Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells

Podoplanin mRNA expression in the human osteoblastic cell lines MG63 and Saos-2

To identify cell lines which are positive for podoplanin expression, reverse transcription PCRs were performed with a series of human cell lines of different origin (Fig. 1A). The osteoblastic osteosarcoma cell line MG63 exhibited a strong RT-PCR signal, whereas Saos-2 osteosarcoma cells lacked it. This result was confirmed by real-time PCR analyses showing that MG63 cells contained three logs more podoplanin mRNA transcripts than Saos-2 cells. While in MG63 cells the podoplanin mRNA population represented approximately 3% of GAPDH mRNA population, it made up just 0.003% in Saos-2 cells (Fig. 1B). A Northern blot analysis revealed the presence of two podoplanin transcript variants in MG63 cells (Fig. 1C). A 2.8 kb transcript corresponding to the full length mRNA was seen, and a shorter species was detected at 1.1 kb. As determined by 3' RACE analysis using MG63-derived cDNA, this short mRNA species represented a transcriptional variant ending after a cryptic polyA-signal at bp 345 of the 2068 bp 3'-UTR (data not shown).

Reverse transcription PCR applying a series of nine ascending 5' primers suggested that in MG63 cells, podoplanin mRNA was initiated at multiple sites mostly located between bp -97 and +1 of the 5'-flanking region (Fig. 1D). In 5' RACE experiments using MG63-derived cDNA, the 5'-length of mRNA transcripts and their frequency of occurrence was determined. The positions and frequencies of cDNA variants derived from 5' RACE analysis are summarized in Fig. 1E. The major transcription start site of podoplanin mRNA was allocated to the position designated +1 (Fig. 1E and 2A).

Isolation and structural analysis of the human PDPN promoter

The human PDPN gene 5' flanking region was isolated by PCR amplifications from a genomic BAC clone and subsequent staggered cloning. When comparing the isolated promoter sequence with the human chromosome 1 sequence, sequence identity was confirmed despite a single base difference at bp -415 (C in BAC clone versus A in the database). DNA sequence analysis using the Transfac database showed that the 5' flanking region lacked a consensus TATA box, whereas a GATA box (GATAAA) was localized 31 nucleotides upstream of the main mRNA transcription initiation site (Fig. 2A). The PDPN gene 5'-flanking region was characterized by a high GC content and revealed the existence of several potential Sp1 transcription factor sites. Moreover, putative binding sites for the basic transcription factors AP-2, AP-4, C/EBP, and NF-1, but not for osteoblast-specific factors were detected along the isolated PDPN promoter sequence (Fig. 2A).

The PDPN promoter is transcriptionally regulated in MG63 and Saos-2 cells

Genomic DNA sequencing of the promoter sequence in Saos-2 cells confirmed that lack of PDPN transcription was not due to deletion mutations (data not shown). Thus, MG63 and Saos-2 cells proved to be a suitable cell system for investigation of transcriptional podoplanin regulation. To assess which portions of the promoter were involved in regulation of podoplanin expression, reporter plasmids harboring progressive deletions from the 5' end of the bp -1885/+171 PDPN flanking region were set according to putative binding sites of transcription factors (Fig. 2A). The sixteen promoter fragments were inserted upstream of a luciferase reporter gene (Fig. 2B) and were evaluated in transient expression analyses. Luciferase transcription was consistently 30 times more active in MG63 than in Saos-2 cells (Fig. 2C). In MG63 cells, bp -1104/+171 construct provided highest promoter activity (761 Luc/Ren × 100), whereas Saos-2 cells exhibited highest normalized luciferase activity with bp -498/+171 construct (27 Luc/Ren × 100). Transcriptional activity diminished progressively with further deletion of the 5' region, showing three activity minima with the constructs bp -666/+171, -261/+171 and -76/+171 in MG63 cells, and with constructs bp -186/+171 and -76/+171 in Saos-2 cells, which indicated the loss of positive regulatory elements (Fig. 2C). The low promoter activity of the smallest bp -26/+171 construct in both cell lines suggested that the 171 bp spanning 5'-UTR of podoplanin mRNA was not involved in upregulation of promoter activity. These data showed that the 1028 bp 5' region contained motifs providing basic PDPN promoter activity, whereas rather silencing motifs bound to the more upstream bp -1885/-1104 region.

Nuclear proteins of MG63 and Saos-2 cells bind to the PDPN promoter

To more precisely define cis-acting DNA sequences responsible for cell-type specific PDPN transcription, seven overlapping DNA probes encompassing bp -1169 to +171 of the PDPN promoter (Fig. 3A) were used for in vitro DNaseI footprinting assays. With MG63 nuclear extracts, eight protected regions, fp8 – fp1, were identified with five probes (Fig. 3B), while probes EB and SB exhibited no differential DNaseI digestion pattern (data not shown). Complexes fp6, fp5 and fp4 were detected on both the coding and the noncoding DNA strand (Fig. 3B), and footprinting region fp1 became also visible with Saos-2 nuclear extracts (Fig. 3B). Moreover, digestional modified regions flanking the protected regions emerged along the probes (Fig. 3, arrowheads), additionally indicating the presence of DNA-binding proteins.

