SEARCH

SEARCH BY CITATION

Keywords:

  • centromere protein H (CENPH);
  • nasopharyngeal carcinoma;
  • Sp1;
  • Sp3;
  • transcriptional regulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information

The overexpression of centromere protein H (CENPH), one of the fundamental components of the human active kinetochore, has been shown to be closely associated with human cancers. However, the mechanism of its transcriptional regulation has not been reported. The aim of the present study was to investigate the regulatory elements for the transcriptional regulation of CENPH in nasopharyngeal carcinoma cells. To characterize the CENPH promoter and identify regulatory elements, we cloned 1015 bp (−975/+40 bp) of the 5′-flanking region of the CENPH gene from immortalized normal nasopharyngeal epithelial cells (Bmi-1/NPEC). Functional analysis established a minimal region (−140/−87 bp) involved in the regulation of human CENPH promoter activity. Through site-directed mutagenesis, a transactivation assay, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we found that the Sp1/Sp3 transcription factors could bind to the CENPH promoter in vitro and in vivo, and that they regulated CENPH promoter activation in human nasopharyngeal carcinoma cells. Furthermore, Sp1 and Sp3 were highly expressed in nasopharyngeal carcinoma cells. Knockdown of Sp1 and Sp3 by small interfering RNA or inhibition of Sp1 and Sp3 activity by mithramycin A decreased CENPH mRNA expression, whereas the exogenous expression of Sp1 and Sp3 upregulated CENPH mRNA expression. Taken together, our results indicate that Sp1 and Sp3 bind to the CENPH minimal promoter and function as a regulator of the transcription of CENPH in human nasopharyngeal carcinomas.


Abbreviations
CENPH

centromere protein H

ChIP

chromatin immunoprecipitation

CIN

chromosomal instability

EGFP

enhanced green fluorescent protein

EMSA

electrophoretic mobility shift assay

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

NPC

nasopharyngeal carcinoma

NPEC

normal nasopharyngeal epithelial cell

siRNA

small interfering RNA

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information

Centromere protein H (CENPH) was initially recognized as a component of the mouse centromere [1]. Human CENPH was then isolated and shown to localize in the inner plate together with centromere protein A and centromere protein C throughout the cell cycle [2]. As a fundamental component of the active centromere complex [3], CENPH plays a central role in linking centromeres to the spindle microtubule [4]. In addition, CENPH has been indicated to play a critical role in centromere structure, kinetochore formation, and sister chromatid separation [2,5–7]. For example, knockdown of CENPH causes severe mitotic phenotypes, such as misaligned chromosomes and multipolar spindles, indicating that CENPH has an important impact on the architecture and function of the human kinetochore complex [5]. A recent study reported that CENPH preferentially attached to the plus end of kinetochore microtubules, and regulated their turnover rate and promoted accurate chromosome alignment at the metaphase plate, suggesting that centromeric localization of CCAN members depends on CENPH [8]. CENPH is required for the localization of most CCAN components and Mis12, but not for endogenous CENPA centromeric localization in interphase [3,9,10].

Deregulation of CENPH has been observed in various human cancers. It was first reported to be upregulated in colorectal cancers, and the ectopic expression of CENPH induced chromosome instability (CIN) in diploid cell lines [11]. Shigeishi and our laboratory reported that CENPH was also upregulated in oral squamous cell carcinoma, nasopharyngeal carcinoma (NPC), breast cancer, esophageal carcinoma, non-small-cell lung cancer, and tongue cancer [12–17]. Moreover, higher CENPH expression is associated with poor prognosis for patients [12–17]. We have previously demonstrated that CENPH is more highly expressed in NPC tumors and cell lines and immortalized nasopharyngeal epithelial cells than in normal tissues and normal nasopharyngeal epithelial cells (NPECs). More importantly, higher CENPH expression is associated with poor overall survival of NPC patients [14]. Therefore, CENPH may be used as a prognostic biomarker for patients with these tumors. However, the molecular mechanism for the upregulation of CENPH in cancer cells is still unclear.

Sp1 and Sp3 are transcription factors that enhance or repress the activity of promoters of genes such as those encoding cyclins, cell cycle inhibitors, c-Myc, and vesicular epithelial growth factors, and they are involved in differentiation, cell cycle regulation, tumor growth, and metastasis [18]. Although Sp1 and Sp3 have a similar structure and bind to the same cognate Sp1-binding sites, their DNA-binding properties and regulatory functions are different, depending on the promoter context and the cellular background [19,20]. The protein expression levels of Sp1 and Sp3 are often greater in cancer cells than in normal cells. Sp1 levels are greater in breast carcinoma, thyroid cancer, hepatocellular carcinoma, pancreatic cancer, colorectal cancer, gastric cancer and lung cancer than in normal tissues or cells [18,21–23].

In this study, we characterized the CENPH promoter and investigated the regulatory transcription factors for the regulation of CENPH overexpression in NPC cells. We demonstrated that Sp1 and Sp3 bind to the CENPH promoter and are required for the full activity of CENPH transcription in NPC cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information

Cloning and characterization of the CENPH promoter

The human CENPH gene (GenBank: NC_000005.9) is located on chromosome 5p15.2 and spans 20 810 bp. It has nine exons, and encodes a 33-kDa protein of 247 amino acids (GenBank: NP_075060.1). To examine its transcriptional regulation, a fragment of the 5′-flanking region of the human CENPH gene was cloned from human genomic DNA. The fragment spanned nucleotides located at positions from −975 to +40 bp (the numbering is based on the translation initiation site ATG being designated as +1). The fragment was then cloned into the pGL3-Basic vector containing the corresponding firefly luciferase reporter gene (named pGL3-975/40), and a luciferase assay was conducted in CNE2 cells. As shown in Fig. 1, the pGL3-975/40 construct had significantly higher luciferase activity than the vector control. This phenomenon was also observed in CNE1 cells (Fig. S1). The result indicated that the fragment corresponding to −975 to +40 bp of the 5′-flanking region of the CENPH gene contained the functional promoter for human CENPH gene transcription.

image

Figure 1.  Analysis of the human CENPH promoter. A series of firefly luciferase reporter systems containing different length of the 5′-upstream CENPH promoter were generated in a pGL3-Basic vector. Luciferase reporter constructs containing the indicated promoter fragments of the CENPH promoter and Renilla were transiently transfected into CNE2 cells. At 24 h post-transfection, cells were lysed, and the reporter gene activity was measured and normalized to Renilla activity, as indicated in Experimental procedures. The values represent the mean ± standard deviation of three independent experiments.

