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Identification and characterization of human PCDH10 gene promoter
Gene 475 (2011) 49–56
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
Identification and characterization of human PCDH10 gene promoter Zesong Li a, Jun Xie a, Wenjie Li a, Aifa Tang a, Xianxin Li a, Zhimao Jiang a, Yonghua Han a, Jiongxian Ye a, Jie Jing b, Yaoting Gui a,⁎, Zhiming Cai a,c,⁎ a
Guangdong Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, 1120, Lianhua Road, Futian District, Shenzhen, Guangdong 518036, PR China Peking University First Hospital, Peking University, Beijing 100034, PR China c Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, PR China b
a r t i c l e
i n f o
Article history: Accepted 3 January 2011 Available online 13 January 2011 Received by Prescott Deininger Keywords: TATA-less promoter NF-Y Sp1/Sp3 CAAT box
a b s t r a c t Recent studies have suggested roles for PCDH10 as a novel tumor suppressor gene. In our previous work, we located the core promoter of PCDH10 to a 462-bp segment of 5′-flanking region characterized by a high GC content. Here we further identified and characterized the promoter for PCDH10. Transient transfection of PC3 and LNCaP cells with a series of deleted promoter constructs indicated that the minimal promoter region was between nucleotides −144 and −99. This segment contained a CAAT box, a GT box, and a putative transcription factor binding site for AP-4. Mutational analysis identified that the CAAT box and GT box are necessary for promoter activity. Ectopic expression of NF-Ys increased reporter gene activity, whereas expression of a dominant-negative NF-YA decreased reporter gene activity. Co-transfection of Sp1/Sp3 expression plasmids enhanced reporter gene activity in a dose-dependent manner. Mithramycin A, an inhibitor of Sp–DNA interaction, reduced PCDH10 promoter activity. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp1/Sp3 to the promoter region in vitro and in vivo. Our data show that Sp1/Sp3 and CBF/NF-Y transcription factors play a crucial role in the basal expression of the human PCDH10 gene. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cadherins are transmembrane glycoproteins of a large superfamily, including the classic cadherins, desmosomal cadherins, protocadherins, atypical cadherins, and cadherin-related neuronal receptors (Nollet et al., 2000; Yagi and Takeichi, 2000; Angst et al., 2001). Protocadherins are a subfamily of the cadherin superfamily (Frank and Kemler, 2002; Hirayama and Yagi, 2006; Zou et al., 2007). The protocadherin gene subfamily consists of more than 60 genes, many of which are highly specific to nervous tissue (Sano et al., 1993). A number of protocadherin genes function as tumor suppressor genes (Waha et al., 2005; Imoto et al., 2006; Yu et al., 2008). Protocadherin-10 (PCDH10) belongs to δ2 subgroup of protocadherin subfamily. The human PCDH10 gene, also known as OL-PCDH or KIAA1400, is located at 4q28.3 on the long arm of chromosome 4 (Wolverton and Lalande, 2001). PCDH10 is richly expressed in the Abbreviations: AP-4, activating enhancer-binding protein 4; cDNA, DNA complementary to RNA; ChIP, chromatin immunoprecipitation; PCDH10, Protocadherin10; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; NE, nuclear extract; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; s.d., standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; s.e.m., standard error of the mean; TSS, transcription start site. ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Gui), [email protected] (Z. Cai). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.01.001
central nervous system and is important for the development and functioning of the central nervous system (Uemura et al., 2007; Morrow et al., 2008; Yagi, 2008). PCDH10 plays a role in the establishment and function of specific cell–cell connections in the brain (Frank and Kemler, 2002) and is essential both for growth of striatal axons and for higher ordered neural circuit formation in the ventral telencephalon (Uemura et al., 2007). Recently, PCDH10 has been implicated as tumor suppressor gene (Ying et al., 2006; Yu et al., 2009). PCDH10 is downregulated or lost in multiple human cancer types (Ying et al., 2006; Ying et al., 2007; Narayan et al., 2009; Wang et al., 2009; Yu et al., 2009). Promoter methylation and chromatin remodeling have emerged as the main mechanisms for the downregulation or loss of PCDH10 in cancers (Ying et al., 2006; Ying et al., 2007; Narayan et al., 2009; Wang et al., 2009; Yu et al., 2009; Cheung et al., 2010). Re-expression of PCDH10 can reduce tumor formation and tumor invasiveness in vivo and in vitro (Ying et al., 2006; Yu et al., 2009; Yu et al., 2010). Although the PCDH10 cDNA was already cloned in 2000 and much is known about how PCDH10 functions, the mechanistic basis governing the basal expression of PCDH10 has not been fully elucidated yet. We previously located the core promoter of PCDH10 to a 462-bp segment of 5′-flanking region characterized by a high GC content. Here we further identified and characterized the promoter and elements that regulate PCDH10 expression in human prostate cancer cells.
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2. Materials and methods 2.1. In silico analyses of putative transcription factor binding sites AliBaba2.1 (www.gene-regulation.com) and Promoter 2.0 (http:// www.cbs.dtu.dk/services/Promoter/) predication software were used to identify the transcription factor binding sites within the 462-bp segment of Human PCDH10 promoter (Knudsen, 1999; Grabe, 2002). The segment was aligned with genomic sequences upstream of PCDH10 orthologues using ClustalW within Lasergene software (DNASTAR, Madison, WI, USA) with default parameters. 2.2. Construction of promoter reporter plasmids and site-directed mutagenesis For construction of a series of PCDH10 promoter-driven luciferase reporter plasmids, various lengths of DNA fragments from the 5′-flanking CpG island of PCDH10 were amplified by PCR with flanking 5′-KpnI and 3′-BglII enzyme restriction sites using Hotstart DNA polymerase (Qiagen, Hilden, Germany). The PCR products were enzyme-digested and then cloned into pGL3-basic vector (Promega, Madison, WI). The inserts were identified by restriction digestion, and the integrity of the constructs was confirmed by sequencing. Promoter constructs harboring nucleotide substitutions in putative transcription factor binding elements were prepared using an overlapping extension PCR protocol. The mutant amplicons were subcloned into pGL3-basic vector. All the site-directed mutagenesis constructs were sequenced to confirm the intended mutations. 2.3. Cell culture, transfection, and luciferase assay The prostate cancer cell lines PC3 and LNCaP (ATCC, Manassas, VA) were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 4.5 mM glutamine. PC3 and LNCaP cells were plated in 24-well culture plate at 1 × 105/well and transiently transfected with the pGL3 promoter reporter and pRLSV40 (Renilla luciferase, Promega, Madison, WI, USA) (50 ng/well) by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). When indicated, pCMV-Sp1, pCMV-Sp3 expression plasmids (OriGene, Rockville, MD, USA), pSG5-CBF/NF-Ys or dominant-negative CBF/NF-YA plasmid (kindly provided by Dr. R. Mantovani) were co-transfected into the cells (Mantovani et al., 1994). After 4 h of incubation, the culture medium was replaced with fresh medium and incubated for a further 48 h, and then cellular extracts were prepared for luciferase activity measurement. Mithramycin A (Sigma-aldrich, St. Louis, MO, USA) was incubated for 24 h before luciferase assays. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instructions and normalized with respect to Renilla luciferase activity. Each experiment was performed at least three times, in triplicate. Activity was defined as Firefly/Renilla ratio. 2.4. Real-time quantitative PCR Total RNA was isolated from prostate cancer cell lines using TRIzol (Invitrogen, Carlsbad, CA, USA). 1 μg of total RNA was reverse transcribed to first-strand cDNA with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using oligo(dT)18 primers, according to the manufacturer's protocol. Real-time PCR was performed on the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using Platinum SYBER Green qPCR Supermix UDG (Invitrogen, Carlsbad, CA, USA). The assay was carried out in triplicate using primer sequences for Sp1 (forward primer 5′-CAGAACCCACAAGCCCAAACAATC-3′; reverse primer 5′-ATGGAGGAGAGTTGAGCAGCATTC-3′), Sp3 (forward primer 5′-GTCAGCAGATGGTCAGCAGGTTC-3′; reverse primer 5′-AAGGTGTTCCAGAGGCAAGTAAGG-3′), and GAPDH