To determine whether the detected elements indeed bound nuclear proteins, gel shift assays were performed using eight double stranded DNA oligonucleotides spanning the DNase I footprint regions (Table 1). Probes alone produced no shifted bands (data not shown). Probes fp7, fp5, fp2 and fp1 produced a similar pattern of low-mobility DNA-protein binding (complexes I – III) when adding nuclear extract of MG63 cells (Fig. 4, lanes -). The same bands became faintly visible with Saos-2 extracts except for probe fp7, which did not show complexes I and II. Consistent with the footprints, oligos fp6 and fp4 bound nuclear proteins strongly from MG63, but weak from Saos-2 cells (Fig. 4, lanes -). Although covering a broad footprint region, probe fp4 only yielded a binding pattern of several rapidly migrating proteins, and fp8 and fp3 did not reveal significant DNA-protein complexes (data not shown).

Specificity of the gel shifted bands was demonstrated by competition assays in which nuclear extracts were preincubated with a 100-molar excess of unlabeled specific (Fig. 4, lanes s) or unrelated probe (Fig. 4, lanes ns) as competitor. As all bands of the gel shifts were eliminated or attenuated by excess of unlabeled probe, these moieties may bind the respective oligos in a manner requiring concomitant association. A computer analysis revealed consensus motifs for transcription factors NF-1 (fp7, fp6), C/EBP-α (fp5, fp4), HNF-3 (fp4), Oct-1 (fp4) and AP-4 (fp1) in the applied probes. However, a panel of antibodies against these factors did not interfere with the observed low molecular weight DNA-protein complexes in supershift assays (data not shown). Nonetheless, these data suggested the presence of several sequence-specific proteins bound to the proximal PDPN promoter in MG63 cells.

Sp proteins bind to fp7, fp5, fp2 and fp1in the PDPN promoter

Probes fp7, fp5, fp2 and fp1 contained GC-rich regions exhibiting high homology to the consensus Sp1-binding motif. To test the binding of Sp-proteins to these elements, supershift analyses with anti-Sp1 and anti-Sp3 antibodies were performed. When anti-Sp1 antibody was added, complex I disappeared in favor of a supershifted band, which showed the predominant contribution of Sp1 (Fig. 5A, lanes Sp1). Addition of anti-Sp3 antibody blocked protein-DNA binding, which resulted in loss of complex II, whereas no supershifted complex appeared (Fig. 5A, lanes Sp3). When used together, Sp1 and Sp3 antibodies supershifted or inhibited both complexes (Fig. 5A, lanes Sp1+Sp3). To further test whether the core Sp1 sequence was required for binding of nuclear proteins, gel shift assays with the same oligonucleotide probes, but mutated at residues known to be critical for Sp-binding (Table 1), were performed with nuclear extracts of MG63 cells. The mutant oligos failed to form the observed complexes, indicating that the chosen mutations were sufficient to eliminate the binding capacities of these sites (Fig. 5B). These studies demonstrated that four elements within the PDPN promoter, herewith designated Sp.4 – Sp.1, bound Sp1/Sp3 proteins from MG63 and also weakly from Saos-2 cells. The results furthermore indicated that Sp1 and Sp3 were not bound coordinately to the DNA stretches, since the anti-Sp1 antibody did not shift Sp3-DNA complexes and vice versa. This suggested that Sp1 and Sp3 occupied the same or overlapping sites of the PDPN promoter.

GC boxes Sp.4 and Sp.2 are essential for PDPN promoter activity

To assess the functional role of Sp binding elements Sp.4 – Sp.1, PCR-directed mutagenesis was performed at the same residues that had blocked Sp1/Sp3 binding in gel shift assays and were tested for their reporter activity. The four Sp binding sites were altered individually and in combination (Fig. 5C), and mutations were introduced into the promoter luciferase variant -857/+171 that supported basal PDPN transcription. The transactivation pattern was similar for MG63 and Saos-2 cells, indicating that Sp1/Sp3 promotion took place through the same elements in both cell lines (Fig. 5D). The utmost relevance was seen for Sp-binding site Sp.2 at bp -100/-95, showing an activity reduction by 73% in MG63 and by 52% in Saos-2 cells upon mutation. Deletion of Sp-binding site Sp.4 at bp -700/-696 decreased luciferase activity by 57% in MG63 and by 16% in Saos-2 cells, whereas mutation of Sp-binding site Sp.3 (bp -570/-564) did not alter luciferase activity substantially. Mutation of site Sp.1 (bp -48/-43) lead to a slight activating trend, while combined mutation of all four sites reduced activity to more than 80% in both cell lines. Taken together, these data suggested that Sp-binding sites Sp.4 and Sp.2 alone, but especially in concert, were central regulatory elements necessary for constitutive PDPN promoter activity in both MG63 and Saos-2 cells, whereas Sp.3 and Sp.1 provided no single activating importance.