Download figure to PowerPoint

To functionally analyze the CENPH promoter and to identify the minimal promoter region needed for CENPH gene expression, a series of 5′-deletion reporter constructs containing sections that spanned from −533, −320, −140 and −87 bp of the promoter region to +40 bp downstream were generated by PCR with the template pGL3-975/40. A luciferase assay was conducted, and similar promoter activity was observed with serial deletion of the promoter region until −140 bp. Moreover, the luciferase activity was abruptly and significantly reduced when the promoter was further deleted to −87 bp upstream (Fig. 1). Similar results were observed in CNE1 cells (Fig. S1). These data collectively indicated that the promoter region located at −140 to −87 bp was the major determinant in the CENPH proximal promoter.

Identification of the transcription factor that binds to the CENPH promoter

Because the promoter region of −140 to −87 bp was the major determinant in the CENPH proximal promoter, this region may contain cis-elements that are essential to drive the transcription of CENPH. We used Transcription Factor Binding Sites software (tfsearch v. 1.3, Computational Biology Research Center, AIST, Japan; alibaba 2.1, BIOBASE, Beverly, MA, USA; and genomatix software suite V2.4, Munich, Germany) to predict the potential cis-elements in the region from −140 to −87 bp. We found four most probable transcription factor sites, which are indicated by red frames in Fig. 2A. To test the functional importance of these binding sites in the CENPH promoter, site-directed mutagenesis was carried out with the pGL3-140/40 promoter construct, which contained a region from −140 to +40 bp. These mutants were constructed harboring the mutations indicated in Fig. 2B. The luciferase assay was then conducted. The pGL3-140/40 ΔSp mutant construct showed ∼ 33% of pGL3-140/40 transcriptional activity in CNE2 cells, but the other three mutant constructs had no obvious effect on transcriptional activity (Fig. 2C). We also found the pGl3-140/40 ΔSp mutant construct activity to be significantly decreased in CNE1 cells (Fig. S2). These results indicated that the consensus sequence of the Sp1-binding site was involved in the full transactivation activity of this region in NPC cells.

image

Figure 2.  Identification of the transcription factor that binds to the CENPH promoter. (A) Transcription factor prediction. Three types of software for prediction of transcription factor-binding sites were used to analyze the region from −140 to −83 bp in the CENPH proximal promoter. The red frames indicate the most probable transcription factors. (B, C) Site-directed mutagenesis of the predicted transcription factor-binding sites. (B) Schematic representation of the substitution mutation in the predicted transcription factor-binding sites of the promoter. (C) A luciferase assay was performed with the mutation constructs that were transfected into CNE2 cells. Asterisks indicate statistically significant results (P < 0.05) as compared with the results for wild-type pGL3-140/40. The data represent the mean ± standard deviation from at least three independent experiments.

Download figure to PowerPoint

In vivo binding of Sp1/Sp3 to the promoter region of the human CENPH gene

To investigate whether Sp1 and Sp3 bind to the human CENPH promoter region in human CNE2 cells in vivo, a chromatin immunoprecipitation (ChIP) assay was performed. A markedly higher amount of chromatin containing the CENPH promoter region was immunoprecipitated by antibodies against Sp1 (260-fold) and Sp3 (90-fold) than by the antibody against FLAG used as a control (Fig. 3A). In addition, real-time PCR was performed with both positive and negative control primers. The positive control was the human IRF-3 gene promoter, which has been reported to be an Sp1/Sp3 target [24], and showed similar binding affinity as the CENPH promoter. The negative control primers CENPH NC F and CENPH NC R were used to amplify a genomic region ∼ 6 kb upstream of the CENPH promoter, which lacks Sp1-binding sites. With respect to this negative control, the binding affinity for Sp1 was 12-fold greater (Sp1 ChIP) and that for Sp3 was three-fold greater (Sp3 ChIP) (Fig. 3B).

image

Figure 3.  ChIP assay and EMSA. (A) A ChIP assay was performed to determine the binding of Sp1 and Sp3 to the CENPH promoter in vivo. Quantitative real-time PCR results for amplification of the CEPH promoter are shown, with antibodies against Sp1, Sp3, or FLAG. The protein–DNA complexes were incubated with polyclonal antibodies directed against Sp1 or Sp3, and isolated by immunoprecipitation (lanes 2 and 3). All immunoprecipitated DNA fragments were analyzed. Input before immunoprecipitation was used as the positive control (lane 1). As the negative controls, the protein–DNA complexes were incubated with an antibody against FLAG (M2, lane 4). The values were normalized, and are expressed as fold changes in comparison with the amount of DNA binding with the control M2 antibody. (B) Real-time PCR was also performed with control primers. The positive control was amplified with the primers IRF-3 F and IRF-3 R. The negative control was amplified with the primers CENPH NC F and CENPH NC R. PCR with the negative control primers was used to normalize quantitative results from different immunoprecipitations. (C) EMSA analysis of the CENPH promoter. The 32P-labeled −114/−90 oligonucleotides were incubated with nuclear extracts from CNE2 cells. Lane 1 lacks nuclear extract. CNE2 cell nuclear extracts were incubated with the labeled probe (lane 2). Competition analyses were performed in the presence of a 100-fold excess of unlabeled −114/−90 oligonucleotide (lane 3). A supershift analysis was performed in the presence of 4 μg of antibodies against Sp1 (lane 4), Sp3 (lane 5), or FLAG (lane 6). CNE2 cell nuclear extracts with overexpressed Sp1 and Sp3 were incubated with the labeled probe (lanes 7–9). A supershift analysis was performed in the presence of 4 μg of antibodies against Sp1 (lane 8) or Sp3 (lane 9) with overexpressed nuclear extract. Arrowheads indicate the supershifted bands. The red asterisk indicates the specific probe–protein complexes. The red triangle indicates the unspecific probe–protein complexes. WT, wild-type.