(forward primer 5′-CGCTCTCTGCTCCTCCTGTTC-3′; reverse primer 5′-ATCCGTTGACTCCGACCTTCAC-3′). The relative expression levels of Sp1 and Sp3 was determined using the 2−ΔΔCt method (Livak and Schmittgen, 2001). 2.5. Electrophoretic mobility shift and supershift Nuclear extracts were prepared using NE-PERTM nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Electrophoretic mobility shift assays (EMSAs) were carried out using a lightshift chemiluminescent EMSA kit (Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The sequence of the sense strands of oligonucleotides were as follows: wild type, 5′-CGGCAGCTCGGTGGGTGGTGCTCC-3′, and mutated type, 5′-CGGCAGCTCAAGGCCTGGTGCTCC-3′. The biotin 3′-end-labeled oligonucleotide and its reverse complement were annealed and purified. Standard EMSA reactions contained 5 μg of nuclear extracts and 20 fmol of biotin 3′end-labeled DNA in a 20 μl binding reaction mixture in the presence of 2.5% glycerol, 5 mM MgCl2, 50 ng/μl of poly(dI·dC), and 0.05% Nonidet P-40. When indicated, 200-fold molar excess of unlabeled oligonucleotide or nonspecific oligonucleotide was introduced to the reaction mixture for competition assay. For supershift assays, nuclear extracts were preincubated with rabbit anti-Sp1 (sc-59X, Santa Cruz Biologicals, Santa Cruz, CA, USA) or rabbit anti-Sp3 antibody (sc-644X, Santa Cruz Biologicals, Santa Cruz, CA, USA) for 40 min on ice, followed by the addition with the indicated probe. For assays in the presence of mithramycin A (Sigma, St. Louis, MO, USA), the DNA probe was preincubated with different concentrations of compound (100, 200 nM final concentration) for 40 min on ice, and then nuclear extracts were added. DNA–protein complexes were separated on 4% polyacrylamide gels. Reaction products were then transferred to a Biodyne B membrane (Pierce, Rockford, IL, USA) and fixed by UV cross-linking. The biotin-labeled reaction products were then visualized by incubation with streptavidin horseradish peroxidase conjugate and subsequent incubation with ECL chemiluminescent reagents. 2.6. Chromatin immunoprecipitation assay Chromatin immunoprecipitation assays were performed using the EZChIP kit (Upstate, Charlottesville, VA, USA) following the manufacturer's instructions. Cells were fixed in 1% formaldehyde at room temperature and glycine was added to quench unreacted aldehydes. Cells were washed, scraped, and collected by centrifugation at 4 °C. Cells were resuspended and sonicated (10 s pulses×5 at 30% duty cycle, Branson sonifier Cell disruptor 250, MA) to shear DNA. Protein–DNA cross-linked products were enriched by immunoprecipitation with 5 μg of anti-Sp1 (sc-59X) or anti-Sp3 antibody (sc-644X). DNA–protein crosslinks were reversed by adding 8 μl 5 M NaCl and incubating the mixture at 65 °C for 5 h. After DNA purification, the extent of enrichment was monitored by PCR using primers (forward primer 5′-TGCCGCGTGACGTGTCTGT-3′; reverse primer, 5′-GCCCTCATTCTGCCAACCAA-3′) specific to PCDH10 promoter fragments. The products were resolved on 2% agarose gel and visualized by ethidium bromide staining. Each of the experiments was repeated at least three times with similar results. 2.7. Western blot analysis Cells were lysed and extracted by RIPA buffer (R0278; Sigma, St. Louis, MO, USA) supplement with protease inhibitor cocktails and phosphatase inhibitors. 40 μg of total protein was separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). After blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 3 h, membrane was incubated with rabbit anti-Sp1 (sc-59X), rabbit anti-Sp3 antibody (sc-644X) or rabbit anti-GAPDH
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antibody (G9545, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. After washed with TBST for three times, the membrane was incubated with HRP-labeled secondary antibody (sc-2030, Santa Cruz Biologicals, Santa Cruz, CA, USA) at room temperature for 1 h. The blotting signals were visualized by using Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA). 3. Results 3.1. Identification of regulatory elements in the PCDH10 promoter In our previous work, we located the core promoter of PCDH10 to a 462-bp (−450/+12) segment of 5′-flanking region characterized by a high GC content. DNA sequence analysis revealed the presence of a CACA box, a CAAT box, a GT box, and several putative binding sites for the basic transcription factors Sp, AP-4 and Creb, but lacked a typical TATA box (Fig. 1A). We aligned the 462 bp sequence with corresponding sequences from Chimpanzee, cattle, rat and mouse, which revealed that sequences from the − 368 to −7 were evolutionarily conserved (Supplementary Fig. 1). A small part of most-conserved DNA segment is shown in Fig 1, which contained a CAAT box, a GT box and several putative binding sites (Fig. 1B). In order to assess which portions of the promoter were involved in the regulation of PCDH10 expression, a series of deletion constructs were generated according to putative binding sites of transcription factors and used to transiently transfect PC3 cells and LNCaP cells. Luciferase transcription was consistently more active in PC3 than in LNCaP cells (Fig. 2). Both in PC3 and LNCaP cells, −160/+12 construct provided highest promoter activity whereas −450/−131 construct and −99/+12 construct are clearly of little importance to PCDH10 expression. These data showed that the region containing the sequence between −144 and −99 was largely dispensable to the basal promoter activity in these two cells. This region contained the CAAT box, the GT box and AP-4 elements. 3.2. Reporter assay analysis of CAAT-box, GT-box, and AP-4 elements mutations To examine the contribution of CAAT box, GT box, and AP-4 elements to the expression of PCDH10, we constructed a series of pGL3-F2 plasmids with substitution mutations in the CAAT box, GT box, and/or AP-4 elements (Fig. 3). They were transfected into PC3 cells and LNCaP cells, and their activities were compared to wild-type constructs. The mutations in the CAAT box resulted in about 40% and 23% reduction of promoter activity in PC3 cells and LNCaP cells, respectively (Fig. 3). The mutations in the GT box resulted in about 56% and 50% reduction of promoter activity in PC3 cells and LNCaP cells, respectively. The mutations in the CAAT box and GT box resulted in very little promoter activity remained. But the mutations in the AP-4 elements did not result in the reduction of promoter activity (Fig. 3). These results strongly suggested that the PCDH10 promoter was cooperatively regulated by the CAAT box and GT box in these two cell lines.
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or pGL3-F2△3 with pCMV-Sp1 or pCMV-Sp3 (0.4 μg). The overexpression of Sp1/Sp3 had no effects on promoter activities in the construct pGL3-F0, which does not contain the GT box, or pGL3-F2△3, which contains the mutated Sp1 binding sites (Fig. 4F). The roles of Sp1/Sp3 in the regulation of the PCDH10 gene were further examined by inhibiting their binding to the promoter. PC3 cells and LNCaP cells were untreated or treated 1 h after transfection with 200 nM of mithramycin A for 24 h, which recognizes GT-rich promoter regions and interferes with the binding of the Sp family (Blume et al., 1991). Mithramycin A decreased promoter activity in the constructs containing the GT box but had no effect on construct pGL3-F0 or pGL3-F2△3, which do not contain the GT box or contain the mutated GT box (Fig. 5A). The CBF/NF-Y in the regulation of the PCDH10 gene was also examined by the overexpression of CBF/NF-Ys or the dominantnegative CBF/NF-YA subunit, which interact with the CBF/NF-YB and CBF/NF-YC subunits to form CBF/NF-Y. As expected, the overexpression of CBF/NF-Ys resulted in a significant stimulation of the activity of pGL3-F2 (Fig. 5B). The overespression of the dominant-negative CBF/NF-YA subunit decreased the activity of pGL3-F2, which contains the CAAT box, but had no effect on the construct pGL3-F2△1, which do contain the mutated CAAT box (Fig. 5B). 3.4. EMSA analysis of GT-box-binding proteins Electrophoretic Mobility Shift Assays (EMSA) and antibody supershift assays were used to determine whether Sp1/Sp3 protein binding occurred within the PCDH10 promoter by using nuclear extracts from PC3 and LNCaP cells, which express higher levels of Sp1 and Sp3 proteins (Tang et al., 2004). As shown in Fig. 6, the Sp1/Sp3 EMSA banding pattern was observed. The specificity of the transcription factor binding was proven by inhibition of binding of biotinylated probe by excess unlabeled wild-type probe (Fig. 6. lane 3). Upon incubation with antibodies to either Sp1 or Sp3, the shift of the specific bands was reduced, which indicated the binding of Sp1/Sp3 to the consensus site. The binding was drastically reduced upon by pretreatment of cells with Mithramycin A (Fig. 6). 3.5. Chromatin immunoprecipitation analysis of Sp1/Sp3 binding to the PCDH10 promoter Further validation of Sp1/Sp3 protein binding to the PCDH10 promoter was obtained by ChIP analysis in native chromatin isolated from PC3 cell and LNCaP cells. The precipitated chromosomal DNA was subjected to PCR amplification of the PCDH10 promoter region harboring the GT-box site. Sp1/Sp3 indeed bound to the PCDH10 promoter region containing the GT-box site. The amplification of input DNA was equal in all the samples and no amplification product was seen in the presence of control rabbit IgG or in the absence of antibody (Fig. 7). Thus, specific amplification was achieved by precipitation with Sp1/Sp3 only. Taken together, these results showed that Sp1/Sp3 binds specifically to PCDH10 promoter to augment PCDH10 transcription. 4. Discussion
3.3. Functional analysis of Sp1/Sp3 and CBF/NF To determine the effects of Sp1/Sp3 on PCDH10 promoter, PC3 and LNCaP cells were co-transfected with the constructs pGL3-F2 and pCMV-Sp1 or pCMV-Sp3, and luciferase activities were measured. The production of Sp1 and Sp3 was confirmed by RT-PCR analysis (Fig. 4A and B) and Western blot analysis (Fig. 4C and D). Furthermore, co-transfection with pCMV-Sp1 or pCMV-Sp3 resulted in a significant stimulation of PCDH10 promoter activity in a plasmid dose-dependent manner in PC3 and LNCaP cells (Fig. 4E). To confirm that Sp1/Sp3 elements indeed contributed to the Sp1/ Sp3-mediated upregulation, we cotransfected the construct pGL3-F0
We previously defined the core promoter of PCDH10 to a 462-bp segment of 5′-flanking region of PCDH10 gene. This segment has at least 88% identity with corresponding sequence of Chimpanzee, cattle, rat, and mouse PCDH10 promoter (Supplementary Fig. 1), which is consistent with the idea that evolutionary conservation of non-coding sequences is an indication of functional transcription factor binding sites (Hardison, 2000). In silico analyses of genomic sequence showed that PCDH10 promoter belonged to the TATA-less, CpG island-associated class of mammalian promoters (Fig. 1). This type of promoter typically contains multiple GC-boxes or GT boxes (Carninci et al., 2006; Purvis et al., 2010).
