Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

Abstract

Background: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene.

Results: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines.

Conclusion: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.

Figure 1

Transient transfection of deletion mutants of the 5'-flanking region of the human podocalyxin gene. Description of the construction of the pGL3-Podxl-pr reporter plasmids and transient transfection experiments are described under "Methods". Luciferase activity was corrected for transfection efficiency with the values obtained with Renilla and was expressed as percent of the maximal activity of the -364 construct. The results are mean ± SEM of three experiments performed in duplicates.

Figure 2

Western blots of human podocalyxin and Sp1 transcription factor. Cell lysates (20 μg) were resolved on SDS-7.5% polyacrylamide gels under reducing conditions, transferred to PVDF membranes and immunoblotted with either 1 μg/ml of anti-podxl or anti Sp1 antibodies. β-Actin content was blotted as a control of gel loading. The experiment was repeated three times.

Figure 3

Effect of Sp1 on podocalyxin gene promoter activity in insect cells. Drosophila-derived SL2 cells were transiently cotransfected with 5 μg of pGL3/Podxl-pr constructs and with either 0.5 μg of pPAC void plasmid or 0.5 μg of pPAC-Sp1 plasmid. Luciferase activities were normalized by protein concentration and are shown as the fold increases relative to the basal activity of void pGL3-basic plasmid (control). Values reported in the figure are mean ± SEM of three independent experiments in duplicates.

Figure 4

Gel mobility-shift analysis of DNA-protein complexes using the human podocalyxin promoter fragment -243/-18. A) Western blots were performed as described under Fig. 2; B) Five μg of nuclear extracts (NE) from Tera-1 and Meg0l cells were incubated with the probe. Competitive gel shift assay was done in the presence of 100-fold molar excess of unlabeled double-stranded oligonucleotides corresponding to the pointed out transcription factors consensus and unrelated nonspecific (Non Sp) motifs. Antibodies (2 μg) for AP2 or non-specific were preincubated with the Tera-1 and Meg0l NE before the addition of the Podxl probe.

Figure 5

Identification of Sp1-DNA complexes by supershift assays. A) Different probes were designed as described under "Methods" in order to identify functional Sp1 sites in the Podxl promoter. B) Antibodies (2 μg) for Sp1 and non-specific, were preincubated with 5 μg of Meg01 nuclear extracts (NE) before the addition of the wild or mutated Sp1-containing Podxl probes. BSA (2 μg) was used as a control. An arrow points to the supershifted Sp1 bands.

Figure 6

Effect of mutating the Sp1 recognition sites on the activity of podocalyxin promoter. Transient transfections were performed into HEK293 cells. Results represent mean ± SEM from four independent experiments performed in duplicates. Promoter activity of each transfection was expressed as a ratio of luciferase/renilla activities. Data are expressed as percent of the maximal activity of the -210 construct *p < 0.05 vs. non Sp1-site-mutated construct.

Figure 7

State of methylation of CpG dinucleotides in the human podocalyxin promoter. A) Schematic representation of a fragment of the Podxl-pr comprising 208 nts upstream the transcription start site (indicated with an arrow) and the 5'UTR. Grey boxes denote the positions of CpG dinucleotides. Underlined nts indicate Sp1 recognition sites. B) Genomic DNA from Tera-1 or Meg0l cells were extracted and subjected to bisulfite treatment, PCR amplification and DNA sequencing as described in Methods. CpG sites are numbered from 1 to 27 relative to the transcription start site. Three bisulfite treatments were performed. Data is expressed as % of methylation, calculated as percentage of conserved C in at least 10 clones obtained after each bisulfite treatment.

Figure 8

Expression of human podocalyxin in HEK293 and Meg0l cells treated with 5-AzaC. Cells were treated with 5-AzaC for 48 hrs. Cell lysates were resolved on SDS-7.5% polyacrylamide gels under non-reducing conditions, transferred to PVDF membranes and immunoblotted with A) 1 μg/ml of anti-podocalyxin mAb and B) 1 μg/ml of anti-Sp1 mAb. β-Actin was blotted as a loading control. The experiment was repeated three times.

Figure 9

Effect of in vitro DNA methylation on the podocalyxin promoter-driven luciferase expression. Transient transfections were performed in HEK293 cells. Results represent mean ± SEM from three independent experiments performed in duplicates. Promoter activity of each transfection was expressed as a ratio of luciferase/renilla activities. Data are expressed as arbitrary units calculated as fold increases over void pGL-3 enhancer vector activity, which is assigned a value of 1.0. *p < 0.05 vs. mock methylated control.