amount of PCR product determined by the change in the emission intensity of the reporter dye divided by the emission intensity of a passive reference dye after subtraction of the base line. The x-axis represents the PCR cycle number. C T was determined by drawing a perpendicular line from the threshold line (dashed line) to the x-axis at the point where the amplification line crosses the threshold line. Similar data were obtained for TIEG2 in HepG2 cells and for Sp3 in SH-SY5Y and HepG2 cells (data not shown). C, association of TIEG2 or Sp3 with MAO B core promoter or 5Ј-irrelevant locus analyzed in both SH-SY5Y and HepG2 cells. The relative differences between input sample and TIEG2 or Sp3 ChIP assay or negative control were determined by using the ⌬C T method (see "Materials and Methods"). These values were presented as percent input in which a DNA cross-link input sample was taken as 100%. Data were the mean Ϯ S.D. from triplicate samples of three independent experiments. Sp3 in SL2 cells, similar activation of MAO B promoter (Fig.  3B, construct 2, compare TIEG2 or Sp3 to basal) was observed.
To confirm that TIEG2 or Sp3 repressed MAO B promoter activity through CACCC element, the Sp1 sites were mutated on MAO B core promoter (Fig. 3, C and D) to further study the repressor function of CACCC element. Cotransfecting TIEG2 or Sp3 with proximal Sp1 site-mutated MAO B core promoter, the luciferase activity was reduced slightly in SH-SY5Y cells (Fig. 3C, construct 3, compare TIEG2 or Sp3 to basal) but significantly in SL2 cells (Fig. 3D, construct 3, compare TIEG2 or Sp3 to basal). Cotransfecting TIEG2 or Sp3 with both Sp1 sites mutated MAO B core promoter, the luciferase activity was decreased significantly in both cell lines (Fig. 3, C and D,  construct 4, compare TIEG2 or Sp3 to basal). These results provided further evidence that the CACCC element was a repressor element for both TIEG2 and Sp3.
TIEG2 and Sp3 Are Activators at the Proximal Sp1 Overlapping Sites of MAO B Promoter-When both CACCC element and distal overlapping Sp1 sites were mutated, the function of TIEG2 and Sp3 were reduced slightly in both cell lines (Fig. 3,  A and B, construct 3 versus construct 2), suggesting that TIEG2 and Sp3 bind weakly to the distal Sp1 sites. However the mutation of the proximal overlapping Sp1 sites together with CACCC element occurred, the activation of TIEG2 and Sp3 were blocked (Fig. 3, A and B, construct 4 versus construct 2) in both cell lines, suggesting that the proximal Sp1 sites are important for TIEG2 and Sp3 activation.
To confirm that the proximal Sp1 sites are responsible for TIEG2 and Sp3 activation, either distal Sp1 sites or proximal Sp1 sites were mutated in MAO B core promoter. Mutation of the distal Sp1 sites had slight effect on TIEG2 activation ( The effects of TIEG2 and Sp3 on the human MAO B 2-kb promoter were also studied by transient transfection assay using different deletion mutants of MAO B 2-kb promoter. The effects of TIEG2 and Sp3 on the MAO B 2-kb promoter (data not shown) are similar to the effect on Ϫ246 to Ϫ99 bp MAO B core promoter (Fig. 3).
All of the transient transfection experiments in SH-SY5Y cell line described above were also performed in HepG2 cells. The results were the same as SH-SY5Y cells (data not shown).
TIEG2 and Sp3 Interacted with CACCC Directly, as Shown by Gel-shift Assay-To confirm the presence of TIEG2 and Sp3 in the binding to MAO B core promoter region, a gel-shift assay was performed using a 23-bp DNA fragment spanning the region Ϫ222 to Ϫ201 containing the CACCC element. The radio-labeled DNA probe was incubated with nuclear extracts isolated from SH-SY5Y and HepG2 cells. Two to three major DNA-protein complexes were observed in both cell lines (Fig.  4A, lane 2 and 6). Excess of cold oligonucleotides reduced all bindings (Fig. 4A, lane 3 and 7), suggesting that these DNAprotein complexes were specific. The lower complex was completely supershifted by incubation with anti-TIEG2 antibody (Fig. 4A, lane 4 and 8), suggesting that this DNA-protein complex contained TIEG2. The upper complex was completely supershifted by incubating with anti-Sp3 antibody (Fig. 4A, lane  5 and 9), suggesting that the upper DNA-protein complex contained Sp3. These results indicated that transcriptional factors TIEG2 and Sp3 bound to CACCC element in both SH-SY5Y and HepG2 cells.
To ensure that TIEG2 and Sp3 were indeed bound to CACCC element, the CACCC element in the 23-bp DNA fragment was FIG. 6. Binding affinity of TIEG2 factor to CACCC and proximal Sp1 sites in the human MAO B core promoter. Nuclear extracts from TIEG2over-expressed SL2 cells were incubated with increasing concentrations of radiolabeled Sp1 sites (Ϫ198 bp to 177 bp) (A) or CACCC element (Ϫ221 bp to Ϫ201 bp) (B). Free and bound probes were resolved by non-denaturing PAGE and visualized by autoradiography. Arrows, positions of TIEG2 protein-DNA complexes. C, bound and free radiolabeled Sp1 sites and CACCC element in panels A and B were quantitated by phosphorimaging analysis, and the K d of TIEG2 binding to radiolabeled Sp1 sites and CACCC element was determined by Scatchard analysis. A Scatchard plot is displayed together with the K d values calculated from three independent experiments. mutated to CttCC and radiolabeled for gel-shift assay. DNAprotein complex was not found in either cell line (data not shown).
