Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter
© Butta et al; licensee BioMed Central Ltd. 2006
Received: 08 February 2006
Accepted: 09 May 2006
Published: 09 May 2006
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.
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 ~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.
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.
Podocalyxin (podxl) is an integral membrane protein originally identified by Kerjaschki et al  in the foot processes of the rat kidney glomerular podocytes. It was described as a sialoprotein of 140-kDa based on its staining with alcian blue. The high sulfate content of this protein contributes in part to its heavily negative charge  that is supposed to maintain open the interdigitations of the podocyte foot for the urine filtration. The importance of the negative charge of this protein is indicated by the loss of the structure and function of the podocytes by treatment with polycations or desialylation with neuraminidase [3, 4]. Null mice for podxl die soon after birth due to failure to develop foot processes in the glomerular epithelial cells . Podxl like proteins have been cloned from the rabbit, human, chicken and mouse [6–9].
The podxl is also expressed in the luminal face of the vascular endothelia, hematopoietic cells and platelets [10, 11], among other tissues. The function of this protein in non-renal cells has not yet been elucidated. It has been suggested that it may function as a ligand for leucocyte adhesion molecule L-selectin  due to its location in the postcapillary venules of the lymph node, high endothelial venules, and the fact that most of the L-selectin ligands in the vascular endothelium are sialomucins. Thus, the podxl of the vascular endothelia may contribute to the migration of lymphocytes toward the lymph nodes.
The podxl proteins show a poorly conserved aminoterminal, extracellular, mucin-like domain containing multiple N- and O-linked glycosylation sites, a disulfide containing domain and conserved transmembrane and cytoplasmic domains. The cytoplasmic tails showed the highest identities among these proteins and interaction with cytosolic proteins have been reported. Based on structural similarities and their high content in sialic acid, Sassetti et al  proposed that CD34, podxl and endoglycan, would form a family named the CD34 family of sialomucins. The three proteins are found in the vascular endothelia and also in early hematopoietic precursors, suggesting that their function may go from either cell anti-adherence, as it is the case in the renal podocytes, to selective adherence or control of cellular differentiation.
Despite the interest of podxl no much is known about the transcriptional regulation of its gene in different cells. The binding of the Wilms Tumor suppressor factor (WT1) to conserved elements within the Podxl gene promoter results in a potent transcriptional activation  that seems to be relevant only for the induction and development of the kidney; however, a detailed analysis of the human Podxl promoter (Podxl-pr) has not yet been performed. In the present study we have further characterized the 5' regulatory region of this gene. We found absence of a TATA-box and a transcriptional regulation supported primarily by Sp1 site(s). In addition, the role of cytosine methylation of the promoter region has been analyzed to elucidate whether this epigenetic mechanism might contribute to control the specific regulation of the expression of this gene.
Features of the 5' flanking region of the human podocalyxin
The human Podxl gene and 250 bps upstream of the putative start methionine was previously cloned and mapped to chromosome 7q32-q33 by Kershaw et al [7, 15]. We amplified by PCR a 1528 bp-DNA fragment of the 5' regulatory region of the human Podxl gene comprising 1297 bp from the transcription start site plus 231 bp of 5'-untranslated region, using the primers sense: -1297/-1275 and antisense: 231/208. We started numbering from the transcription start site [GenBank:AF395890]. Computer analysis using the software "Transcription Factor Binding Site Profile Database" (TFSEARCH program) , revealed a high G+C content of the proximal promoter region and a lack of CCAAT and TATA-like elements, a feature commonly found in the house-keeping genes. We also detected potential binding sites for the transcription factors AP2 (-247/-236 and -114/-93 nts) (two overlapped sites); overlapped Sp1 sites (167/-140, -146/-128 and -91/-68 nts); overlapped Sp1 and AP2 sites (-279/-266 nts), overlapped Sp1 and NFKB sites (-204/-189 nts), three GATA-1 sites (-1138/-1129, -1057/-1051 and -444/-435 nts) and three WT-1 sites (two overlapped at -1233/-1216 and one at -507/-498 nts).
Regulatory role of Sp1 on Podxl promoter activity
Effect of promoter CpG methylation on the cellular expression of podxl
In this work we have analyzed the structure and function of the Podxl gene promoter. This protein was originally described in the podocytes of the epithelial glomerular cells . Podxl is also present in extra-renal tissues, particularly in the vascular endothelial and hematopoietic cells. Moreover, podxl has also been found in tumor cells [21–23] and its expression has been considered as a good marker of mieloproliferative disorders . The interest on podxl has been recently focused on its putative capacity to regulate the intercellular contacts.