Download figure to PowerPoint

We also conducted a ChIP assay in CNE1 cells and C666 cells, and the results were similar (Fig. S3). These results suggested that Sp1 and Sp3 bound to the CENPH promoter region in NPC cells in vivo.

Sp1 and Sp3 bind to the CENPH promoter in vitro

Electrophoretic mobility shift assays (EMSAs) were carried out with nuclear proteins from CNE2 cells and radiolabeled double-stranded oligonucleotide probes carrying wild-type CENPH promoter fragments from −114 to −90 bp. The complex formation was analyzed in a nondenaturing polyacrylamide gel. As shown in Fig. 3C, incubation of CNE2 cell nuclear extracts with the probe resulted in the formation of retarded complexes (lane 2). The retarded complexes were effectively abrogated by a 100-fold molar excess of unlabeled wild-type oligonucleotide (lane 3). To resolve the composition of the complexes, we performed supershift EMSAs by incubating nuclear extracts from CNE2 cells with the radiolabeled probe in the presence of antibodies against Sp1 or Sp3. A supershifted band was detected on incubation with antibodies against Sp1 or Sp3 (Fig. 3, lanes 4 and 5). An antibody against FLAG was used as a negative control (Fig. 3, lane 6). However, the supershifted band with the antibody against Sp3 was not very clear. The specific binding activity of Sp1 and Sp3 was further confirmed with the CNE2 nuclear extract containing overexpressed Sp1 and Sp3, and supershifted bands with the antibodies against Sp1 and Sp3 (Fig. 3, lanes 8 and 9) could be observed more clearly. The above results indicated that Sp1 and Sp3 bound to the CENPH promoter in vitro.

Sp1 and Sp3 regulate CENPH basal expression

To further confirm the involvement of Sp1 and Sp3 in regulating CENPH transcription, we examined the inhibitory effect of mithramycin A, which blocks the binding of Sp1 family members to GC-rich regions [25]. Mithramycin A was added to the CNE2 cell culture medium at different concentrations. After 36 h, the RNA and proteins of disposed cells were harvested and analyzed, and this showed that CENPH expression was dose-dependently inhibited at both the RNA and protein levels by mithramycin A (Fig. 4A,B).

image

Figure 4.  Sp1 and Sp3 regulated CENPH basal expression. (A, B) Mithramycin A reduces endogenous CENPH expression. CNE2 cells were treated with mithramycin A for 36 h at different final concentrations before the cells were harvested. CENPH expression was analyzed with real-time PCR (A) and western blotting (B). (C, D) RNA silencing of Sp1 and Sp3 reduces endogenous CENPH expression. CNE2 cells were transfected with 50 μm siRNAs specific for Sp1, Sp3, or Sp1 + Sp3, and 24 h later, they were transfected with luciferase reporter plasmids containing the −140/40-bp fragment. After 24 h, cell lysates were harvested. The mRNA and protein levels were then determined (C), and the promoter activity was measured (D). The mRNA levels were assessed by real-time RT-PCR normalized to GAPDH, and protein levels were assessed by immunoblotting normalized to a-tubulin. The arrowhead indicates the main Sp3 protein band. The promoter activity was normalized for transfection efficiency by cotransfection with the Renilla vector. NC: scrambled siRNA control. Asterisks indicate statistically significant results (P < 0.05) as compared with the results for the scrambled siRNA control. The data represent the mean ± standard deviation from at least three independent experiments.

Download figure to PowerPoint

In addition, we tested whether knockdown of Sp1 or Sp3 might affect CENPH expression, by using small interfering RNAs (siRNAs) specific for Sp1 and Sp3. The siRNA treatment induced a decrease in the cellular levels of Sp1 and Sp3 mRNA and proteins. In addition, the RNA and protein levels of the endogenously expressed CENPH were also reduced (Fig. 4C). Moreover, the promoter activity was also reduced, coincident with the reduction of these regulatory factors, but the promoter activity of the Sp mutant construct was not reduced (Fig. 4D).

Futhermore, we wanted to test whether the exogenous expression of Sp1 or Sp3 might affect CENPH expression. CNE2 cells were transfected with control vector, Sp1, Sp3 or Sp1 plus Sp3 expression plasmids, together with enhanced green fluorescent protein (EGFP)-expressing vector. Thirty-six hours later, EGFP-positive cells were sorted for further analysis of mRNA expression from the cotransfected cells. The Sp1, Sp3 and CENPH mRNA levels were detected by real-time RT-PCR. As shown in Fig. 5, overexpression of either Sp1, Sp3 or both Sp1 and Sp3 mRNA was confirmed by RT-PCR. Consequently, CENPH transcription was also upregulated in concert with the increase in these regulatory factors. These results indicated that Sp1 and Sp3 could regulate CENPH expression. Although Sp3 is known to function as either an activator or repressor, depending on the target genes, our observation that the inhibition of Sp3 reduced CENPH expression raised the possibility that Sp3 might activate CENPH expression. Treatment with siRNA against Sp3 decreased CENPH expression slightly more than Sp1 interference treatment, suggesting that both Sp1 and Sp3 function in the regulation of expression of CENPH in NPC cells.

image

Figure 5.  Forced expression of Sp1 and Sp3 upregulates CENPH expression. CNE2 cells were transfected with either control vector, Sp1, Sp3 or Sp1 + Sp3 expression plasmids together with EGFP-expressing vector, with the FuGENE HD transfection reagent. Thirty-six hours later, EGFP-positive cells were sorted from the cotransfected cells, and the sorted cells were used for detection of RNA level. The figure shows the mRNA levels of Sp1, Sp3 and CENPH as determined by real-time PCR. 6B-Sp1 represents the Sp1 expression vectors (pcDNA6/myc-His B-Sp1); 6B-Sp3 represents the Sp3 expression vectors (pcDNA6/myc-His B-Sp3); Vector represents the void vector (pcDNA6/myc-His B).