Stimulatory Sp1 and repressive Sp3 coordinatively activate the PDPN promoter

To directly confirm the effect of Sp1 and Sp3 on PDPN promoter activation, bp -857/+171 wild-type promoter luciferase construct was cotransfected with increasing amounts of CMV-promoter driven Sp1 and Sp3 expression plasmids alone and in combination into MG63 and Saos-2 cells (Fig. 6A). In both cell types, Sp1 overexpression resulted in a weak but dose dependent stimulation of luciferase activity with 1.8-fold induction in MG63 and 1.6-fold induction in Saos-2 cells relative to Sp1 mock-transfected cells. Similarly, Sp3 overexpression lead to a 1.4-fold and 1.8-fold induction in MG63 and Saos-2 cells, respectively. When both factors were coexpressed, no further activation above 1.8-fold promoter activity was seen. In contrast, luciferase activity of the combined mutational variant Sp.4-1mut was not altered in these experiments. These results suggested that Sp1 and Sp3 were able to stimulate PDPN transcription through Sp1/Sp3 binding sites in the proximal promoter region independently.

To confirm the effect of Sp1 and Sp3 on the activity of endogenous PDPN gene expression in MG63 and Saos-2 cells, real-time PCR was performed after transfection with Sp1- and Sp3-expression plasmids. The results corresponded to the observed potency of the respective factors to stimulate promoter activity. Podoplanin mRNA contents were clearly increased by overexpressed Sp1 and Sp3 (Fig. 6B). Overexpression of both, Sp1 and Sp3, did not further change mRNA levels in MG63 cells but strongly increased them in Saos-2 cells. These results showed that Sp1 and Sp3 indeed were able to upregulate podoplanin expression.

The roles of Sp1/Sp3 function on the PDPN promoter were more clearly established in Drosophila SL2 cells, which lack endogenous Sp-proteins. Drosophila-specific pPac-Sp1 and pPac-Sp3 expression plasmids were cotransfected with the wild-type and Sp-site mutant promoter variant Sp.4-1mut (Fig. 6C) and fold promoter activation versus pPac-empty transfections was evaluated. Cotransfection of Sp1 lead to a 16-fold luciferase activity increase of the wild-type promoter. In contrast to the results seen with MG63 and Saos-2 cells, overexpression of Sp3 reduced promoter activity 8-fold, which discovered a strong repressive action of solely present Sp3. Nevertheless, coexpression of both, Sp1 and Sp3, further increased promoter activity up to 21-fold. Since Sp1 activity was not decreased in the presence of pPac-Sp3, Sp3 seemingly did not compete with Sp1 for binding to the same site. The same effects were visible on a reduced level with mutant variant Sp.4-1mut and pGL3-Basic, indicating background activity of these constructs. Basically, these results suggested opposed action of single Sp1 or Sp3 on the PDPN promoter, while presence of both factors caused a concerted activation.

Mutation of site Sp.1 at bp -48/-43 (Sp.1mut) showed a slight increase in promoter activity (Fig. 5D), which corresponded to lowered activity of bp -76/+171 versus bp -38/+171 deletion construct (Fig. 2C). In order to explore a potential repressive role of Sp3 on this site, reporter constructs bp -76/+171 and -38/+171 were cotransfected with Sp1 and Sp3 expression plasmids and the multiple of promoter activation was determined (Fig. 6D). Consistently with luciferase reporter assays (Fig. 2C), construct -38/+171 conferred a roughly 2-fold higher promoter activity than -76/+171 in both cell lines (data not shown). Sp1 activated both constructs up to 1.4-fold in both cell lines. Consistent with the overexpression studies shown in Fig. 6A, Sp3 overexpression did not exert repressive effects on the bp -76/+171 construct. In MG63 cells, activity of the bp -76/+171 construct was not altered by additional Sp3-overexpression, while it even increased in Saos-2 cells. Therefore, we were not able to demonstrate a repressive Sp3 action on site Sp.1. We rather suggest a suppressive interaction of Sp-proteins bound to Sp.1 in combination with another yet unknown factor located more downstream from bp -38, which is abolished through shortening of the promoter or by deletion mutation of site Sp.1. A more detailed analysis of this site will be of future interest.

Conclusively, these results clearly demonstrated the pivotal importance of Sp1 and the auxiliary importance of Sp3 for basal transactivation of the PDPN promoter.

Saos-2 cells exhibit reduced nuclear Sp protein levels and low Sp protein amounts bound to the PDPN promoter in vivo

To further clarify the transcriptional difference between MG63 and Saos-2 cells, we focussed on the observation that nuclear extracts of Saos-2 cells produced weak Sp1/Sp3 EMSA signals compared to those derived with MG63 extracts (Fig. 5B). A comparative Western blot analysis showed little difference in overall Sp1/Sp3 protein contents between MG63 and Saos-2 cells (Fig. 7A, lanes 1 and 2, and densitometric analysis), whereas nuclear Sp1 and Sp3 amounts were lowered about 82 and 88%, respectively, in Saos-2 versus MG63 cells (Fig. 7A, lane 3 and 4, and densitometric analyis).