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Fig. 1. Characterization and evolutionary conservation of the 5′ flanking region of the PCDH10 gene. A, The sequence corresponds to nts 58,617,741 to 58,618,202 of human chromosome 4 (GenBank accession no. NT_016354.19). Putative transcription factor binding sites are boxed and labeled above. B, Promoter sequences from Chimpanzee (nts 1,245,852–1,246,043, GenBank accession no. NW_001234084.1), cattle (nts 349,455–349,211, GenBank accession no. NW_001493497.2), rat (nts 3,177,214–3,177,405, GenBank accession no. NW_001084802.1) and mouse (nts 4,727,630–4,727,821, GenBank accession no. NT_039229.7) is aligned to the human PCDH10 promoter region. Homologous bases are highlighted in grey. Putative consensus-binding elements for transcription factors are indicated above.
By transient transfections of PC3 and LNCaP cells with a series of 5′- and 3′-deletions promoter constructs, we found that the sequence between −144 and −99 was sufficient for the basal transcriptal activity
of the human PCDH10 gene in PC3 and LNCaP cells (Fig. 2). Substitution mutation showed that both CAAT box and GT box within the region are essential for promoter activity. But GT box seemed to play a more
Fig. 2. Promoter activity of human PCDH10 deletion constructs in PC3 and LNCaP. The cells were transfected with 0.6 μg pGL3-basic or luciferase reporter constructs containing different size promoter fragments (indicated in bp relative to the transcription start site (+ 1)). All of the constructs were cotransfected with pRLSV40 vector as an internal control for transfection efficiency. Promoter activity of each construct was measured as firefly luciferase activity normalized to Renilla luciferase, and the results were expressed as fold relative to the activity of pGL3-basic (which was assigned an activity value of 1.0). The means and S.E. are shown (n = 3). The data are representative of at least three independent experiments. Transfections were carried out in triplicate for each experiment.
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Fig. 3. Identification of functional sites within the human PCDH10 promoter region under basal conditions in PC3 and LNCaP cells. Schematic representation of the PCDH10 promoter site-directed mutagenesis constructs used for transient transfections is shown on the left (nts 58,618,070–58,618,202, GenBank accession no. NT_016354.19). Promoter activity was measured as described in Fig. 2. The results are expressed as the means ± S.E. (n = 3). The data are representative of at least three independent experiments. Transfections were carried out in triplicate for each experiment. Student's t-test, *p b 0.01; **p b 0.001.
Fig. 4. Effects of Sp1/Sp3 on reporter gene expression. PC3 and LNCaP cells were transfected with pCMV-Sp1/Sp3 expression plasmids, and the empty vector was cotransfected as a control in each group. (A and B) Relative mRNA levels of Sp1 and Sp3 in PC3 and LNCaP cells were determined by using real-time quantitative RT-PCR and SYBR Green dye. The relative expression level of PCDH10 was determined by using the 2−ΔΔCt method. The expression of control in PC3 or LNCaP cells was set as 1. Student's t-test, **p b 0.001. (C and D) The expression of SP1 and SP3 was assessed using Western blot. (E) PC3 and LNCaP cells were co-transfected with 0.6 μg of pGL3-F2 and the indicated concentrations of pCMV-Sp1/ Sp3 expression plasmids. The activity of the control in PC3 or LNCaP cells was set as 1, and the remaining activities were expressed as the fold induction against the control. The data are expressed as the means ± S.E. (n = 3). Student's t-test,*p b 0.01; **p b 0.001. (F) PC3 and LNCaP cells were co-transfected with 0.6 μg of pGL3-F0 or pGL3-F2Δ3, and 0.4 μg of pCMV-Sp1/Sp3 expression plasmids. All of the constructs were cotransfected with pRLSV40 vector as an internal control for transfection efficiency. Luciferase activity is expressed as the percent activity of the control. The data are expressed as the means ± S.E. (n = 3).
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Fig. 5. The functional analysis by inhibition of the binding of Sp1/3 and CBF/NF-Y to PCDH10 promoter. (A) PC3 and LNCaP cells were transfected with 0.6 μg of pGL3-F2, pGL3-F0, pGL3-F2△3. The cells were treated for 40 h with or without 200 nM of mithramycin. Luciferase activity is expressed as the percent activity of the mithramycin-untreated cells. (B) PC3 and LNCaP cells were co-transfected 0.6 μg of pSG5-CBF/NF-Ys or dominant-negative CBF/NF-YA plasmid and 0.6 μg of pGL3-F2 or pGL3-F2△1. The empty vector was cotransfected as a control in each group. All of the constructs were cotransfected with pRLSV40 vector as an internal control for transfection efficiency. Luciferase activity is expressed as the percent activity of the control. The data are expressed as the means ± S.E. (n = 3). Student's t-test, *p b 0.01.
Fig. 6. Binding of Sp1/Sp3 to the promoter region of the PCDH10 gene. Electrophoretic mobility shift assay (EMSA) was performed without or with nuclear extracts (8 μg) from (A) PC3 and (B) LNCaP cells in the presence of the various competitors. Lane 1, biotin-labeled probe only; lanes 2–8, labeled probe with 8 μg nuclear extract; lane 3, 100-fold molar excess of unlabeled probe; lanes 4, biotin-labeled mutated probe; lanes 5–6, the nuclear extracts were preincubated with anti-Sp1 (lane 5) or anti-Sp3 (lane 6) antibodies for 30 min and then added to the reaction mixture; lanes 7–8, the nuclear extracts were preincubated with 100 nM (lane 7) or 200 nM mithramycin A (lane 8) for 30 min and then added to the reaction mixture. Arrows indicated the formation of specific protein–DNA complexes. Each of the experiments was repeated at least three times with similar results.