Download figure to PowerPoint

Coexpression analysis of Sp1, Sp3, and CENPH

To further investigate the relevance of Sp1 and Sp3 to CENPH, we evaluated their expression levels in nasopharyngeal epithelial cell lines. The following cell lines were used: NPECs (NPEC1 and NPEC2) and NPC cells (6-10B, C666, CNE1, and CNE2). All four NPC cell lines showed higher levels of expression of Sp1 and Sp3 than the NPEC lines (Fig. 6). Consistently, the levels of CENPH mRNA and CENPH protein in NPC cell lines were high, whereas they were weakly detected in NPECs (Fig. 5A,B). These results indicated that Sp1, Sp3 and CENPH were highly expressed in NPC cell lines as compared with NPECs.

image

Figure 6.  Expression analysis of Sp1, Sp3 and CENPH in NEPCs and NPC cells by real-time PCR and western blotting. (A) Expression of CENPH by real-time PCR. (B) Expression of Sp1, Sp3 and CENPH by western blotting. 6-10B, C666, CNE1, CNE2 are NPC cell lines. The RNA levels were assessed by real-time RT-PCR normalized to GAPDH, and protein levels were assessed by immunoblotting normalized to a-tubulin. The arrowhead indicates the main Sp3 protein band.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information

CENPH, which is a CCAN component, has been recognized as a constitutive centromeric coiled-coil protein [1,2]. CENPH is the basic component of the human active kinetochore, and inappropriate expression of the centromere proteins could be a major cause of CIN. Overexpression of CENPH has been shown to cause chromosome mis-segregation and aneuploidy in diploid cell lines. CIN is induced by continuous chromosome mis-segregation during mitosis, and is recognized as a hallmark of human cancer. CIN is significant in the development and progression of cancer, because aneuploidy is found in the earliest stages of tumorigenesis [26,27]. Therefore, it is important to study the molecular mechanism underlying the regulation of CENPH overexpression in cancer.

We previously reported that CENPH was overexpressed in NPC cells and tumor tissue samples. The overexpression of CENPH was correlated with poor prognosis of NPC patients [14]. It has also been reported that CENPH is overexpressed in many cancers, such as colon, lung, oral and esophageal carcinoma [11,13]; in addition, its amplification is related to increased CIN. However, the molecular mechanism of CENPH regulation in cancer is unknown. Here, we characterized the CENPH core promoter and demonstrated that Sp1 and Sp3 were the essential transcription factors that regulated the overexpression of CENPH in NPC.

Transcriptional deregulation of tumor-associated genes is one of the most frequently observed cellular phenomena during cell transformation [28]. Transcription factors of the Sp1 family are frequently involved in the basal expression of many TATA-less promoters [29], and several predicted binding sites for Sp1 family members are present in the CENPH core promoter region. Sp3 also binds to the same Sp1 consensus sequence [30]. Thus, we postulate that transcription of CENPH should be closely regulated by Sp1 and Sp3 family proteins. Using ChIP and EMSA assays, we confirmed that Sp1/Sp3 transcription factors indeed bind to the CENPH promoter in vivo and in vitro. The importance of Sp1/Sp3 binding sites in mediating the transcriptional activity of the CENPH promoter was demonstrated by significantly reduced core promoter activity after disruption of Sp1/Sp3 binding with point mutations in pGL3-140/40. More importantly, the influence of Sp1 and Sp3 in the regulation of CENPH promoter activity or endogenous CENPH was further confirmed by RNA interference knockdown of Sp1/Sp3 expression or by mithramycin A, which inhibits the binding of Sp1/Sp3 to DNA. As the transcriptional activity of the pGL3-140/40 ΔSp mutant construct (one-third reduction as compared with pGL3-140/40; Fig. 2) is significantly higher than that of pGL3-87/40 (almost 100-fold reduction as compared with pGL3 140/40, Fig. 1), we cannot exclude the possibility that there are other, undetermined, transcription factor(s) that are important for the transcriptional regulation of CENPH.

Sp1 is reported to be an important transcription factor involved in human cancers. By cooperating with other oncogenes or tumor suppressors, Sp1 can modulate these target genes and act directly as an oncogene [31]. Sp1 and Sp3, which are ubiquitously expressed in mammalian cells, may enhance or repress promoter activity and regulate the expression of multiple target genes by binding and acting through GC boxes. Dysregulation of Sp1 and Sp3 is observed in many cancers and diseases. Sp1 and Sp3 have been demonstrated to regulate Ras association domain-containing protein 1, vascular endothelial growth factor and matrix metallopeptidase 9 in NPC [32,33]. Inhibition of Sp1 activity resulted in a reduction in the migration and invasion ability of NPC cells [33]. As overexpression of CENPH is correlated with local invasion of the primary tumor in different cancer types, the role of Sp1 in tumor invasion might be associated with CENPH under some circumstances. Because of the amino acid sequence similarity of the DNA-binding domains, Sp1 and Sp3 recognize and associate with the same DNA elements with similar affinity. Several studies suggest that Sp1 is responsible for basal transcription, and Sp3 is important for induced transcription activation [34–36]. One recent study demonstrated that Sp1 and Sp3 selectively bind to different Sp1/Sp3 site(s) on the same promoter [37]. Our studies revealed that both Sp1 and Sp3 can bind to the CENPH promoter and enhance the promoter activity of CENPH, possibly through cooperative regulation. A recent study showed that Sp1 together with Sp3 may function as the main regulator of the basal and serum-induced transcription of CENP-W in a human cervical carcinoma cell line [38], which, to some degree, supports the findings of our study.