In order to assess whether Sp proteins bound to the identified DNA stretches in vivo, chromatin immunoprecipitations were performed using antibodies against Sp1 and Sp3 and crosslinked chromatin from MG63 and Saos-2 cells (Fig. 7B). After immunoprecipitation with anti-Sp1 antibody, extensive PCR amplifications from the Sp-binding sites relative to control input DNA were seen with MG63 derived chromatin (Fig. 7B, upper panels). In contrast, Saos-2 cells exhibited weak amplification levels versus input DNA (Fig. 7B, lower panels), which indicated low Sp1 occupation of these positions. PCRs from anti-Sp3 antibody precipitated chromatin resulted in overall weaker amplification results (Fig. 7C), which corresponded to the low DNA-bound Sp3 amounts seen in EMSAs (Fig. 5A). Site Sp.4 was stronger occupied by Sp3 in MG63 than in Saos-2 cells, while in both cell types site Sp.3 was scarcely occupied by Sp3. Sites Sp.2 and Sp.1 were acquainted with Sp3 to an equal, yet moderate extent in both cell lines. These data raised the possibility that drastically reduced nuclear Sp protein concentrations were responsible for weak PDPN promoter activity in Saos-2 compared to MG63 cells.

The PDPN promoter is strongly methylated in MG63 but not in Saos-2 cells

To further unravel the transcriptional difference between podoplanin expression in MG63 and Saos-2 cells, epigenetic effects were considered. The PDPN promoter contained a high CG content which provided putative methylation sites. Correspondingly, two CpG islands were detected [28], one encompassing bp -183/+16 that spanned the core promoter, and the other stretching from bp +76 to +517, including exon 1 (bp +203 to +267) plus 249 bp of intron 1 (Fig. 8A). A Pst I genomic DNA-fragment that covered bp -2799 to +1191 of the PDPN promoter and gene was used to analyze a potential methylation-sensitive restriction pattern. The fragment contained 121 palindromic CpG dinucleotides, sixteen of which represented isoschizomeric Hpa II/Msp I restriction sites accumulating in three clusters. Five Southern blotting probes were situated beside and between these clusters (Fig. 8A). Digestion of genomic DNA with Pst I alone produced the anticipated 3990 bp fragment (Fig. 8B, lanes P), and double-digestion with Pst I plus methylation-insensitive enzyme Msp I yielded the smallest possible fragments (1047, 1044, 399, 257 and 700 bp, respectively) (Fig. 8B, lanes M). When using Pst I together with methylation-sensitive Hpa II, complete digested bands were observed with Saos-2 derived genomic DNA, while in MG63 cells the distal probes P/B and F2/K mainly detected undigested 2.2 and 2.7 kb bands besides small portions of complete digested fragments (Fig. 8B, lanes H). Moreover, with probe F5/Bs only the digestion resistant 2.7 kb fragment was visible. These results indicated strong methylation of region bp -2799/-464 in MG63 cells, while it was totally unmethylated in Saos-2 cells.

To clarify the PDPN promoter methylation status in MG63 cells in detail, genomic DNA was analyzed by bisulfite sequencing. Genomic DNA was treated with sodium bisulfite under conditions where cytosines are converted to uracils, while methylated cytosines remain unmodified [29]. Three PCR reactions were set to inspect the methylation level of the Hpa II/Msp I restriction site clusters (Fig. 8C). When PCR1 (bp -198 to +131) was considered, the methylation level was found to be less than 5%. With PCR2 (bp -759 to -485), except CpG sites 32 and 33 that were methylated to 25% and 50%, respectively, all CpG sites were fully methylated. PCR3 (bp -2005 to -1572) comprised three CpG sites, which were highly (site 8 to 86%, site 9 to 57%) or fully (site 10) methylated. The observed methylation pattern corresponded to the fragmentation results seen in Southern blotting and identified a highly methylated DNA status of the PDPN promoter upstream of bp -485 in MG63 cells.

Methylation of GC boxes Sp.4 and Sp.2 does not affect Sp-protein binding

In MG63 cells, the heavy methylation pattern stretched accross functional Sp1/Sp3 binding site Sp.4 (CpG number 23, Fig. 8C), whereas binding site Sp.2 was situated in the virtually unmethylated proximal promoter region (CpG number 52, Fig. 8C). To test whether this situation gave rise to modulatory binding of Sp-proteins, we compared the capabilities of respective oligos fp7 (site Sp.4) and fp2 (site Sp.2) to inhibit the formation of Sp1-/Sp3-DNA complexes with nuclear extracts of MG63 cells in methylated as well as unmethylated form. As shown in Fig. 9A, protein-DNA complexes were competed by increasing concentrations of both, methylated and unmethylated forms of probes fp7 and fp2 to an equal extent. These data suggested that Sp binding sites Sp.4 and Sp.2 were able to interact with Sp1/Sp3 even when methylated.