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Fig. 7. In vivo association between Sp1/Sp3 and human PCDH10 gene promoter. Chromatin immunoprecipitation assay was used to analyze the interaction of Sp3 and Sp1 to the PCDH10 promoter in PC3 and LNCaP cells. The immunoprecipitated DNA obtained from ChIP is amplified utilizing PCR primers specific for the region containing the CAAT box and GT box in the PCDH10 gene promote. PCR products were separated on a 2% agarose gel containing ethidium bromide and detected via ultraviolet illumination. This figure shows a representative of at least three independent experiments. Lane 1, input DNA; lane 2, H2O; lane 3, normal rabbit IgG control; lanes 4–5, anti-Sp1 and anti-Sp3 antibodies.
important role in regulating the PCDH10 expression because mutation in GT box resulted in more decrease of PCDH10 promoter activity (Fig. 3), which was supported by the fact that the sequence of GT box was more conserved than that of CAAT box in evolution (Fig. 1B). CAAT boxes are found in the promoter region of 30% of genes, including housekeeping genes, inducible and cell-cycle regulated genes (Mantovani, 1999). The role of CAAT boxes depends on their ability to interact with a variety of transcription factors (Mantovani, 1999). NF-Y is a ubiquitous transcription factor involved in transcription of various promoters harboring CAAT box (Sun et al., 2009). Previous studies showed that dominant-negative NF-YA mutant can inhibit the interaction of NF-Y with CAAT boxes (Mantovani et al., 1994; Mantovani, 1999; van Wageningen et al., 2008; Luan et al., 2010; Pallai et al., 2010). In the present study, the expression of this dominant-negative NF-YA mutant repressed the human PCDH10 gene promoter activity in PC3 and LNCaP cells (Fig. 5B), which confirmed the interaction between NF-Y and human PCDH10 promoter. GT box is a consensus binding site for the Sp family of transcription factors (Sp1–Sp4). Sp1 and Sp3 recognize the same promoter elements (GC- and GT-boxes) with similar specificity and affinity (Hagen et al., 1992). Various reports have shown that Sp1 is a transcriptional activator, whereas Sp3 can act as either an activator or a repressor of transcription depending on the cell and promoter context (Yu et al., 2003; Safe and Abdelrahim, 2005). Our data indicated that overexpression of Sp1 or Sp3 was able to activate the human PCDH10 promoter in PC3 and LNCaP cells (Fig. 4E). This observation was consistent with the findings that Sp1 or Sp3 is known to play an important role in TATA-box less promoters and was associated with multiple transcription initiation sites (Melton et al., 1984; Muckenfuss et al., 2007). Sp family of transcription factors has been shown to regulate the expression of cadherin genes such as E-cadherin (Liu et al., 2005), N-cadherin (Le Mee et al., 2005) and vascular endothelial cadherin gene transcription (Gory et al., 1998). Together with our data, this indicated that Sp family of transcription factors played an important role in regulating the expression of cadherin gene family. Our ChIP and EMSA data strongly indicated that the transcription factors Sp1 and Sp3 could bind specifically to the GT box and therefore regulated PCDH10 transcription (Figs. 6 and 7). Supershift complex I and III were stronger than complex II in PC3 (Fig. 6), but the situation was converse in LNCaP cells, possibly due to the different cells. It has been reported that mithramycin A inhibits transcription of many genes through suppression of Sp1/Sp3 binding to their promoters (Blume et al., 1991). We also found that mithramycin A inhibited PCDH10 promoter activity by using combination of luciferase reporter assay and EMSA (Fig. 6).
In summary, the present results showed that the binding sites for NF-Y and Sp1/Sp3 were critical for PCDH10 transcription and thus the expression of PCDH10 might be modulated by the levels or activities of these transcription factors. This study extends our knowledge of the transcriptional regulation of PCDH10 and may therefore contribute to better understand the role of PCDH10 in tissue development and during pathophysiological processes. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2011.01.001. Acknowledgments This work was supported by the National Natural Science Foundation of China (30700824, 30972992), the Research Fund for the Doctoral Program of Higher Education of China (200800010106), the Medical Scientific Research Foundation of Guangdong Province (A2010548), and the Scientific Research Foundation of the State Human Resource Ministry for Returned Chinese Scholars, China. We thank Dr. Robert Mantovani at the University of Milan, Italy for the expression plasmid of NF-YA, -YB, -YC and the dominant-negative NF-YA mutant expression vector. References Angst, B.D., Marcozzi, C., Magee, A.I., 2001. The cadherin superfamily: diversity in form and function. J. Cell Sci. 114, 629–641. Blume, S.W., Snyder, R.C., Ray, R., Thomas, S., Koller, C.A., Miller, D.M., 1991. Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J. Clin. Invest. 88, 1613–1621. Carninci, P., et al., 2006. Genome-wide analysis of mammalian promoter architecture and evolution. Nat. Genet. 38, 626–635. Cheung, H.H., Lee, T.L., Davis, A.J., Taft, D.H., Rennert, O.M., Chan, W.Y., 2010. Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer. Br. J. Cancer 102, 419–427. Frank, M., Kemler, R., 2002. Protocadherins. Curr. Opin. Cell Biol. 14, 557–562. Gory, S., Dalmon, J., Prandini, M.H., Kortulewski, T., de Launoit, Y., Huber, P., 1998. Requirement of a GT box (Sp1 site) and two Ets binding sites for vascular endothelial cadherin gene transcription. J. Biol. Chem. 273, 6750–6755. Grabe, N., 2002. AliBaba2: context specific identification of transcription factor binding sites. In Silico Biol. 2, S1–S15. Hagen, G., Muller, S., Beato, M., Suske, G., 1992. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res. 20, 5519–5525. Hardison, R.C., 2000. Conserved noncoding sequences are reliable guides to regulatory elements. Trends Genet. 16, 369–372. Hirayama, T., Yagi, T., 2006. The role and expression of the protocadherin-alpha clusters in the CNS. Curr. Opin. Neurobiol. 16, 336–342. Imoto, I., et al., 2006. Frequent silencing of the candidate tumor suppressor PCDH20 by epigenetic mechanism in non-small-cell lung cancers. Cancer Res. 66, 4617–4626. Knudsen, S., 1999. Promoter2.0: for the recognition of PolII promoter sequences. Bioinformatics 15, 356–361.
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Le Mee, S., Fromigue, O., Marie, P.J., 2005. Sp1/Sp3 and the myeloid zinc finger gene MZF1 regulate the human N-cadherin promoter in osteoblasts. Exp. Cell Res. 302, 129–142. Liu, Y.N., Lee, W.W., Wang, C.Y., Chao, T.H., Chen, Y., Chen, J.H., 2005. Regulatory mechanisms controlling human E-cadherin gene expression. Oncogene 24, 8277–8290. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408. Luan, X., Ito, Y., Zhang, Y., Diekwisch, T.G., 2010. Characterization of the mouse CP27 promoter and NF-Y mediated gene regulation. Gene 460, 8–19. Mantovani, R., 1999. The molecular biology of the CCAAT-binding factor NF-Y. Gene 239, 15–27. Mantovani, R., Li, X.Y., Pessara, U., Hooft van Huisjduijnen, R., Benoist, C., Mathis, D., 1994. Dominant negative analogs of NF-YA. J. Biol. Chem. 269, 20340–20346. Melton, D.W., Konecki, D.S., Brennand, J., Caskey, C.T., 1984. Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene. Proc. Natl Acad. Sci. USA 81, 2147–2151. Morrow, E.M., et al., 2008. Identifying autism Loci and genes by tracing recent shared ancestry. Science 321, 218–223. Muckenfuss, H., et al., 2007. Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene. Nucleic Acids Res. 35, 3784–3796. Narayan, G., Scotto, L., Neelakantan, V., Kottoor, S.H., Wong, A.H., Loke, S.L., Mansukhani, M., Pothuri, B., Wright, J.D., Kaufmann, A.M., Schneider, A., Arias-Pulido, H., Tao, Q., Murty, V.V., 2009. Protocadherin PCDH10, involved in tumor progression, is a frequent and early target of promoter hypermethylation in cervical cancer. Genes Chromosomes Cancer. Nollet, F., Kools, P., van Roy, F., 2000. Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members. J. Mol. Biol. 299, 551–572. Pallai, R., Simpkins, H., Chen, J., Parekh, H.K., 2010. The CCAAT box binding transcription factor, nuclear factor-Y (NF-Y) regulates transcription of human aldo-keto reductase 1 C1 (AKR1C1) gene. Gene 459, 11–23. Purvis, T.L., et al., 2010. Transcriptional regulation of the Alstrom syndrome gene ALMS1 by members of the RFX family and Sp1. Gene 460, 20–29. Safe, S., Abdelrahim, M., 2005. Sp transcription factor family and its role in cancer. Eur. J. Cancer 41, 2438–2448. Sano, K., Tanihara, H., Heimark, R.L., Obata, S., Davidson, M., St John, T., Taketani, S., Suzuki, S., 1993. Protocadherins: a large family of cadherin-related molecules in central nervous system. EMBO J 12, 2249–2256. Sun, F., Xie, Q., Ma, J., Yang, S., Chen, Q., Hong, A., 2009. Nuclear factor Y is required for basal activation and chromatin accessibility of fibroblast growth factor receptor 2 promoter in osteoblast-like cells. J. Biol. Chem. 284, 3136–3147.