Mithramycin A can modulate gene transcription by binding to GC-rich domains in gene promoters. It has been shown to have antitumor activity in combination with hydroxurea or interferon-α [39,40]. We showed here that mithramycin A inhibited CENPH expression in a dose-dependent manner in CNE2 cells. At a concentration of 125 nm without observed cytotoxicity, the expression of CENPH was reduced to ∼ 15% of that in mock-treated cells (Fig. 4A,B), which is similar to the expression level in normal cells. As normalized centromere proteins are essential for chromosome separation [5], and mithramycin A has been shown to have antitumor activity in various studies, our results suggest that mithramycin A should be investigated in the treatment of NPC, to protect against CIN by normalizing CENPH expression. Most recently, it has been reported that CIN can induce a cancer stem cell phenotype in NPC cells [41], suggesting that it is worth investigating whether mithramycin A could be used in NPC treatment for controlling cancer stem cells.

In conclusion, we have identified the CENPH proximal promoter region, and have shown that Sp1 and Sp3 bind to the CENPH minimal promoter region and function as the activator elements for CENPH expression in NPC. To our knowledge, this is the first report on the transcriptional regulation of CENPH. Our results may be useful to illustrate the mechanism of modulation of CENPH gene expression and to identify novel therapeutic strategies, especially for the treatment of NPC patients.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information

Materials

RPMI-1640 medium and keratinocyte/serum-free medium were purchased from Life Technologies (Carlsbad, CA, USA), mithramycin A was purchased from Sigma (St Louis, MO, USA), FuGENE HD and Lipofectamine RNAiMAX transfection reagent were purchased from Roche (Mannheim, Germany), the dual-luciferase reporter assay system was purchased from Promega (Madison, WI, USA), ECL Plus was purchased from Amersham Biosciences (Piscataway, NJ, USA), pcDNA6/myc-His B was purchased from Invitrogen (Carlsbad, CA, USA), the promoterless pGL3 basic vector and the phRL-SV40 vector were purchased from Promega, the QuikChange II Site-Directed Mutagenesis Kit was purchased from Stratagene (La Jolla, CA, USA), TRIzol reagent was purchased from Invitrogen, restriction endonucleases and T4 DNA ligase were purchased from New England BioLabs (Beverly, MA, USA), and PrimeSTAR HS DNA polymerase was purchased from TaKaRa (Dalian, China). The polyclonal rabbit antibodies against human Sp3 and CENPH for western blotting were purchased from Bethyl Laboratories (Montgomery, TX, USA), the polyclonal rabbit antibodies against human Sp1 for western blotting were purchased from Millipore (MA, USA), the polyclonal rabbit antibodies against human Sp1 and Sp3 for ChIP and EMSA were purchased from Santa-Cruz Biotechnology (Santa Cruz, CA, USA), the polyclonal mouse antibodies against human a-tubulin and FLAG(M2) were purchased from Sigma, and the secondary antibodies peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Amersham Biosciences (Pittsburg, PA, USA).

Cell culture

Primary NPEC cultures were established as described previously [42], and grown in keratinocyte/serum-free medium. All NPC cell lines were maintained in RPMI-1640 medium (Invitrogen) supplemented with 5% heat-inactivated fetal bovine serum (Invitrogen). All cell cultures were maintained at 37 °C in 5% CO2.

Construction of chimeric plasmids and site-directed mutagenesis

PCR was used to generate constructs of the CENPH promoter. PCR was performed with sets of oligonucleotide primers specific for the human CENPH gene sequence; the forward primer was BglII-site-linked and the reverse primer was HindIII-site-linked. The genomic DNA from an immortalized nasopharyngeal epithelial cell line, Bmi-1/NPEC, was used to generate the pGL3-975/40 construct with reference to the starting codon ATG, which was further used as the template for other constructs. These PCR products were subcloned into the BglII–HindIII sites of the pGL3 basic vector (Promega). Substitution mutation constructs were generated with a QuikChange II mutagenesis kit, according to the manufacturer’s protocol and using the pGL3-140/40 plasmid as a template. Full-length Sp1 and Sp3 coding sequences were amplified by using cDNA from an immortalized cell, Bmi-1/NPEC, and cloned into the pcDNA6/myc-His B vector. The oligonucleotide primers were synthesized by Invitrogen (Guangzhou, China), and all constructs and mutants were confirmed by sequencing. The following primers were used: −975F, −533F, −320F, −87F, 40R, Sp1-Mut, NF1-Mut, MOK2-Mut, ZKSCAN3-Mut, 6B-Sp1 F, 6B-Sp1 R, 6B-Sp3 F, and 6B-Sp3 R (Table S1).

Luciferase assay and transient transfection

Approximately 1.2 × 105 CNE2 cells were cultured for 24 h before transfection in 12-well plates. Plasmid DNA (0.5 μg) was then transfected into these cells with the FuGENE HD transfection reagent (Roche). The plasmid phRL-SV40 vector (Promega) was always cotransfected as an internal control for the normalization of transfection efficiency. Twenty-four hours after transfection, the cells were lysed in a passive lysis buffer and subjected to a luciferase assay. Luciferase assays were performed with the Dual-Luciferase Reporter Assay System (Promega): a 20-μL aliquot was used for luminescence measurements with a luminometer (GloMax Multi Jr; Promega), according to the manufacturer’s protocol. The data were represented as the ratio of firefly to Renilla luciferase activity (relative luciferase activity). The overexpression transfection experiments were performed as follows: 5 × 105 CNE2 cells were seeded per 100-mm dish, and transfected on the next day with 20 μg of control vector, Sp1, or Sp3, or 10 μg of Sp1 plus 10 μg of Sp3 expression plasmids, together with 2 μg of EGFP-expressing vector, with the FuGENE HD transfection reagent. EGFP-positive cells were sorted from the cotransfected cells 36 h later with a Moflo XDP (Beckman Coulter, Fullerton, CA, USA), and the cells were then used for RNA expression analysis. These experiments were performed three times in duplicate, and the results are expressed as the mean ± standard deviation of three independent experiments.