Distal promoter methylation enhances PDPN promoter activity

To further test the role of DNA methylation and histone acetylation for control of podoplanin expression in vivo, MG63 and Saos-2 cells were treated with methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaCdR) and histone deacetylase inhibitor trichostatin A (TSA). Cell viability was tested by increasing concentrations of the agents over a cultivation period of 3 days to exclude mRNA downregulation effects upon apoptosis. Then, cells were treated either with 5-azaCdR and TSA alone or with 5-azaCdR followed by TSA, and podoplanin mRNA amounts were analyzed by quantitative Northern blot analysis. In Saos-2 cells, the drugs were not able to initiate de novo synthesis of podoplanin mRNA (data not shown), which demonstrated that lack of PDPN transcription was not under epigenetic control. In MG63 cells, variations of both podoplanin mRNA bands seen in Northern blot were evaluated (Fig. 9B). 5-azaCdR treatment did not change PDPN transcription levels when compared to untreated cells, while 0.6 μM TSA lead to a respective 28% and 48% reduction of PDPN transcription. Moreover, a combination of 1 μM 5-azaCdR and 0.6 μM TSA decreased PDPN transcription by 50% and 57%, respectively. These data indicated that in MG63 cells, in vivo modification of the promoter chromatin structure through combined demethylation and histone hyperacetylation rather repressed than enhanced PDPN transcription.

To investigate whether methylation of the distal PDPN promoter portion indeed assisted activation of gene transcription, further luciferase reporter gene assays were performed (Fig. 9C). Complete in vitro methylation of the promoter plasmids pGL3(-1885/+171) and pGL3(-857/+171) lead to more than 90% loss of promoter activity in MG63 and Saos-2 cells, indicating that the vectors were susceptible to inhibition by methylation. To further assess promoter activity upon selective methylation of defined promoter regions, fragments bp -1885/+171 and -857/+171, or fragments bp -1885/-189 and -825/-189, which comprised the region that wasmethylated in vivo in MG63 cells, were generated by restriction digest with Sac I/Xho I or Sac I/Apa I, respectively (Fig. 9C). The four fragments were methylated by Sss I methylase or mock-methylated, religated into the unmethylated reporter plasmid backbone and transfected into MG63 and Saos-2 cells. In vitro methylation of all promoter CpGs until bp +171 revealed a weak trend towards transcriptional deactivation being more pronounced in Saos-2 than in MG63 cells (Fig. 9C). This result supported the observation that methylation of site Sp1.2 did not affect Sp1/Sp3 binding (Fig. 9A). Conversely, selective methylation of CpG sites upstream of bp -189 containing 35 or 24 CpG sites, respectively, led to considerable promoter activity increase versus mock methylated constructs, which added up to more than 40% and 30% in MG63 and to more than 25% and 60% in Saos-2 cells. These data indicated that a putative methylation-dependent enhancement mechanism, possibly efficiently altering chromatin structure, was able to act on PDPN transcription.

Cell culture

Human osteoblast-like osteosarcoma cell lines MG63 and Saos-2 were obtained from American Type Culture Collection (ATCC CRL-1427 and HTB-85, respectively). Cells were maintained at 37°C with 5% CO₂ and were grown as described in the ATCC protocol. Drosophila Schneider SL-2 embryo cells (ATCC CRL-1963), a kind gift from Dr. Hans Rotheneder, were maintained in Drosophila-SFM (Gibco) supplemented with 2 mM glutamine without antibiotics.

Reverse transcription and Real-time PCR, Northern blotting and RACE

Total RNA was extracted from cells lines with TriReagent (Molecular Research Center, Inc.). For RT-PCR analysis of cell lines, total RNA of twelve different cell lines was transcribed into cDNA using RT-for-PCR-Kit (Clontech), and PCRs were carried out with Taq DNA Polymerase (Amersham Biosciences) and the RNA-specific primers 12B1-F1 and LF3-AT, which create a 322 bp PCR fragment. For real-time PCR, cDNA was synthesized with the Sprint PowerScript Kit (Clontech). PCR then was performed with the PDPN-specific primers Hs 01089982-g1 and the GAPDH-specific primers Hs 9999905-m1 (Applied Biosystems) in an ABI Prism 7000 Taqman. For analyzation of multiple potential start sites, total RNA was transcribed into cDNA by Superscript II RT (Invitrogen) using the PDPN gene specific 3' primer hPP (+207/+178) located at the 3'-end of the 5' untranslated region. RT-PCR was then performed with nine 5' primers (Table I) in combination with the 3' primer hPP (+114/+96). For Northern blotting, total RNA was fractionated on 1.2% formaldehyde-denaturing agarose gels, transferred onto a nylon membrane (Zeta-probe GT genomic membrane, BioRad) and fixed by UV cross-linking. Blots then were incubated with a probe covering bp +206 to +723 of podoplanin mRNA and with a GAPDH probe for normalization of RNA amount. Densitometric quantification was perfomed with the LumiAnalyst 3.0 program (Roche). 5'- and 3'-RACE were performed using respective RACE systems (Gibco) and total RNA derived from MG63 cells. PCR products derived from both procedures were cloned into vector pCR2.1 (Invitrogen) and the sequencing results of randomly picked clones were employed to construct a map of transcriptional variants.