Tang, S., et al., 2004. Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells. Oncogene 23, 6942–6953. Uemura, M., Nakao, S., Suzuki, S.T., Takeichi, M., Hirano, S., 2007. OL-protocadherin is essential for growth of striatal axons and thalamocortical projections. Nat. Neurosci. 10, 1151–1159. van Wageningen, S., Nikoloski, G., Vierwinden, G., Knops, R., van der Reijden, B.A., Jansen, J.H., 2008. The transcription factor nuclear factor Y regulates the proliferation of myeloid progenitor cells. Haematologica 93, 1580–1582. Waha, A., Guntner, S., Huang, T.H., Yan, P.S., Arslan, B., Pietsch, T., Wiestler, O.D., 2005. Epigenetic silencing of the protocadherin family member PCDH-gamma-A11 in astrocytomas. Neoplasia 7, 193–199. Wang, K.H., Liu, H.W., Lin, S.R., Ding, D.C., Chu, T.Y., 2009. Field methylation silencing of the protocadherin 10 gene in cervical carcinogenesis as a potential specific diagnostic test from cervical scrapings. Cancer Sci. Wolverton, T., Lalande, M., 2001. Identification and characterization of three members of a novel subclass of protocadherins. Genomics 76, 66–72. Yagi, T., 2008. Clustered protocadherin family. Dev. Growth Differ. 50 (Suppl 1), S131–S140. Yagi, T., Takeichi, M., 2000. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev. 14, 1169–1180. Ying, J., et al., 2007. Frequent epigenetic silencing of protocadherin 10 by methylation in multiple haematologic malignancies. Br. J. Haematol. 136, 829–832. Ying, J., et al., 2006. Functional epigenetics identifies a protocadherin PCDH10 as a candidate tumor suppressor for nasopharyngeal, esophageal and multiple other carcinomas with frequent methylation. Oncogene 25, 1070–1080. Yu, B., Datta, P.K., 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, 5368–5376. Yu, B., et al., 2010. High-resolution melting analysis of PCDH10 methylation levels in gastric, colorectal and pancreatic cancers. Neoplasma 57, 247–252. Yu, J., et al., 2009. Methylation of protocadherin 10, a novel tumor suppressor, is associated with poor prognosis in patients with gastric cancer. Gastroenterology 136, 640–651. Yu, J.S., et al., 2008. PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer. Oncogene 27, 4657–4665. Zou, C., Huang, W., Ying, G., Wu, Q., 2007. Sequence analysis and expression mapping of the rat clustered protocadherin gene repertoires. Neuroscience 144, 579–603.
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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e
Identification and characterization of human PCDH10 gene promoter Zesong Li a, Jun Xie a, Wenjie Li a, Aifa Tang a, Xianxin Li a, Zhimao Jiang a, Yonghua Han a, Jiongxian Ye a, Jie Jing b, Yaoting Gui a,⁎, Zhiming Cai a,c,⁎ a
Guangdong Key Laboratory of Male Reproductive Medicine and Genetics, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, 1120, Lianhua Road, Futian District, Shenzhen, Guangdong 518036, PR China Peking University First Hospital, Peking University, Beijing 100034, PR China c Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, PR China b
a r t i c l e
i n f o
Article history: Accepted 3 January 2011 Available online 13 January 2011 Received by Prescott Deininger Keywords: TATA-less promoter NF-Y Sp1/Sp3 CAAT box
a b s t r a c t Recent studies have suggested roles for PCDH10 as a novel tumor suppressor gene. In our previous work, we located the core promoter of PCDH10 to a 462-bp segment of 5′-flanking region characterized by a high GC content. Here we further identified and characterized the promoter for PCDH10. Transient transfection of PC3 and LNCaP cells with a series of deleted promoter constructs indicated that the minimal promoter region was between nucleotides −144 and −99. This segment contained a CAAT box, a GT box, and a putative transcription factor binding site for AP-4. Mutational analysis identified that the CAAT box and GT box are necessary for promoter activity. Ectopic expression of NF-Ys increased reporter gene activity, whereas expression of a dominant-negative NF-YA decreased reporter gene activity. Co-transfection of Sp1/Sp3 expression plasmids enhanced reporter gene activity in a dose-dependent manner. Mithramycin A, an inhibitor of Sp–DNA interaction, reduced PCDH10 promoter activity. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp1/Sp3 to the promoter region in vitro and in vivo. Our data show that Sp1/Sp3 and CBF/NF-Y transcription factors play a crucial role in the basal expression of the human PCDH10 gene. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Cadherins are transmembrane glycoproteins of a large superfamily, including the classic cadherins, desmosomal cadherins, protocadherins, atypical cadherins, and cadherin-related neuronal receptors (Nollet et al., 2000; Yagi and Takeichi, 2000; Angst et al., 2001). Protocadherins are a subfamily of the cadherin superfamily (Frank and Kemler, 2002; Hirayama and Yagi, 2006; Zou et al., 2007). The protocadherin gene subfamily consists of more than 60 genes, many of which are highly specific to nervous tissue (Sano et al., 1993). A number of protocadherin genes function as tumor suppressor genes (Waha et al., 2005; Imoto et al., 2006; Yu et al., 2008). Protocadherin-10 (PCDH10) belongs to δ2 subgroup of protocadherin subfamily. The human PCDH10 gene, also known as OL-PCDH or KIAA1400, is located at 4q28.3 on the long arm of chromosome 4 (Wolverton and Lalande, 2001). PCDH10 is richly expressed in the Abbreviations: AP-4, activating enhancer-binding protein 4; cDNA, DNA complementary to RNA; ChIP, chromatin immunoprecipitation; PCDH10, Protocadherin10; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; NE, nuclear extract; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; s.d., standard deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; s.e.m., standard error of the mean; TSS, transcription start site. ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Gui), [email protected] (Z. Cai). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.01.001
central nervous system and is important for the development and functioning of the central nervous system (Uemura et al., 2007; Morrow et al., 2008; Yagi, 2008). PCDH10 plays a role in the establishment and function of specific cell–cell connections in the brain (Frank and Kemler, 2002) and is essential both for growth of striatal axons and for higher ordered neural circuit formation in the ventral telencephalon (Uemura et al., 2007). Recently, PCDH10 has been implicated as tumor suppressor gene (Ying et al., 2006; Yu et al., 2009). PCDH10 is downregulated or lost in multiple human cancer types (Ying et al., 2006; Ying et al., 2007; Narayan et al., 2009; Wang et al., 2009; Yu et al., 2009). Promoter methylation and chromatin remodeling have emerged as the main mechanisms for the downregulation or loss of PCDH10 in cancers (Ying et al., 2006; Ying et al., 2007; Narayan et al., 2009; Wang et al., 2009; Yu et al., 2009; Cheung et al., 2010). Re-expression of PCDH10 can reduce tumor formation and tumor invasiveness in vivo and in vitro (Ying et al., 2006; Yu et al., 2009; Yu et al., 2010). Although the PCDH10 cDNA was already cloned in 2000 and much is known about how PCDH10 functions, the mechanistic basis governing the basal expression of PCDH10 has not been fully elucidated yet. We previously located the core promoter of PCDH10 to a 462-bp segment of 5′-flanking region characterized by a high GC content. Here we further identified and characterized the promoter and elements that regulate PCDH10 expression in human prostate cancer cells.