The information about the regulation of the Podxl gene is scarce. The Wilms tumor suppressor factor (WT1) is a potent regulator of kidney induction and nephrogenesis [25, 26] that controls the expression of Podxl . However, the WT1 factor is downregulated in all tissues except in the glomerular epithelial cells where it is expressed throughout life. Mutations in the WT1 gene are associated with glomerulosclerosis in patients of Denys Drash syndrome indicating the importance of this factor in the renal function in adulthood. Moreover, nephrogenesis can be rescued in WTl-null mice by reintroducing the gene . However, regulatory factors other than WT1 in renal or extra-renal tissues are undefined. The lack of TATA or CAAT boxes in the Podxl promoter, its high content of C+Gs and the control by Sp1 sites are typical features of house-keeping genes. The transcriptional activity of the progressive 5' deletions of this promoter indicated that its basic activity was located in the region 66 to -111 nts increasing as the length of the DNA fragment increased (Fig. 1). The progressive rise in the promoter activity directly correlates with the number of recognition sites for Sp1, suggesting its functional importance. This interpretation is supported by the absence of transcriptional activity of the Podxl promoter constructs in insect cells (Drosophila SL2) that lack Sp1 transcription factor and the initiation of transcriptional activity by cotransfection of an expression plasmid encoding Sp1 (Fig. 3). Despite differences in endogenous content of podxl, the activity of the Podxl promoter was similar whether Tera-1, HepG2 or HEK293 cells were used in the transfection experiments (Fig. 1). This observation may find an explanation in the similar levels of Sp1 factor found in these cells (Fig. 2).
The nucleotide competition assays gave positive results for Sp1 and AP2 whether nuclear extracts from Tera-1 or from Meg0l cells were used; however, no supershift was observed when monoclonal against AP2 was utilized (Fig. 4B). The competitive effect of AP2 oligonucleotide in the formation of protein-DNA complexes may be related to the sequence similarities between Sp1 and AP2 recognition sites. The EMSA studies indicate that overlapped Sp1 sites -167/-140 nts, -91/-68 nts and -204/-189 nts were able to interact with Sp1, since some of the formed complexes were supershifted by anti-Sp1 Ab and their formation was-prevented by mutating the recognition sites. Moreover, the mutation of the Sp1 sites reduced the transcriptional activity of Podxl.
DNA methylation is associated with malignant transformation  of cells but knowledge of the epigenetic events in the initiation and progression of human cancer is limited. The abundant number of CpG islands in the Podxl promoter suggested that methylation could be a regulatory factor. Transcriptional repression of methylated genes may be mediated by the binding of certain proteins, such as MeCP2 [28–30]. Moreover, DNA methylation may interfere with the binding of Sp1 to DNA .
Demethylation of the promoter DNA may be a necessary step in the epigenetic reprogramming of somatic cell nuclei . Demethylation is a selective process, operating only on the promoter, but not on enhancers; both a putative Sp1/Sp3 and a GGGAGGG binding site are required for demethylation and the initiation of transcription. We analyzed whether methylation of the Podxl promoter could influence its transcriptional activity: firstly, using demethylating agents in vivo and in second place by studying the state of methylation of the CpG islands in cultured cells expressing different amounts of Podxl. As a demethylating agent we used 5-azaC, structural analog of citidine, that inactivates methyltransferases upon its incorporation into DNA with the result of activation of DNA transcription. This inhibitor has been used successfully as a therapeutic agent in acute myeloid leukemia and myelodysplastic syndrome . In our case, treatment of low podxl-expressing cells with 5-azaC induced a dose-dependent increase on podxl content whereas no effects could be detected in cells, like HEK293, expressing higher levels of podxl. However, the reported effect of 5-azaC in inducing the activity of genes in the absence of methylation of CpG islands indicates that these results should be taken cautiously . The treatment with 5-azaC may stabilize and increase the endogenous content of Sp1  nevertheless in our case 5-azaC did not modify Sp1 cellular content. On the other hand, bisulfite treatment of genomic DNA from high (Tera-1) or low (Meg0l) podxl-expressing cells showed a direct relationship between the degree of methylation of the promoter region and expression of podxl. The influence of the CpG methylation was further assessed by treating the promoter constructs -111,-181 and -210 with the methylase Sss I prior to transfection into HEK293 cells. These results add further evidence indicating that methylation may inhibit the activity of the Podxl promoter.