Isolation of the PDPN promoter

The genomic BAC clone 53F6 was derived from the Human RPCI-11 library (Research Genetics) [60]. By Southern blotting of several restriction digests, utilizing a unique Sac II site at bp +125 as a size reference point, the clone was assayed for containing a large portion of the 5'-upstream region of the human PDPN gene. 4.6 kb of the PDPN 5'-flanking region were sequenced by primer walking applying the Sanger dideoxy chain termination method. According to these results, four subsequent PCR primer pairs were designed, which enabled the generation of four fragments each covering ~1.2 kb of the 5'-flanking region, and, by the insertion of appropriate restriction sites, stepwise cloning of the promoter. The PCR products were cloned separately into the T/A cloning site of pCR 2.1 (Invitrogen) and subjected to sequence analysis. Potential transcription factor binding sites were predicted using the Alibaba and Gene Viewer programs, which employ TRANSFAC 5.0 matrices [61] with a core similarity of 1.0 and matrix similarity 0.9. Potential CpG islands were identified by the EMBOSS CpGplot program package [62].

Plasmid constructs

Two PDPN promoter constructs were derived from the original cloning procedure with 5' primers FII, FI and 3' primer RI (Table 1). Additional deletions were prepared using fourteen 5' primers (Table 1) in combination with 3' primer RI. PCR products were generated by standard PCR procedure and cloned into the T/A cloning site of the pCR 2.1 vector. 5'-flanking Sac I and Xho I sites, which had been synthesized at the upper and lower primers, were used for unidirectional cloning of fragments upstream from the luciferase gene into the pGL3-Basic vector (Promega). Mutations at Sp1/Sp3 consensus sites were introduced using mutation primers (Table 1) and the QuikChange II site-directed mutagenesis kit (Stratagene). A Sp1 expression plasmid was derived from cDNA clone Nr. 7030772 (ATCC, Molecular Genomics Resources), and a loss of function mutation in this clone was corrected by site directed mutagenesis as described above. After digestion with Xma I, the Sp1 coding region was cloned into the correct reading-frame of expression vector pIRES-neo (Stratagene), which contains a CMV promoter. pMCS-Sp3, pPac-Sp1, pPac-Sp3 and pPac-empty were a kind gift from Dr. Guntram Suske, Marburg, Germany [63]. All DNA constructs were purified using Endo-free Midiprep Kit (Promega), and correct identities were confirmed by sequencing.

Transient transfections and luciferase reporter assay

MG63 and Saos-2 cells were transiently transfected in 24-wells using Effectene transfection reagent (Qiagen), as recommended by the manufacturer. For transfections, 0.05 pmol of each reporter pGL3-promoter construct or the external control vector pGL3-Control (Promega) were used per well. In each well, 0.005 pmol thymidine kinase driven Renilla luciferase vector pRL-TK (Promega) served as an internal standard for transfection efficiency. For triple-transfection experiments, wells were transfected with 0.1 μg of reporter constructs and 0.01 μg of pRL-TK together with increasing amounts of Sp1 or Sp3 expression plasmid. Luciferase activities were normalized for transfection efficiency according to Renilla activities. For testing effects of Sp-protein overexpression on PDPN transcription, MG63 and Saos-2 cells were transfected in 12-wells with 0.6 μg pCMV-Sp1 or pMCS-Sp3, or with 0.3 μg of each vector. SL-2 Drosophila cells were transiently transfected in 12-wells with 0.25 μg of pPac vectors together with 0.25 μg of luciferase reporter using FuGene6 (Roche) according to the manufacturer's recommendations. After 48 h, cells were lysed in 1 × passive lysis buffer and luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega) per the manufacturer's instructions. When using single Luciferase signals, activities were normalized to protein amounts of the lysates as determined by BCA assay (Pierce). Measurements were made using a Lumat LB9507 luminometer (EG&G Berthold).

Preparation of nuclear extracts

Nuclear extracts were prepared from MG63 and Saos-2 cells with minor variations of the method of Dignam et al. [64]. Briefly, cells were washed twice with ice-cold phosphat-buffered saline, scraped off in PBS and centrifuged 5 min at 1500 × g at 4°C. Cells were resuspended in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF), left on ice for 20 min and were lyzed by passing them ten times through a 22 gauge needle. The nuclei were recovered by centrifugation for 6 min at 4500 × g, washed with buffer A, and the nuclear proteins were extracted with high salt buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT and 0.5 mM PMSF) on a rotating wheel for 30 min at 4°C. Extracts were centifuged for 15 min at 10000 g, and supernatants were stored in aliquots at -78°C until use. Protein concentrations were measured by BCA protein assay (Pierce) giving typical protein yields of 3 – 4 μg/μl.