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2. Materials and methods 2.1. In silico analyses of putative transcription factor binding sites AliBaba2.1 (www.gene-regulation.com) and Promoter 2.0 (http:// www.cbs.dtu.dk/services/Promoter/) predication software were used to identify the transcription factor binding sites within the 462-bp segment of Human PCDH10 promoter (Knudsen, 1999; Grabe, 2002). The segment was aligned with genomic sequences upstream of PCDH10 orthologues using ClustalW within Lasergene software (DNASTAR, Madison, WI, USA) with default parameters. 2.2. Construction of promoter reporter plasmids and site-directed mutagenesis For construction of a series of PCDH10 promoter-driven luciferase reporter plasmids, various lengths of DNA fragments from the 5′-flanking CpG island of PCDH10 were amplified by PCR with flanking 5′-KpnI and 3′-BglII enzyme restriction sites using Hotstart DNA polymerase (Qiagen, Hilden, Germany). The PCR products were enzyme-digested and then cloned into pGL3-basic vector (Promega, Madison, WI). The inserts were identified by restriction digestion, and the integrity of the constructs was confirmed by sequencing. Promoter constructs harboring nucleotide substitutions in putative transcription factor binding elements were prepared using an overlapping extension PCR protocol. The mutant amplicons were subcloned into pGL3-basic vector. All the site-directed mutagenesis constructs were sequenced to confirm the intended mutations. 2.3. Cell culture, transfection, and luciferase assay The prostate cancer cell lines PC3 and LNCaP (ATCC, Manassas, VA) were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 4.5 mM glutamine. PC3 and LNCaP cells were plated in 24-well culture plate at 1 × 105/well and transiently transfected with the pGL3 promoter reporter and pRLSV40 (Renilla luciferase, Promega, Madison, WI, USA) (50 ng/well) by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). When indicated, pCMV-Sp1, pCMV-Sp3 expression plasmids (OriGene, Rockville, MD, USA), pSG5-CBF/NF-Ys or dominant-negative CBF/NF-YA plasmid (kindly provided by Dr. R. Mantovani) were co-transfected into the cells (Mantovani et al., 1994). After 4 h of incubation, the culture medium was replaced with fresh medium and incubated for a further 48 h, and then cellular extracts were prepared for luciferase activity measurement. Mithramycin A (Sigma-aldrich, St. Louis, MO, USA) was incubated for 24 h before luciferase assays. Luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instructions and normalized with respect to Renilla luciferase activity. Each experiment was performed at least three times, in triplicate. Activity was defined as Firefly/Renilla ratio. 2.4. Real-time quantitative PCR Total RNA was isolated from prostate cancer cell lines using TRIzol (Invitrogen, Carlsbad, CA, USA). 1 μg of total RNA was reverse transcribed to first-strand cDNA with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using oligo(dT)18 primers, according to the manufacturer's protocol. Real-time PCR was performed on the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using Platinum SYBER Green qPCR Supermix UDG (Invitrogen, Carlsbad, CA, USA). The assay was carried out in triplicate using primer sequences for Sp1 (forward primer 5′-CAGAACCCACAAGCCCAAACAATC-3′; reverse primer 5′-ATGGAGGAGAGTTGAGCAGCATTC-3′), Sp3 (forward primer 5′-GTCAGCAGATGGTCAGCAGGTTC-3′; reverse primer 5′-AAGGTGTTCCAGAGGCAAGTAAGG-3′), and GAPDH
(forward primer 5′-CGCTCTCTGCTCCTCCTGTTC-3′; reverse primer 5′-ATCCGTTGACTCCGACCTTCAC-3′). The relative expression levels of Sp1 and Sp3 was determined using the 2−ΔΔCt method (Livak and Schmittgen, 2001). 2.5. Electrophoretic mobility shift and supershift Nuclear extracts were prepared using NE-PERTM nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Electrophoretic mobility shift assays (EMSAs) were carried out using a lightshift chemiluminescent EMSA kit (Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The sequence of the sense strands of oligonucleotides were as follows: wild type, 5′-CGGCAGCTCGGTGGGTGGTGCTCC-3′, and mutated type, 5′-CGGCAGCTCAAGGCCTGGTGCTCC-3′. The biotin 3′-end-labeled oligonucleotide and its reverse complement were annealed and purified. Standard EMSA reactions contained 5 μg of nuclear extracts and 20 fmol of biotin 3′end-labeled DNA in a 20 μl binding reaction mixture in the presence of 2.5% glycerol, 5 mM MgCl2, 50 ng/μl of poly(dI·dC), and 0.05% Nonidet P-40. When indicated, 200-fold molar excess of unlabeled oligonucleotide or nonspecific oligonucleotide was introduced to the reaction mixture for competition assay. For supershift assays, nuclear extracts were preincubated with rabbit anti-Sp1 (sc-59X, Santa Cruz Biologicals, Santa Cruz, CA, USA) or rabbit anti-Sp3 antibody (sc-644X, Santa Cruz Biologicals, Santa Cruz, CA, USA) for 40 min on ice, followed by the addition with the indicated probe. For assays in the presence of mithramycin A (Sigma, St. Louis, MO, USA), the DNA probe was preincubated with different concentrations of compound (100, 200 nM final concentration) for 40 min on ice, and then nuclear extracts were added. DNA–protein complexes were separated on 4% polyacrylamide gels. Reaction products were then transferred to a Biodyne B membrane (Pierce, Rockford, IL, USA) and fixed by UV cross-linking. The biotin-labeled reaction products were then visualized by incubation with streptavidin horseradish peroxidase conjugate and subsequent incubation with ECL chemiluminescent reagents. 2.6. Chromatin immunoprecipitation assay Chromatin immunoprecipitation assays were performed using the EZChIP kit (Upstate, Charlottesville, VA, USA) following the manufacturer's instructions. Cells were fixed in 1% formaldehyde at room temperature and glycine was added to quench unreacted aldehydes. Cells were washed, scraped, and collected by centrifugation at 4 °C. Cells were resuspended and sonicated (10 s pulses×5 at 30% duty cycle, Branson sonifier Cell disruptor 250, MA) to shear DNA. Protein–DNA cross-linked products were enriched by immunoprecipitation with 5 μg of anti-Sp1 (sc-59X) or anti-Sp3 antibody (sc-644X). DNA–protein crosslinks were reversed by adding 8 μl 5 M NaCl and incubating the mixture at 65 °C for 5 h. After DNA purification, the extent of enrichment was monitored by PCR using primers (forward primer 5′-TGCCGCGTGACGTGTCTGT-3′; reverse primer, 5′-GCCCTCATTCTGCCAACCAA-3′) specific to PCDH10 promoter fragments. The products were resolved on 2% agarose gel and visualized by ethidium bromide staining. Each of the experiments was repeated at least three times with similar results. 2.7. Western blot analysis Cells were lysed and extracted by RIPA buffer (R0278; Sigma, St. Louis, MO, USA) supplement with protease inhibitor cocktails and phosphatase inhibitors. 40 μg of total protein was separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore, Bedford, MA, USA). After blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 3 h, membrane was incubated with rabbit anti-Sp1 (sc-59X), rabbit anti-Sp3 antibody (sc-644X) or rabbit anti-GAPDH
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antibody (G9545, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. After washed with TBST for three times, the membrane was incubated with HRP-labeled secondary antibody (sc-2030, Santa Cruz Biologicals, Santa Cruz, CA, USA) at room temperature for 1 h. The blotting signals were visualized by using Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA). 3. Results 3.1. Identification of regulatory elements in the PCDH10 promoter In our previous work, we located the core promoter of PCDH10 to a 462-bp (−450/+12) segment of 5′-flanking region characterized by a high GC content. DNA sequence analysis revealed the presence of a CACA box, a CAAT box, a GT box, and several putative binding sites for the basic transcription factors Sp, AP-4 and Creb, but lacked a typical TATA box (Fig. 1A). We aligned the 462 bp sequence with corresponding sequences from Chimpanzee, cattle, rat and mouse, which revealed that sequences from the − 368 to −7 were evolutionarily conserved (Supplementary Fig. 1). A small part of most-conserved DNA segment is shown in Fig 1, which contained a CAAT box, a GT box and several putative binding sites (Fig. 1B). In order to assess which portions of the promoter were involved in the regulation of PCDH10 expression, a series of deletion constructs were generated according to putative binding sites of transcription factors and used to transiently transfect PC3 cells and LNCaP cells. Luciferase transcription was consistently more active in PC3 than in LNCaP cells (Fig. 2). Both in PC3 and LNCaP cells, −160/+12 construct provided highest promoter activity whereas −450/−131 construct and −99/+12 construct are clearly of little importance to PCDH10 expression. These data showed that the region containing the sequence between −144 and −99 was largely dispensable to the basal promoter activity in these two cells. This region contained the CAAT box, the GT box and AP-4 elements. 3.2. Reporter assay analysis of CAAT-box, GT-box, and AP-4 elements mutations To examine the contribution of CAAT box, GT box, and AP-4 elements to the expression of PCDH10, we constructed a series of pGL3-F2 plasmids with substitution mutations in the CAAT box, GT box, and/or AP-4 elements (Fig. 3). They were transfected into PC3 cells and LNCaP cells, and their activities were compared to wild-type constructs. The mutations in the CAAT box resulted in about 40% and 23% reduction of promoter activity in PC3 cells and LNCaP cells, respectively (Fig. 3). The mutations in the GT box resulted in about 56% and 50% reduction of promoter activity in PC3 cells and LNCaP cells, respectively. The mutations in the CAAT box and GT box resulted in very little promoter activity remained. But the mutations in the AP-4 elements did not result in the reduction of promoter activity (Fig. 3). These results strongly suggested that the PCDH10 promoter was cooperatively regulated by the CAAT box and GT box in these two cell lines.