The in vitro transcription of the human Podxl promoter is dependent on the presence of Sp1 sites. This conclusion is supported by the following observations: 1) No activity was detected when the promoter constructs were transfected into Sp1-lacking insect cells in which the transcription was initiated by cotransfection of an Sp1 expression plasmid; 2) The promoter function was impaired by mutation of the Sp1 sites.
Methylation of the CpG promoter islands seems to be a significant regulatory mechanism in controlling the transcription of Podxl. The latter observation may provide an explanation for the different rates of podxl expression in different tissues of the organism.
Cell culture media was from GIBCO, Invitrogen (Scotland, UK). Fetal bovine serum was from ICN Biochemicals (Irvine, CA, USA). Restriction endonucleases were obtained from Roche Diagnostics, SL (Barcelona, Spain). Antibody against β-actin was from Sigma RBI (Madrid, Spain); mAbs against Sp-1 (sc-420x and sc-420) or AP2 (sc-25343x) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse mAb against human podxl (clone "3D3") was a generous gift of Dr. David B. Kershaw from the Department of Pediatrics, University of Michigan. [α-32P]dCTP was from Amersham Iberica (Spain). The Dual-Luciferase Reporter Assay System, pGL3-basic vector and Wizard DNA Clean-Up System were from Promega (Madison, WI). Sss I (CpG) methylase was from New England Biolabs (Beverly, MA). Oligonucleotides were obtained from Invitrogen (Paisley, UK). All other reagents were of analytical grade and were purchased from commercial sources.
Construction of reporter plasmids
A 1528-bp DNA fragment was amplified by PCR from human genomic DNA using the primers Podxl-pr -1297/-1275 (5'-GAGCGGGGGAGGGGAAAGGAA-3') and Podxl-pr 231/208 (5'-GGAGCAGGTGGCTGCGGTGC-3') as sense and antisense oligonucleotides, respectively. The PCR product was subcloned directly into a T vector (Topo TA cloning, Invitrogen) to introduce Xho I and Hind III restriction sites in 5' and 3' ends, respectively. The Xho I/Hind III (-1297/231) fragment was subcloned into Xho I/Hind III sites of pGL3-basic to yield the construct -1297. Construct -1234 was produced by partial digestion with EcoR I and religation of the -1297 construct. Constructs -111, -181, -210, -364, -851, and -1108 were obtained by PCR amplification from construct -1297 using as antisense primer Podxl-pr 231/212 (5'-GCCTC GAGCAGGTGGCTGCGGT-3', underlined bp were mutated to generate a Xho I site) and one of the following sense primers:
Podxl-pr -111/-93 (5'-TGGCGA GCTCCTGGGGGAGGG-3'), Podxl-pr -181/-161 (5'-GCTCAGA GC TCACGGCCCAGCG-3'), Podxl-pr -210/-189 (5'-GTCGA GCTCCAGGGCGGAGTTTC-3'), Podxl-pr -364/343 (5'-GGGAG CT CGCCGGGCCCACTTA-3'), Podxl-pr -851/-830 (5'-CTGAG CTCAGAGGCAGGTTTGC-3'), Podxl-pr -1108/-1087 (5'-TTG AGC TC CCCAACAACCCCCT-3'). Underlined nucleotides in sense oligonucleotides were mutated to generate Sac I sites. pGL3-constructs with mutated Sp1 sites were obtained by PCR from DNA template and antisense oligonucleotide mentioned above and the following sense primers: Podxl-pr -90/-79 mutSp1-A (5'-GAGCTCCGGT GGT GGGGGT GGGGG-3'), Podxl-pr -146/-126 mutSp1-B (5'-CA GCT CCGGCCTT GCCCGGCC-3'), Podxl-pr -178/-150 mutSp1-C (5'-TCATTGTTCAAA GCCCAGA GCCCA CCCAG-3'), Podxl-pr -207/-184 mutSp1-C (5'-GAGCTCCAGGTT GGAGTTTCC-3'). Underlined nucleotides show mutated nucleotides. The resulting PCR products were subcloned into a T vector and sequenced in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The TOPO constructs were digested with Sac I and Xho I and ligated to the Sac I and Xho I sites of pGL3-basic plasmid. Restriction enzyme digestion, sequence analysis, or both were used to verify the constructions. A computer analysis for potential binding sites on DNA sequences was carried out with the TFSEARCH program .
HepG2 (hepatoma cells) and HEK293 (human embryonic kidney cells) were grown in DMEM, Tera-1 (testes embryonal carcinoma cells) in McCoy medium, Meg0l (human megakaryoblastic cells) in RPMI medium, all obtained from GIBCO (Madrid, Spain), and Drosophila Schneider's SL2 cells in Schneider's insect medium (Sigma, Madrid, Spain). Culture media were supplemented with 10% fetal bovine serum. SL2 cells were grown at 25°C and all other cell lines at 37°C.