DNaseI footprint analysis

Footprinting experiments were performed with the Core Footprinting System Kit (Promega). DNA probes were dephosphorylated, labeled at both ends with (γ³²P)-ATP (Hartmann Analytic) by T4 polynucleotide kinase, and digested with an restriction enzyme that released one of the labeled ends. Probes were purified on an agarose gel and DNA was recovered with the Qiaquick Gel Extraction Kit (Qiagen). 5 ng of the labeled probe were incubated with 5 μg nuclear extract in 50 μl of 25 mM Tris-HCl (pH 8.0), 50 mM KCl, 6.25 mM MgCl₂, 1 mM EDTA, 20% glycerol and 1 mM DTT for 20 min on ice and then, 50 μl of a 5 mM CaCl₂/10 mM MgCl₂ solution was added. After 1 min at room temperature, various amounts (0.15 – 1.5 units) of RQ1 RNase-free DNase I were added in order to obtain an even DNase I ladder from top to the bottom of the gel, mixed gently, and after 2 min of incubation the reaction was terminated with 90 μl stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 μg/ml yeast RNA). DNA was extracted with phenol/chloroform/isoamylalcohol 1:1:1, precipitated with ethanol, and the pellet was dissolved in loading solution (1:2 0.1 M NaOH:formamide (v/v), 0.1% cylene cyanol, 0.1% bromphenol blue). After heating the probes 2 min at 95°C and chilling on ice, they were loaded onto a 6% polyacrylamide sequencing gel containing 8 M urea and run in 1 × TBE buffer at 1500 Volt. Gels were dried on a filter paper and exposed to a Kodak Phosphor screen. A sequencing reaction of the respective DNA probe was carried out with the Sequenase Version 2.0 DNA Sequencing Kit (USB Corp.) using (α³⁵S)-dATP (New England Nuclear) in the reaction mixture and was run side by side with the footprint assay. Images were analyzed with a STORM 830 facility (Molecular dynamics).

Electrophoretic mobility shift assays (EMSAs)

Double-stranded oligonucleotide probes were 5'-endlabeled with (γ³²P)-ATP and T4 polynucleotide kinase. EMSAs were carried out in a 10 μl reaction containing 3 μg of nuclear extract, 250 ng poly(dI-dC), 2 μl of 5 × binding buffer (50 mM HEPES, pH 7.9, 50 mM KCl, 25 mM MgCl₂, 2.5 mM EDTA, 40% glycerol, 5 mM DTT, 0.1% NP40, 200 μg/ml BSA) and labeled oligo probe (80 fmoles, 20.000 cpm) for 15 min on ice. Competitive EMSAs were performed under identical conditions by adding the 50 or 100 fold amount of the same unlabeled doublestranded oligo or an unspecific oligo as negative control. For supershift experiments, 1.5 or 1 μl of anti-human Sp1-, or Sp3-specific antibodies (Santa Cruz) or unspecific rabbit IgG were added to the reaction mix and incubated for 20 min on ice. Protein-DNA complexes were resolved on a 4.5% nondenaturing polyacryamide gel with Tris-EDTA buffer (45 mM Tris, 44.5 mM borate, 1 mM EDTA, pH 8.0) at 350 V at 4°C. Gels were dried on filter paper, exposed 1 to 5 days to a Kodak Phosphor screen and scanned with a STORM 830 facility.

Western blot analysis

Nuclear extracts or total lysates from MG63 and Saos-2 cells were applied to 10% SDS-PAGE and transferred onto nitrocellulose membrane. Western analysis was performed by incubating the membrane with rabbit anti-Sp1, -Sp3, -TFIIB, or -GAPDH antibodies (Santa Cruz), followed by HRP-conjugated secondary antibodies. Signals were detected by enhanced chemiluminescence using an ECL kit (Amersham Pharmacia). Densitometric quantification was perfomed with the LumiAnalyst 3.0 program (Roche).