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or pGL3-F2△3 with pCMV-Sp1 or pCMV-Sp3 (0.4 μg). The overexpression of Sp1/Sp3 had no effects on promoter activities in the construct pGL3-F0, which does not contain the GT box, or pGL3-F2△3, which contains the mutated Sp1 binding sites (Fig. 4F). The roles of Sp1/Sp3 in the regulation of the PCDH10 gene were further examined by inhibiting their binding to the promoter. PC3 cells and LNCaP cells were untreated or treated 1 h after transfection with 200 nM of mithramycin A for 24 h, which recognizes GT-rich promoter regions and interferes with the binding of the Sp family (Blume et al., 1991). Mithramycin A decreased promoter activity in the constructs containing the GT box but had no effect on construct pGL3-F0 or pGL3-F2△3, which do not contain the GT box or contain the mutated GT box (Fig. 5A). The CBF/NF-Y in the regulation of the PCDH10 gene was also examined by the overexpression of CBF/NF-Ys or the dominantnegative CBF/NF-YA subunit, which interact with the CBF/NF-YB and CBF/NF-YC subunits to form CBF/NF-Y. As expected, the overexpression of CBF/NF-Ys resulted in a significant stimulation of the activity of pGL3-F2 (Fig. 5B). The overespression of the dominant-negative CBF/NF-YA subunit decreased the activity of pGL3-F2, which contains the CAAT box, but had no effect on the construct pGL3-F2△1, which do contain the mutated CAAT box (Fig. 5B). 3.4. EMSA analysis of GT-box-binding proteins Electrophoretic Mobility Shift Assays (EMSA) and antibody supershift assays were used to determine whether Sp1/Sp3 protein binding occurred within the PCDH10 promoter by using nuclear extracts from PC3 and LNCaP cells, which express higher levels of Sp1 and Sp3 proteins (Tang et al., 2004). As shown in Fig. 6, the Sp1/Sp3 EMSA banding pattern was observed. The specificity of the transcription factor binding was proven by inhibition of binding of biotinylated probe by excess unlabeled wild-type probe (Fig. 6. lane 3). Upon incubation with antibodies to either Sp1 or Sp3, the shift of the specific bands was reduced, which indicated the binding of Sp1/Sp3 to the consensus site. The binding was drastically reduced upon by pretreatment of cells with Mithramycin A (Fig. 6). 3.5. Chromatin immunoprecipitation analysis of Sp1/Sp3 binding to the PCDH10 promoter Further validation of Sp1/Sp3 protein binding to the PCDH10 promoter was obtained by ChIP analysis in native chromatin isolated from PC3 cell and LNCaP cells. The precipitated chromosomal DNA was subjected to PCR amplification of the PCDH10 promoter region harboring the GT-box site. Sp1/Sp3 indeed bound to the PCDH10 promoter region containing the GT-box site. The amplification of input DNA was equal in all the samples and no amplification product was seen in the presence of control rabbit IgG or in the absence of antibody (Fig. 7). Thus, specific amplification was achieved by precipitation with Sp1/Sp3 only. Taken together, these results showed that Sp1/Sp3 binds specifically to PCDH10 promoter to augment PCDH10 transcription. 4. Discussion
3.3. Functional analysis of Sp1/Sp3 and CBF/NF To determine the effects of Sp1/Sp3 on PCDH10 promoter, PC3 and LNCaP cells were co-transfected with the constructs pGL3-F2 and pCMV-Sp1 or pCMV-Sp3, and luciferase activities were measured. The production of Sp1 and Sp3 was confirmed by RT-PCR analysis (Fig. 4A and B) and Western blot analysis (Fig. 4C and D). Furthermore, co-transfection with pCMV-Sp1 or pCMV-Sp3 resulted in a significant stimulation of PCDH10 promoter activity in a plasmid dose-dependent manner in PC3 and LNCaP cells (Fig. 4E). To confirm that Sp1/Sp3 elements indeed contributed to the Sp1/ Sp3-mediated upregulation, we cotransfected the construct pGL3-F0
We previously defined the core promoter of PCDH10 to a 462-bp segment of 5′-flanking region of PCDH10 gene. This segment has at least 88% identity with corresponding sequence of Chimpanzee, cattle, rat, and mouse PCDH10 promoter (Supplementary Fig. 1), which is consistent with the idea that evolutionary conservation of non-coding sequences is an indication of functional transcription factor binding sites (Hardison, 2000). In silico analyses of genomic sequence showed that PCDH10 promoter belonged to the TATA-less, CpG island-associated class of mammalian promoters (Fig. 1). This type of promoter typically contains multiple GC-boxes or GT boxes (Carninci et al., 2006; Purvis et al., 2010).
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Fig. 1. Characterization and evolutionary conservation of the 5′ flanking region of the PCDH10 gene. A, The sequence corresponds to nts 58,617,741 to 58,618,202 of human chromosome 4 (GenBank accession no. NT_016354.19). Putative transcription factor binding sites are boxed and labeled above. B, Promoter sequences from Chimpanzee (nts 1,245,852–1,246,043, GenBank accession no. NW_001234084.1), cattle (nts 349,455–349,211, GenBank accession no. NW_001493497.2), rat (nts 3,177,214–3,177,405, GenBank accession no. NW_001084802.1) and mouse (nts 4,727,630–4,727,821, GenBank accession no. NT_039229.7) is aligned to the human PCDH10 promoter region. Homologous bases are highlighted in grey. Putative consensus-binding elements for transcription factors are indicated above.
By transient transfections of PC3 and LNCaP cells with a series of 5′- and 3′-deletions promoter constructs, we found that the sequence between −144 and −99 was sufficient for the basal transcriptal activity
of the human PCDH10 gene in PC3 and LNCaP cells (Fig. 2). Substitution mutation showed that both CAAT box and GT box within the region are essential for promoter activity. But GT box seemed to play a more
Fig. 2. Promoter activity of human PCDH10 deletion constructs in PC3 and LNCaP. The cells were transfected with 0.6 μg pGL3-basic or luciferase reporter constructs containing different size promoter fragments (indicated in bp relative to the transcription start site (+ 1)). All of the constructs were cotransfected with pRLSV40 vector as an internal control for transfection efficiency. Promoter activity of each construct was measured as firefly luciferase activity normalized to Renilla luciferase, and the results were expressed as fold relative to the activity of pGL3-basic (which was assigned an activity value of 1.0). The means and S.E. are shown (n = 3). The data are representative of at least three independent experiments. Transfections were carried out in triplicate for each experiment.
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Fig. 3. Identification of functional sites within the human PCDH10 promoter region under basal conditions in PC3 and LNCaP cells. Schematic representation of the PCDH10 promoter site-directed mutagenesis constructs used for transient transfections is shown on the left (nts 58,618,070–58,618,202, GenBank accession no. NT_016354.19). Promoter activity was measured as described in Fig. 2. The results are expressed as the means ± S.E. (n = 3). The data are representative of at least three independent experiments. Transfections were carried out in triplicate for each experiment. Student's t-test, *p b 0.01; **p b 0.001.
Fig. 4. Effects of Sp1/Sp3 on reporter gene expression. PC3 and LNCaP cells were transfected with pCMV-Sp1/Sp3 expression plasmids, and the empty vector was cotransfected as a control in each group. (A and B) Relative mRNA levels of Sp1 and Sp3 in PC3 and LNCaP cells were determined by using real-time quantitative RT-PCR and SYBR Green dye. The relative expression level of PCDH10 was determined by using the 2−ΔΔCt method. The expression of control in PC3 or LNCaP cells was set as 1. Student's t-test, **p b 0.001. (C and D) The expression of SP1 and SP3 was assessed using Western blot. (E) PC3 and LNCaP cells were co-transfected with 0.6 μg of pGL3-F2 and the indicated concentrations of pCMV-Sp1/ Sp3 expression plasmids. The activity of the control in PC3 or LNCaP cells was set as 1, and the remaining activities were expressed as the fold induction against the control. The data are expressed as the means ± S.E. (n = 3). Student's t-test,*p b 0.01; **p b 0.001. (F) PC3 and LNCaP cells were co-transfected with 0.6 μg of pGL3-F0 or pGL3-F2Δ3, and 0.4 μg of pCMV-Sp1/Sp3 expression plasmids. All of the constructs were cotransfected with pRLSV40 vector as an internal control for transfection efficiency. Luciferase activity is expressed as the percent activity of the control. The data are expressed as the means ± S.E. (n = 3).
