Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells
Open accessAbstract
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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Single-gene studies have demonstrated that DNA methylation can activate gene expression.
“Single-gene studies have also demonstrated that DNA methylation can activate gene expression47–49.”
From Transcription factors as readers and effectors of DNA methylation (2016) · cites this paper as [48]
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The Osteocyte: An Endocrine Cell … and More
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Transcription factors as readers and effectors of DNA methylation
Zhu, H., Wang, G., & Qian, J. (2016) · Nature Reviews Genetics · doi:10.1038/nrg.2016.83 · Cited by 649 · see citing works
Bahar Halpern, K., Vana, T., & Walker, M. D. (2014) · Journal of Biological Chemistry · doi:10.1074/jbc.m114.573469 · Cited by 119 · see citing works
- Authors
- Hantusch, B., Kalt, R., Krieger, S., Puri, C., & Kerjaschki, D.
- Affiliations
- Medical University of Vienna (AT)
- Corresponding author
- Hantusch, Brigitte
- Venue
- BMC Molecular Biology
- Published
- 2007
- Volume
- 8
- Issue
- 1
- Pages
- 20
- ISSN
- 1471-2199 (ISSN-L)
- PMC
- PMC1828165
- OpenAlex
- W2013211434
- Cited by
- 63
- References
- 65
- Citations by year
- 2025: 1 · 2024: 3 · 2023: 1 · 2022: 1 · 2021: 2 · 2020: 2 · 2018: 3 · 2017: 2 · 2016: 4 · 2015: 4 · 2014: 6 · 2013: 5 · 2012: 9
- FWCI
- 1.86
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by
- Locations
- BMC Molecular Biology (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Universität Wien
- Medizinische Universität Wien
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
- Health Sciences › Medicine › Rheumatology › Bone and Dental Protein Studies
- Keywords
- Sp1, Sp3, DNA methylation, transcriptional regulation, osteoblasts, MG-63 cells, Saos-2 cells, DNase I footprinting, transcription factor binding sites, decitabine, trichostatin A, histone deacetylation, CpG methylation
- MeSH terms
- Podoplanin, Humans, Membrane Glycoproteins, Osteoblasts, Promoter Regions, Genetic, RNA, Messenger, Osteosarcoma, Transcriptional Activation, Sp1 Transcription Factor, DNA Methylation, Electrophoretic Mobility Shift Assay, Cell Line, Tumor, Sp3 Transcription Factor
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