TIEG2 and Sp3 Interacted with the Proximal Sp1 Site Directly, as Shown by Gel-shift Assay-To study whether the proximal Sp1 sites interacted with TIEG2 and Sp3, a gel-shift assay was performed using a 23-bp DNA fragment spanning the region Ϫ196 to Ϫ175 bp, which contains the proximal Sp1 sites. The radiolabeled DNA probe was incubated with nuclear extracts isolated from SH-SY5Y and HepG2 cells. Two or three major DNA-protein complexes were observed with nuclear proteins from SH-SY5Y cells or HepG2 cells, respectively (Fig. 4B,  lane 2 and 6). Excess of cold oligonucleotides reduced all bindings (Fig. 4B, lane 3 and 7), suggesting these DNA-protein complexes were specific. The TIEG2 or Sp3 complex was completely supershifted by incubating with anti-TIEG2 (Fig. 4B,  lane 4 and 8) or anti-Sp3 antibody (Fig. 4B, lane 5 and 9), respectively. These results indicated that transcriptional factors TIEG2 and Sp3 bound to the proximal Sp1 sites directly. On the other hand, TIEG2 or Sp3 protein bound to the distal Sp1 sites very weakly (data not shown).
TIEG2 and Sp3 Bound to the Natural Core Promoter of MAO B-To study whether these transcription factors interacted with the natural human MAO B promoter in vivo, the occupancy of endogenous TIEG2 and Sp3 to the native core promoter of MAO B was determined by ChIP assay combined with quantitative real-time PCR. The core promoter sequence and 5Ј irrelevant region (negative control) were used for occupancy analysis (Fig.  5A). The representative TIEG2 ChIP/quantitative real-time PCR amplification plots (triplicate) were shown in Fig. 5B. C T is the cycle number required to reach the threshold levels of PCR products (the dashed line in Fig. 5B). The average C T for the input sample, TIEG2 cross-linked DNA and 5Ј irrelevant region, were 17.02 Ϯ 1.02, 22.84 Ϯ 0.69, and 29.14 Ϯ 0.92, respectively, in SH-SY5Y cells (Fig. 5B). A ⌬C T value was calculated for each sample using the C T value for the input DNA sample by subtracting the C T of input control and then converted to fold differences (see "Materials and Methods"). The relative differences between input sample and TIEG2 or Sp3 ChIP assay or negative control were summarized in Fig. 5C and presented as the percent of input (which was taken as 100%). The relative differences of TIEG2-or Sp3-conjugated DNA was 1.68 or 1.44%, respectively, compared with the input DNA (Fig. 5C) for SH-SY5Y cells, and 1.51 or 1.25% for HepG2 cells (Fig. 5C). Quantitative PCR analysis of 5Ј irrelevant region yielded negligible values (less than 0.03% input DNA, Fig. 5C). This result indicated that anti-TIEG2 or anti-Sp3 antibody is able to immunoprecipitate the native TIEG2-DNA or Sp3-DNA complex and thus demonstrated that TIEG2 and Sp3 indeed bound to the natural core promoter of MAO B.
The Binding Affinity of TIEG2 for the Proximal Sp1 Sites Was Higher than CACCC Element, but the Affinity of Sp3 Was Similar between the Proximal Sp1 Sites and CACCC Element-To understand why TIEG2 activated overall MAO B gene expression but Sp3 had no effect (Fig. 3A, construct 1), the affinity of TIEG2 for CACCC element and the proximal Sp1 sites was evaluated by gel-shift experiments (Fig. 6). TIEG2 protein was ectopically expressed in SL2 cells. The binding affinity was evaluated by using increasing concentrations of radiolabeled Sp1 sites (Fig. 6A) or CACCC element (Fig. 6B) followed by quantification of bound and free oligonucleotide (Fig. 6C). Scatchard analysis revealed that the dissociation constant (K d ) value was 0.29 ϫ 10 Ϫ9 M for TIEG2 binding to Sp1 sites, 0.46 ϫ 10 Ϫ9 M for TIEG2 binding to CACCC element. Therefore, the K d value of TIEG2 binding to Sp1 sites is significantly lower than that of binding to CACCC element (p Ͻ 0.05). These K d values are in the same range as for Sp1 binding to the Sp1-like sequences (24,25). Nuclear extracts from Sp3-over-expressed SL2 cells were incubated with increasing concentrations of 32 P-labeled 23-bp DNA probe containing CACCC element (Ϫ221 bp to Ϫ201 bp) (A) or 32 P-labeled 23-bp DNA probe containing the proximal Sp1 sites of MAO B (Ϫ198 bp to 177 bp) (B). Free and bound probes were resolved by non-denaturing PAGE and visualized by autoradiography. Arrows, positions of Sp3 protein-DNA complexes. C, bound and free radiolabeled Sp1 sites and CACCC element in panels A and B were quantitated by phosphorimager analysis, and the K d of Sp3 binding to radiolabeled Sp1 sites and CACCC element was determined by Scatchard analysis. A Scatchard plot is displayed together with the K d values calculated from three independent experiments.