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Fig. 5. The functional analysis by inhibition of the binding of Sp1/3 and CBF/NF-Y to PCDH10 promoter. (A) PC3 and LNCaP cells were transfected with 0.6 μg of pGL3-F2, pGL3-F0, pGL3-F2△3. The cells were treated for 40 h with or without 200 nM of mithramycin. Luciferase activity is expressed as the percent activity of the mithramycin-untreated cells. (B) PC3 and LNCaP cells were co-transfected 0.6 μg of pSG5-CBF/NF-Ys or dominant-negative CBF/NF-YA plasmid and 0.6 μg of pGL3-F2 or pGL3-F2△1. The empty vector was cotransfected as a control in each group. All of the constructs were cotransfected with pRLSV40 vector as an internal control for transfection efficiency. Luciferase activity is expressed as the percent activity of the control. The data are expressed as the means ± S.E. (n = 3). Student's t-test, *p b 0.01.
Fig. 6. Binding of Sp1/Sp3 to the promoter region of the PCDH10 gene. Electrophoretic mobility shift assay (EMSA) was performed without or with nuclear extracts (8 μg) from (A) PC3 and (B) LNCaP cells in the presence of the various competitors. Lane 1, biotin-labeled probe only; lanes 2–8, labeled probe with 8 μg nuclear extract; lane 3, 100-fold molar excess of unlabeled probe; lanes 4, biotin-labeled mutated probe; lanes 5–6, the nuclear extracts were preincubated with anti-Sp1 (lane 5) or anti-Sp3 (lane 6) antibodies for 30 min and then added to the reaction mixture; lanes 7–8, the nuclear extracts were preincubated with 100 nM (lane 7) or 200 nM mithramycin A (lane 8) for 30 min and then added to the reaction mixture. Arrows indicated the formation of specific protein–DNA complexes. Each of the experiments was repeated at least three times with similar results.
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Fig. 7. In vivo association between Sp1/Sp3 and human PCDH10 gene promoter. Chromatin immunoprecipitation assay was used to analyze the interaction of Sp3 and Sp1 to the PCDH10 promoter in PC3 and LNCaP cells. The immunoprecipitated DNA obtained from ChIP is amplified utilizing PCR primers specific for the region containing the CAAT box and GT box in the PCDH10 gene promote. PCR products were separated on a 2% agarose gel containing ethidium bromide and detected via ultraviolet illumination. This figure shows a representative of at least three independent experiments. Lane 1, input DNA; lane 2, H2O; lane 3, normal rabbit IgG control; lanes 4–5, anti-Sp1 and anti-Sp3 antibodies.
important role in regulating the PCDH10 expression because mutation in GT box resulted in more decrease of PCDH10 promoter activity (Fig. 3), which was supported by the fact that the sequence of GT box was more conserved than that of CAAT box in evolution (Fig. 1B). CAAT boxes are found in the promoter region of 30% of genes, including housekeeping genes, inducible and cell-cycle regulated genes (Mantovani, 1999). The role of CAAT boxes depends on their ability to interact with a variety of transcription factors (Mantovani, 1999). NF-Y is a ubiquitous transcription factor involved in transcription of various promoters harboring CAAT box (Sun et al., 2009). Previous studies showed that dominant-negative NF-YA mutant can inhibit the interaction of NF-Y with CAAT boxes (Mantovani et al., 1994; Mantovani, 1999; van Wageningen et al., 2008; Luan et al., 2010; Pallai et al., 2010). In the present study, the expression of this dominant-negative NF-YA mutant repressed the human PCDH10 gene promoter activity in PC3 and LNCaP cells (Fig. 5B), which confirmed the interaction between NF-Y and human PCDH10 promoter. GT box is a consensus binding site for the Sp family of transcription factors (Sp1–Sp4). Sp1 and Sp3 recognize the same promoter elements (GC- and GT-boxes) with similar specificity and affinity (Hagen et al., 1992). Various reports have shown that Sp1 is a transcriptional activator, whereas Sp3 can act as either an activator or a repressor of transcription depending on the cell and promoter context (Yu et al., 2003; Safe and Abdelrahim, 2005). Our data indicated that overexpression of Sp1 or Sp3 was able to activate the human PCDH10 promoter in PC3 and LNCaP cells (Fig. 4E). This observation was consistent with the findings that Sp1 or Sp3 is known to play an important role in TATA-box less promoters and was associated with multiple transcription initiation sites (Melton et al., 1984; Muckenfuss et al., 2007). Sp family of transcription factors has been shown to regulate the expression of cadherin genes such as E-cadherin (Liu et al., 2005), N-cadherin (Le Mee et al., 2005) and vascular endothelial cadherin gene transcription (Gory et al., 1998). Together with our data, this indicated that Sp family of transcription factors played an important role in regulating the expression of cadherin gene family. Our ChIP and EMSA data strongly indicated that the transcription factors Sp1 and Sp3 could bind specifically to the GT box and therefore regulated PCDH10 transcription (Figs. 6 and 7). Supershift complex I and III were stronger than complex II in PC3 (Fig. 6), but the situation was converse in LNCaP cells, possibly due to the different cells. It has been reported that mithramycin A inhibits transcription of many genes through suppression of Sp1/Sp3 binding to their promoters (Blume et al., 1991). We also found that mithramycin A inhibited PCDH10 promoter activity by using combination of luciferase reporter assay and EMSA (Fig. 6).
In summary, the present results showed that the binding sites for NF-Y and Sp1/Sp3 were critical for PCDH10 transcription and thus the expression of PCDH10 might be modulated by the levels or activities of these transcription factors. This study extends our knowledge of the transcriptional regulation of PCDH10 and may therefore contribute to better understand the role of PCDH10 in tissue development and during pathophysiological processes. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2011.01.001. Acknowledgments This work was supported by the National Natural Science Foundation of China (30700824, 30972992), the Research Fund for the Doctoral Program of Higher Education of China (200800010106), the Medical Scientific Research Foundation of Guangdong Province (A2010548), and the Scientific Research Foundation of the State Human Resource Ministry for Returned Chinese Scholars, China. We thank Dr. Robert Mantovani at the University of Milan, Italy for the expression plasmid of NF-YA, -YB, -YC and the dominant-negative NF-YA mutant expression vector. References Angst, B.D., Marcozzi, C., Magee, A.I., 2001. The cadherin superfamily: diversity in form and function. J. Cell Sci. 114, 629–641. Blume, S.W., Snyder, R.C., Ray, R., Thomas, S., Koller, C.A., Miller, D.M., 1991. Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J. Clin. Invest. 88, 1613–1621. Carninci, P., et al., 2006. Genome-wide analysis of mammalian promoter architecture and evolution. Nat. Genet. 38, 626–635. Cheung, H.H., Lee, T.L., Davis, A.J., Taft, D.H., Rennert, O.M., Chan, W.Y., 2010. Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer. Br. J. Cancer 102, 419–427. Frank, M., Kemler, R., 2002. Protocadherins. Curr. Opin. Cell Biol. 14, 557–562. Gory, S., Dalmon, J., Prandini, M.H., Kortulewski, T., de Launoit, Y., Huber, P., 1998. Requirement of a GT box (Sp1 site) and two Ets binding sites for vascular endothelial cadherin gene transcription. J. Biol. Chem. 273, 6750–6755. Grabe, N., 2002. AliBaba2: context specific identification of transcription factor binding sites. In Silico Biol. 2, S1–S15. Hagen, G., Muller, S., Beato, M., Suske, G., 1992. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res. 20, 5519–5525. Hardison, R.C., 2000. Conserved noncoding sequences are reliable guides to regulatory elements. Trends Genet. 16, 369–372. Hirayama, T., Yagi, T., 2006. The role and expression of the protocadherin-alpha clusters in the CNS. Curr. Opin. Neurobiol. 16, 336–342. Imoto, I., et al., 2006. Frequent silencing of the candidate tumor suppressor PCDH20 by epigenetic mechanism in non-small-cell lung cancers. Cancer Res. 66, 4617–4626. Knudsen, S., 1999. Promoter2.0: for the recognition of PolII promoter sequences. Bioinformatics 15, 356–361.
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