Tags

Type your tag names separated by a space and hit enter

Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells.
BMC Mol Biol. 2007 Mar 07; 8:20.BM

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.

Authors+Show Affiliations

Institute of Clinical Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. brigitte.hantusch@meduniwien.ac.atNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17343736

Citation

Hantusch, Brigitte, et al. "Sp1/Sp3 and DNA-methylation Contribute to Basal Transcriptional Activation of Human Podoplanin in MG63 Versus Saos-2 Osteoblastic Cells." BMC Molecular Biology, vol. 8, 2007, p. 20.
Hantusch B, Kalt R, Krieger S, et al. Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells. BMC Mol Biol. 2007;8:20.
Hantusch, B., Kalt, R., Krieger, S., Puri, C., & Kerjaschki, D. (2007). Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells. BMC Molecular Biology, 8, 20.
Hantusch B, et al. Sp1/Sp3 and DNA-methylation Contribute to Basal Transcriptional Activation of Human Podoplanin in MG63 Versus Saos-2 Osteoblastic Cells. BMC Mol Biol. 2007 Mar 7;8:20. PubMed PMID: 17343736.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells. AU - Hantusch,Brigitte, AU - Kalt,Romana, AU - Krieger,Sigurd, AU - Puri,Christina, AU - Kerjaschki,Dontscho, Y1 - 2007/03/07/ PY - 2006/10/27/received PY - 2007/03/07/accepted PY - 2007/3/9/pubmed PY - 2007/10/13/medline PY - 2007/3/9/entrez SP - 20 EP - 20 JF - BMC molecular biology JO - BMC Mol Biol VL - 8 N2 - 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. SN - 1471-2199 UR - https://www.unboundmedicine.com/medline/citation/17343736/Sp1/Sp3_and_DNA_methylation_contribute_to_basal_transcriptional_activation_of_human_podoplanin_in_MG63_versus_Saos_2_osteoblastic_cells_ L2 - https://bmcmolbiol.biomedcentral.com/articles/10.1186/1471-2199-8-20 DB - PRIME DP - Unbound Medicine ER -