Small interfering RNAs

Each siRNA targeting the coding region of Sp1 or Sp3 was synthesized by GenePharma (Shanghai, China). The sequences were as follows: si-Sp1, CCAACAGAUUAUCACAAAUdTdT; and si-Sp3, GCGGCAGGUGGAGCCUUCACUdTdT. The scrambled siRNA control (NC) was used as a negative control (Dharmacon, Rockford, USA; D-001220-01-20). To measure the inhibitory effect on promoter activity, siRNAs (50 μm) were transfected into cells with Lipofectamine RNAiMAX, according to the manufacturer’s protocol (Invitrogen). Twenty-four hours later, the luciferase assay was conducted with the corresponding plasmids.

Quantitative RT-PCR analysis

The expression levels of Sp1, Sp3 and CENPH mRNA were determined by SYBR green real-time RT-PCR. Total RNA from different cell lines was extracted with Trizol reagent. cDNA was synthesized from 2 μg of total RNA with oligo(dT)20 (50 μm) and reverse transcriptase (Invitrogen). Then, quantitative PCR was performed with Power SYBR Green qPCR SuperMix-UDG (Invitrogen) and CFX96 Real-Time PCR (BioRad, Hercules, CA, USA). Reactions were run in duplicate in three independent experiments. The geometric mean of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the variability in expression levels. The primers that were used are as follows: Sp1 F, Sp1 R, Sp3 F, Sp3 R, CENPH F, CENPH R, GAPDH F, and GAPDH R. The primer sequences are shown in Table S1.

Western blotting

Sample preparation for immunoblotting was conducted as previously described [43]. Briefly, cells were harvested in 1 × SDS sample buffer [62.5 mm Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol] and were heated for 10 min at 98 °C. The protein concent-ration was determined with the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated electrophoretically on 9% SDS/polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Amersham Biosciences). Nonspecific binding was blocked for 1 h at 20 °C with 5% nonfat milk in NaCl/Tris/Tween-20. The filters were incubated with the primary antibody anti-Sp1 IgG (1 : 1000 dilution), anti-Sp3 IgG (1 : 2000 dilution) or anti-CENPH IgG (1 : 500 dilution) overnight at 4 °C, and this was followed by 1 h of incubation with secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (1 : 2000 dilution). Immunoreactivity was then determined by chemiluminescence detection according to the manufacturer’s instructions (Amersham Biosciences).

ChIP assays

ChIP was performed as previously described [44]. Briefly, 2 × 106 cells were plated per 100-mm-diameter dish and treated with formaldehyde to cross-link chromatin-associated proteins to DNA. The cells were trypsinized and resuspended in lysis buffer, and the nuclei were isolated and sonicated to shear the DNA into fragments of 500 bp–1 kb (verified by agarose gel electrophoresis). Equal aliquots of chromatin supernatants were subjected to overnight immunoprecipitation with different antibodies as indicated, or antibody against FLAG as a negative control. DNA was extracted, and the CENPH promoters were amplified with the primers −154F and −22R by real-time PCR. In addition, we used the primer pair CENPH NC F/CENPH NC R, which amplifies a 128-bp genomic region lacking Sp1-binding sites, as the negative control primers, and we used the primer pair IRF-3 F/IRF-3 R, which can amplify the human IRF-3 gene promoter, as the positive control primers [24]. All ChIP assays were performed three to four times, and representative results are presented. The primers used for real-time PCR are shown in Table S1.

Nuclear extract preparation and EMSA

Nuclear extracts were prepared as described previously [45,46]. The nuclear extract, which contained ectopically expressed Sp1 and Sp3, was obtained from the CNE2 cells transfected with the pcDNA6/myc-His B vector of Sp1 and Sp3 for 48 h with FuGENE HD, according to the manufacturer’s protocol. The following dsDNA oligonucleotides were used in the EMSAs: −114/−90 WT (see Table S1 for sequences). Double-stranded oligonucleotides were annealed and end-labeled with [32P]ATP[γP] (PerkinElmer Life and Analytical Sciences, Wellesley, MA, USA) with T4 polynucleotide kinase (Invitrogen). Binding reactions were performed with 5 μg of nuclear extract for 30 min at 24 °C in binding buffer [0.5 mm dithiothreitol, 0.5 mm MgCl2, 0.5 mm ZnSO4, 0.5 mm EDTA, 50 mm NaCl, 4% glycerol, 50 ng/mL poly(dI-dC), and 10 mm Tris/HCl, pH 7.5] with labeled oligomers (100 000–150 000 c.p.m.) in a total volume of 20 μL. For supershift analysis, 4 μg of antibodies against Sp1, Sp3 and FLAG was incubated with nuclear extracts for 1 h at 4 °C, and this was followed by an additional 30 min of incubation at 24 °C in the presence of labeled probes. For competition analysis, a 100-fold excess of unlabeled double oligonucleotides was incubated with nuclear extracts for 1 h at 4 °C, and this was followed by an additional 30 min of incubation at 24 °C in the presence of labeled probes. The protein–DNA complexes were analyzed on a 4% nondenaturing polyacrylamide gel containing 3% glycerol and 0.25 × Tris/borate/EDTA at 4 °C, vacuum-dried with heat, and photographed.

Transcription factor binding site analyses

The tfsearch (v. 1.3) database, alibaba 2.1, and the geno-matix software suite (v. 2.4) were used to localize putative transcription factor binding sites within the 5′-flanking region of the human CENPH gene.

