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

Hantusch B, Kalt R, Krieger S, Puri C, Kerjaschki D - BMC Mol. Biol. (2007)

Bottom Line: Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells.These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation.Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Clinical Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. brigitte.hantusch@meduniwien.ac.at

ABSTRACT

Background: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells.

Results: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types.

Conclusion: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells.

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Possible model of transcriptional control of podoplanin expression in MG63 versus Saos-2 cells. The PDPN promoter is active, when, accompanied by distal hypermethylation, several factors accumulate near Sp1/Sp3 proteins bound at sites bp -700/-696 (Sp1.4) and bp -100/-95 (Sp1.2). A factor located downstream from bp -38 and associated with Sp1/Sp3 bound to site Sp.1 may exert a repressive effect. The arrow indicates the major transcription start site; the small vertical bars indicate the CpG sites; the diamonds indicate the CpG sites analyzed in this study; fp, footprint detected protein; MDF, methylation dependent factor.
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Figure 10: Possible model of transcriptional control of podoplanin expression in MG63 versus Saos-2 cells. The PDPN promoter is active, when, accompanied by distal hypermethylation, several factors accumulate near Sp1/Sp3 proteins bound at sites bp -700/-696 (Sp1.4) and bp -100/-95 (Sp1.2). A factor located downstream from bp -38 and associated with Sp1/Sp3 bound to site Sp.1 may exert a repressive effect. The arrow indicates the major transcription start site; the small vertical bars indicate the CpG sites; the diamonds indicate the CpG sites analyzed in this study; fp, footprint detected protein; MDF, methylation dependent factor.

Mentions: Chromatin modification by CpG methylation and/or histone deacetylation has been linked inevitably with transcriptional repression [49]. However, our data provide evidence for a positive correlation between DNA methylation/histone modification and PDPN transcription in MG63 cells, as in our hands, induction of this classical "active chromatin state" rather conferred deactivation of gene activity. More importantly, site-specific DNA methylation by no means lead to down-modulation of promoter activity. The theoretical possibility of a repressor binding to the promoter and being competed by DNA methylation in MG63 cells has to be excluded, because demethylated PDPN promoter (i.e. the pGL3-promoter luciferase constructs) was highly active in MG63 cells. In agreement with that, no repressor-DNA interactions could be identified in Saos-2 cells. The mechanistic principle underlying the observed phenomenon could be diverse: When treating MG63 cells with 5-azaCdR in combination with TSA, consecutive conformational changes of chromatin may hinder Sp-protein binding and lead to downregulation of PDPN transcription. Secondly, it is tempting to hypothesize that methylation-dependent protein-DNA interactions being able to stimulate transcription may exist (methylation dependent factor MDF, Fig. 10). For example, the zinc finger protein Kaiso has been found to bind to both, methylated and sequence-specific recognition sites, fulfilling repressive [50] or activating functions [51]. Here, we identify a completely different methylation scenario than has been observed in brain and lung alveolar type I cells of the rat [25]. In these cells, DNA methylation was detected at key site Sp.2 under podoplanin/T1α non-expressing conditions, whereas its transcription could be initiated by addition of methyltransferase-inhibitor. These findings may indicate that the switch between transcriptional silencing and expression of podoplanin in different tissues/organs could depend on varying epigenetic phenomena, apparently driven by basal Sp1/Sp3 activation at conserved promoter sites. Studies using additional cell lines that constitutively or upon stimulation express podoplanin are ongoing to better understand the effect of methylation on this type of promoter. Preliminary data are pointing at again different methylation patterns in other cell lines. Moreover, altered histone modification patterns of the PDPN promoter chromatin have to be considered, as this process is intimately connected with DNA methylation [52]. Presumably, diverse regulatory pathways are mediating these epigenetic patterns in different cell types or even species.


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

Hantusch B, Kalt R, Krieger S, Puri C, Kerjaschki D - BMC Mol. Biol. (2007)

Possible model of transcriptional control of podoplanin expression in MG63 versus Saos-2 cells. The PDPN promoter is active, when, accompanied by distal hypermethylation, several factors accumulate near Sp1/Sp3 proteins bound at sites bp -700/-696 (Sp1.4) and bp -100/-95 (Sp1.2). A factor located downstream from bp -38 and associated with Sp1/Sp3 bound to site Sp.1 may exert a repressive effect. The arrow indicates the major transcription start site; the small vertical bars indicate the CpG sites; the diamonds indicate the CpG sites analyzed in this study; fp, footprint detected protein; MDF, methylation dependent factor.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828165&req=5

Figure 10: Possible model of transcriptional control of podoplanin expression in MG63 versus Saos-2 cells. The PDPN promoter is active, when, accompanied by distal hypermethylation, several factors accumulate near Sp1/Sp3 proteins bound at sites bp -700/-696 (Sp1.4) and bp -100/-95 (Sp1.2). A factor located downstream from bp -38 and associated with Sp1/Sp3 bound to site Sp.1 may exert a repressive effect. The arrow indicates the major transcription start site; the small vertical bars indicate the CpG sites; the diamonds indicate the CpG sites analyzed in this study; fp, footprint detected protein; MDF, methylation dependent factor.
Mentions: Chromatin modification by CpG methylation and/or histone deacetylation has been linked inevitably with transcriptional repression [49]. However, our data provide evidence for a positive correlation between DNA methylation/histone modification and PDPN transcription in MG63 cells, as in our hands, induction of this classical "active chromatin state" rather conferred deactivation of gene activity. More importantly, site-specific DNA methylation by no means lead to down-modulation of promoter activity. The theoretical possibility of a repressor binding to the promoter and being competed by DNA methylation in MG63 cells has to be excluded, because demethylated PDPN promoter (i.e. the pGL3-promoter luciferase constructs) was highly active in MG63 cells. In agreement with that, no repressor-DNA interactions could be identified in Saos-2 cells. The mechanistic principle underlying the observed phenomenon could be diverse: When treating MG63 cells with 5-azaCdR in combination with TSA, consecutive conformational changes of chromatin may hinder Sp-protein binding and lead to downregulation of PDPN transcription. Secondly, it is tempting to hypothesize that methylation-dependent protein-DNA interactions being able to stimulate transcription may exist (methylation dependent factor MDF, Fig. 10). For example, the zinc finger protein Kaiso has been found to bind to both, methylated and sequence-specific recognition sites, fulfilling repressive [50] or activating functions [51]. Here, we identify a completely different methylation scenario than has been observed in brain and lung alveolar type I cells of the rat [25]. In these cells, DNA methylation was detected at key site Sp.2 under podoplanin/T1α non-expressing conditions, whereas its transcription could be initiated by addition of methyltransferase-inhibitor. These findings may indicate that the switch between transcriptional silencing and expression of podoplanin in different tissues/organs could depend on varying epigenetic phenomena, apparently driven by basal Sp1/Sp3 activation at conserved promoter sites. Studies using additional cell lines that constitutively or upon stimulation express podoplanin are ongoing to better understand the effect of methylation on this type of promoter. Preliminary data are pointing at again different methylation patterns in other cell lines. Moreover, altered histone modification patterns of the PDPN promoter chromatin have to be considered, as this process is intimately connected with DNA methylation [52]. Presumably, diverse regulatory pathways are mediating these epigenetic patterns in different cell types or even species.

Bottom Line: Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells.These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation.Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Clinical Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. brigitte.hantusch@meduniwien.ac.at

ABSTRACT

Background: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells.

Results: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types.

Conclusion: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells.

Show MeSH
Related in: MedlinePlus