ChIP assay

ChIP assays were performed with a ChIP kit (Upstate) according to the manufacturer's recommendations. For each chromatin immunoprecipitation assay, 2 – 3 × 10⁷ cells (80 – 90% confluency) were crosslinked with 1% formaldehyde at room temperature for 20 min. Cells were washed twice with ice-cold PBS, scraped from the culture dish and resuspended in SDS lysis buffer (Upstate). Chromatin was fragmented by sonication (Labsonic U, B. Braun) in 20 sec pulses on ice to an average size of 600 bp. The lysates were diluted 1:10 with dilution buffer and pre-cleared by adding 75 μl protein A agarose for 30 min at 4°C. 40 μl of the lysate were removed as control input DNA and microcentrifuged at full speed for 10 sec to pellet residual cell debris. IP was performed by adding 4 μg anti-Sp1 antibody (Upstate), 4 μg anti-Sp3 antibody (Upstate) or 5 μg unspecific rabbit IgG overnight at 4°C under rotation, and an aliquot was incubated without antibody. Immunocomplexes were precipitated by addition of protein A agarose for 1 hr at 4°C. Precipitates were washed once with low salt buffer, once with high salt buffer, once with LiCl buffer and twice with TE buffer (Upstate). Protein/DNA complexes were eluted from the antibodies with 1% SDS, 0.1 M NaHCO₃, and DNA-protein interactions were reversed by addition of 5 M NaCl and heating to 65°C for 4 hrs. Proteins were digested with proteinase K for 1 hr at 45°C. Remaining DNA was purified by phenol/chloroform extraction and subsequent ethanol precipitation. The DNA was finally resuspended in 40 μl 1 × TE, pH 8.0, and 4 μl of the DNA was used for each PCR reaction. Site-specific PCR was carried out using primer pairs located at binding sites Sp1.1/Sp1.2 (193 bp), Sp1.3 (184 bp) and Sp1.4 (115 bp). PCR primers were designed as 19–26 mers with ~55% GC content. PCR was performed with a hot start, followed by 30 – 35 cycles of 30 s at 95°C, 1 min annealing at 63°C, 65°C and 68°C for each Sp1 site, respectively, and 30 s extension at 72°C. Besides that, input DNA was used concentrated (4 μl) and in 1:4 and 1:16 dilutions in PCR reactions. PCR products were run on a 2% agarose gel and visualized by ethidium bromide staining. Each ChIP experiment was carried out at least three times with similar results.

Genomic DNA southern blot analysis

Genomic DNA was extracted from MG63 and Saos-2 cells according to Sambrook et al. [65]. Briefly, genomic DNA was isolated by cell lysis with proteinase K (Promega) digestion and extraction with phenol/chloroform. After precipitation, DNA was digested with the restriction enzyme Pst I to generate a 3989 bp podoplanin promoter fragment ranging from bp -2799 to bp +1190 and then with the isoschizomeric restriction enzymes Hpa II and Msp I (NEB) with the binding site 5'-CCGG-3' to release methylation-sensitive and -insensitive fragments. DNA fragments were separated on a 1.5% agarose gel, transferred onto a nylon membrane and hybridized with five radioactively labeled probes (P/B, F2/K, F5/Bs, Ex1, Int1). After hybridization and subsequent washing, autoradiographs were analyzed with a STORM 830 facility.

Sodium bisulfite genomic DNA sequencing

Bisulfite genomic sequencing was performed as described previously [29]. Briefly, genomic DNA (5 μg) was denatured in 0.3 M NaOH at 37°C for 15 min. Then, 3 M sodium bisulfite (Sigma) and 10 mM hydroquinone (Sigma) were added and samples were incubated at 50°C for 16 h. The modified DNA was purified through the Wizard Clean-Up system (Promega) and denatured by addition of 0.3 M NaOH at 37°C for 15 min. DNA was precipitated, resuspended in 1 mM Tris-HCl, pH 8 and used for PCR reactions. Three regions were amplified with primer pairs specific for bisulfite-reacted upper strands (Table 1). PCR was performed with DNA isolated from four independent bisulfite treatment experiments and PCR products were cloned into pCR2.1-TOPO vector (Invitrogen). Fifty to eighty clones from each reaction were analyzed by sequencing.

5-AzaCdR and TSA treatment

Viability of MG63 and Saos-2 cells under the influence of DNA methyltransferase inhibitor 5-azaCdR was tested up to concentrations of 1, 10 and 50 μM, and for histone deacetylase inhibitor TSA up to concentrations of 1, 2 and 3 μM over a period of 72 hrs. 5-azaCdR was added for 48 h at a final concentration of 1 μM, and TSA was added for 24 h alone, or for additional 24 h at the end of 5-azaCdR treatment final concentrations of 0.3 and 0.6 μM. Cells then were used for RNA isolation and subsequent quantitative Northern blotting analysis as described above. Densitometric quantification was perfomed with the LumiAnalyst 3.0 program (Roche).

In vitro methylation of oligos and reporter plasmids

Sss I methylase (NEB) was used for the methylation of EMSA oligos and of PDPN promoter-luciferase constructs. Oligos, pGL3-II and pGL3-I plasmid DNA were incubated with methylase according to the manufacturers' recommendations. Region-specific methylation was carried out after excision with Sac I/Xho I and Sac I/Apa I to isolate the promoter fragments -1885/+171, -1885/-189 from pGL3-II and -857/+171 and -857/-189 from pGL3-I, as described previously [31]. In each case, half of the DNA was methylated in the absence of S-adenosyl-methionine as mock methylation. The efficiency of methylation was determined with the methylation-sensitive enzyme Hpa II. Methylated and mock-methylated fragments were religated into their descendant plasmids and equal amounts were transfected into MG63 and Saos-2 cells.

Statistical analyses

Statistical relevant differences were assigned to a p-value of at least ≤0.05 using Student's paired t-test.