Statistical analysis

An unpaired t-test was used to determine statistical significance.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information

This study was supported by grants from the National Natural Science Foundation of China (30872931, 81071932).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information
  • 1
    Sugata N, Munekata E & Todokoro K (1999) Characterization of a novel kinetochore protein, CENP-H. J Biol Chem 274, 2734327346.
  • 2
    Sugata N, Li S, Earnshaw WC, Yen TJ, Yoda K, Masumoto H, Munekata E, Warburton PE & Todokoro K (2000) Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere–kinetochore complexes. Hum Mol Genet 9, 29192926.
  • 3
    Fukagawa T, Mikami Y, Nishihashi A, Regnier V, Haraguchi T, Hiraoka Y, Sugata N, Todokoro K, Brown W & Ikemura T (2001) CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells. EMBO J 20, 46034617.
  • 4
    Westermann S, Cheeseman IM, Anderson S, Yates JR III, Drubin DG & Barnes G (2003) Architecture of the budding yeast kinetochore reveals a conserved molecular core. J Cell Biol 163, 215222.
  • 5
    Orthaus S, Ohndorf S & Diekmann S (2006) RNAi knockdown of human kinetochore protein CENP-H. Biochem Biophys Res Commun 348, 3646.
  • 6
    Izuta H, Ikeno M, Suzuki N, Tomonaga T, Nozaki N, Obuse C, Kisu Y, Goshima N, Nomura F, Nomura N et al. (2006) Comprehensive analysis of the ICEN (Interphase Centromere Complex) components enriched in the CENP-A chromatin of human cells. Genes Cells 11, 673684.
  • 7
    Zhao X, Zhao L, Tian T, Zhang Y, Tong J, Zheng X & Meng A (2010) Interruption of cenph causes mitotic failure and embryonic death, and its haploinsufficiency suppresses cancer in zebrafish. J Biol Chem 285, 2792427934.
  • 8
    Amaro AC, Samora CP, Holtackers R, Wang E, Kingston IJ, Alonso M, Lampson M, McAinsh AD & Meraldi P (2010) Molecular control of kinetochore–microtubule dynamics and chromosome oscillations. Nat Cell Biol 12, 319329.
  • 9
    Okada M, Cheeseman IM, Hori T, Okawa K, McLeod IX, Yates JR III, Desai A & Fukagawa T (2006) The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat Cell Biol 8, 446457.
  • 10
    Kwon MS, Hori T, Okada M & Fukagawa T (2007) CENP-C is involved in chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Mol Biol Cell 18, 21552168.
  • 11
    Tomonaga T, Matsushita K, Ishibashi M, Nezu M, Shimada H, Ochiai T, Yoda K & Nomura F (2005) Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy. Cancer Res 65, 46834689.
  • 12
    Liao WT, Feng Y, Li ML, Liu GL, Li MZ, Zeng MS & Song LB (2011) Overexpression of centromere protein H is significantly associated with breast cancer progression and overall patient survival. Chin J Cancer 30, 627637.
  • 13
    Shigeishi H, Higashikawa K, Ono S, Mizuta K, Ninomiya Y, Yoneda S, Taki M & Kamata N (2006) Increased expression of CENP-H gene in human oral squamous cell carcinomas harboring high-proliferative activity. Oncol Rep 16, 10711075.
  • 14
    Liao WT, Song LB, Zhang HZ, Zhang X, Zhang L, Liu WL, Feng Y, Guo BH, Mai HQ, Cao SM et al. (2007) Centromere protein H is a novel prognostic marker for nasopharyngeal carcinoma progression and overall patient survival. Clin Cancer Res 13, 508514.
  • 15
    Guo XZ, Zhang G, Wang JY, Liu WL, Wang F, Dong JQ, Xu LH, Cao JY, Song LB & Zeng MS (2008) Prognostic relevance of centromere protein H expression in esophageal carcinoma. BMC Cancer 8, 233245.
  • 16
    Liao WT, Wang X, Xu LH, Kong QL, Yu CP, Li MZ, Shi L, Zeng MS & Song LB (2009) Centromere protein H is a novel prognostic marker for human nonsmall cell lung cancer progression and overall patient survival. Cancer 115, 15071517.
  • 17
    Liao WT, Yu CP, Wu DH, Zhang L, Xu LH, Weng GX, Zeng MS, Song LB & Li JS (2009) Upregulation of CENP-H in tongue cancer correlates with poor prognosis and progression. J Exp Clin Cancer Res 28, 7481.
  • 18
    Davie JR, He S, Li L, Sekhavat A, Espino P, Drobic B, Dunn KL, Sun JM, Chen HY, Yu J et al. (2008) Nuclear organization and chromatin dynamics – Sp1, Sp3 and histone deacetylases. Adv Enzyme Regul 48, 189208.
  • 19
    Majello B, De Luca P & Lania L (1997) Sp3 is a bifunctional transcription regulator with modular independent activation and repression domains. J Biol Chem 272, 40214026.
  • 20
    Yu B, Datta PK & Bagchi S (2003) Stability of the Sp3–DNA complex is promoter-specific: Sp3 efficiently competes with Sp1 for binding to promoters containing multiple Sp-sites. Nucleic Acids Res 31, 53685376.
  • 21
    Wang XB, Peng WQ, Yi ZJ, Zhu SL & Gan QH (2007) Expression and prognostic value of transcriptional factor sp1 in breast cancer. Ai Zheng 26, 9961000.
  • 22
    Chuang JY, Wu CH, Lai MD, Chang WC & Hung JJ (2009) Overexpression of Sp1 leads to p53-dependent apoptosis in cancer cells. Int J Cancer 125, 20662076.
  • 23
    Kong LM, Liao CG, Fei F, Guo X, Xing JL & Chen ZN (2010) Transcription factor Sp1 regulates expression of cancer-associated molecule CD147 in human lung cancer. Cancer Sci 101, 14631470.
  • 24
    Xu HG, Jin R, Ren W, Zou L, Wang Y & Zhou GP (2012) Transcription factors Sp1 and Sp3 regulate basal transcription of the human IRF-3 gene. Biochimie 94, 13901397.
  • 25
    Blume SW, Snyder RC, Ray R, Thomas S, Koller CA & Miller DM (1991) Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J Clin Invest 88, 16131621.
  • 26
    Manning AL, Longworth MS & Dyson NJ (2010) Loss of pRB causes centromere dysfunction and chromosomal instability. Genes Dev 24, 13641376.
  • 27
    Martinez AC & van Wely KH (2011) Centromere fission, not telomere erosion, triggers chromosomal instability in human carcinomas. Carcinogenesis 32, 796803.
  • 28
    Varmus HE (1987) Oncogenes and transcriptional control. Science 238, 13371339.
  • 29
    Li B, Adams CC & Workman JL (1994) Nucleosome binding by the constitutive transcription factor Sp1. J Biol Chem 269, 77567763.
  • 30
    Noti JD (1997) Sp3 mediates transcriptional activation of the leukocyte integrin genes CD11C and CD11B and cooperates with c-Jun to activate CD11C. J Biol Chem 272, 2403824045.
  • 31
    Black AR, Black JD & Azizkhan-Clifford J (2001) Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 188, 143160.
  • 32
    Lee VH, Chow BK, Lo KW, Chow LS, Man C, Tsao SW & Lee LT (2009) Regulation of RASSF1A in nasopharyngeal cells and its response to UV irradiation. Gene 443, 5563.
  • 33
    Su B, Xiang B, Wang L, Cao L, Xiao L, Li X, Wu M & Li G (2010) Profiling and comparing transcription factors activated in non-metastatic and metastatic nasopharyngeal carcinoma cells. J Cell Biochem 109, 173183.
  • 34
    Ward SV & Samuel CE (2003) The PKR kinase promoter binds both Sp1 and Sp3, but only Sp3 functions as part of the interferon-inducible complex with ISGF-3 proteins. Virology 313, 553566.
  • 35
    Dokmanovic M & Marks PA (2005) Prospects: histone deacetylase inhibitors. J Cell Biochem 96, 293304.
  • 36
    Jaiswal AS, Balusu R & Narayan S (2006) 7,12-Dimethylbenzanthracene-dependent transcriptional regulation of adenomatous polyposis coli (APC) gene expression in normal breast epithelial cells is mediated by GC-box binding protein Sp3. Carcinogenesis 27, 252261.
  • 37
    Kimura N, Takamatsu N, Yaoita Y & Osamura RY (2008) Identification of transcriptional regulatory elements in the human somatostatin receptor sst2 promoter and regions including estrogen response element half-site for estrogen activation. J Mol Endocrinol 40, 7591.
  • 38
    Kim H, Lee S, Park B & Che L (2010) Sp1 and Sp3 mediate basal and serum-induced expression of human CENP-W. Mol Biol Rep 37, 35933600.
  • 39
    Koller CA & Miller DM (1986) Preliminary observations on the therapy of the myeloid blast phase of chronic granulocytic leukemia with plicamycin and hydroxyurea. N Engl J Med 315, 14331438.
  • 40
    Dutcher JP, Coletti D, Paietta E & Wiernik PH (1997) A pilot study of alpha-interferon and plicamycin for accelerated phase of chronic myeloid leukemia. Leuk Res 21, 375380.
  • 41
    Liang Y, Zhong Z, Huang Y, Deng W, Cao J, Tsao G, Liu Q, Pei D, Kang T & Zeng YX (2010) Stem-like cancer cells are inducible by increasing genomic instability in cancer cells. J Biol Chem 285, 49314940.
  • 42
    Song LB, Zeng MS, Liao WT, Zhang L, Mo HY, Liu WL, Shao JY, Wu QL, Li MZ, Xia YF et al. (2006) Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma progression and immortalizes primary human nasopharyngeal epithelial cells. Cancer Res 66, 62256232.
  • 43
    Song LB, Liao WT, Mai HQ, Zhang HZ, Zhang L, Li MZ, Hou JH, Fu LW, Huang WL, Zeng YX et al. (2006) The clinical significance of twist expression in nasopharyngeal carcinoma. Cancer Lett 242, 258265.
  • 44
    Zeng M, Kumar A, Meng G, Gao Q, Dimri G, Wazer D, Band H & Band V (2002) Human papilloma virus 16 E6 oncoprotein inhibits retinoic X receptor-mediated transactivation by targeting human ADA3 coactivator. J Biol Chem 277, 4561145618.
  • 45
    Li M, Linseman DA, Allen MP, Meintzer MK, Wang X, Laessig T, Wierman ME & Heidenreich KA (2001) Myocyte enhancer factor 2A and 2D undergo phosphorylation and caspase-mediated degradation during apoptosis of rat cerebellar granule neurons. J Neurosci 21, 65446552.
  • 46
    Yuan Z, Gong S, Luo J, Zheng Z, Song B, Ma S, Guo J, Hu C, Thiel G, Vinson C et al. (2009) Opposing roles for ATF2 and c-Fos in c-Jun-mediated neuronal apoptosis. Mol Cell Biol 29, 24312442.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgement
  8. References
  9. Supporting Information

Fig. S1. Analysis of the human CENPH promoter in CNE1 cells.

Fig. S2. Functional analysis of the Sp1/Sp3-binding sites in the CENPH promoter in CNE1 cells.

Fig. S3. The ChIP assays were performed to confirm the binding of Sp1 and Sp3 to the CENPH promoter in vivo in CNE1 and C666 cells.

Table S1. Sequences of oligonucleotides used in quantitative RT-PCR, cloning, site-directed mutagenesis, EMSA and ChIP.

Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
FEBS_8654_sm_FigS1-S3andTableS1.zip211KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.