Transient transfection procedure
HepG2, Tera-1 and HEK293 cells were transfected by the calcium phosphate precipitation technique in 60-mm dishes with 2 μg of pGL3/Podxl constructs described above and 0.5 μg of phRL-cmv plasmid for transfection efficiency correction. For negative and positive controls of transfection, pGL3-basic and the 3TP promoter-luciferase construct (p3TP-lux), containing three copies of the TPA response element from the human collagenase promoter plasmids were used. SL2 cells were cotransfected with pGL3/Podxl constructs and either pPAC-Sp1 that expresses human Sp1 driven by the Drosophila actin promoter, or the control vector pPacO, containing only the Drosophila actin promoter. The amount of transfected DNA was maintained constant with void plasmid. Either 6 h (HEK293 and Tera-1 cells) or 16 h (HepG2 and SL2 cells) after transfection, the medium was changed and cells were incubated for another 48 h. The luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) in cell extracts prepared according to manufacturer recommendations in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). Luciferase activities were normalized by Renilla luciferase activity, except for SL2 experiments that were normalized by the protein concentration that was measured using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA, USA).
Gel retardation assay
DNA probes were prepared by PCR amplification from the -1296 construct. The following sense and antisense primers were used: for -243/-18 probe: Podxl-pr -243/-223 (5'-CCAAGGCTCGAGGCCGCCGG-3') and Podxl-pr -18/-38 (5'-CACTGGCGGCTGCGGCTACTC-3'); for-538/-238 probe: Podxl-pr-538/-518 (5'-GGGCTCGAGAGGCAGGTGAAT-3') and Podxl-pr -238/-258 (5'-CCTTGGGGGGCTGTGTGTGCG-3'), for A probe: Podxl-pr -111/-93 and Podxl-pr -18/-38, for mutSp1-A probe: Podxl-pr -90/-79 mutSp1-A and Podxl-pr -18/-38, for B probe: Podxl-pr -146/-136 (5'GAGCTCCGGCCCCGCCCGGCC-3') and Podxl-pr -94/-114 (5'CTCGAGCAGGAGCCCGCCAGC-3'); for mutSp1-B probe: Podxl-pr-146/-126 mutSp1-B and Podxl-pr -94/-114; for C probe: Podxl-pr-181/161 and Podxl-pr -94/-114; for mutSp1-C probe: hPodxl-pr -178/-150 mutSp1-C and Podxl-pr -94/-114; for D probe: Podxl-pr -210/-189 and Podxl-pr -150/-178mutSp1-C (5'-CTGGGTT GGTT CTGGGCTT TGAATGA-3'); for mutSp1-D probe: Podxl-pr -207/-184 mutSp1-D and Podxl-pr -150/-178mutSp1-C. All the probes were labeled using the Klenow fragment of Escherichia coli polymerase and [32P]dCTP. Nuclear extracts from Tera-1 and Meg0l cells were prepared according to standard protocols. Nuclear extracts (5 μg) were preincubated for 15 min at 4°C in 25 μL-binding reaction containing: 10 mM Tris/HCl pH 7.5, 50 mM NaCl, 2.5 mM MgC12, 4% glycerol, 0.5 mM dithiothreitol and 3 μg poly(dI-dC). After adding ~30 pg of labeled DNA, the mixture was incubated for 30 min at room temperature, and the complexes formed resolved on a 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer. Gels were run at 150 V at room temperature, dried and exposed at -80°C to an X-ray film.
For competition assays, 100-fold molar excess of unlabeled double stranded DNA consensus sequences of binding sites for AP2, Sp1, NFkB and GATA transcription factors were included in the binding reaction. These DNA fragments were prepared by hybridization of sense and antisense oligonucleotides of the following sequences: 5'-GATCGAACTGACCGCCCGCGGCCC-3' for AP2, 5'-ATTCGATCGGGGCGGGGCGAG-3' for Sp1, 5'-AGTTGAGGGGACTTTCCCAGGC-3' for NFkB and 5'-CACTTGATAACAGAAAGTGATAACTCT-3' for GATA DNA binding sites. As nonspecific competitor, an unrelated DNA fragment of similar size and base composition was used. In supershift experiments, nuclear extracts were preincubated with either mAbs against Sp1, AP2 or nonspecific antibodies, prior to incubation with the radiolabeled probes.
In Vitro methylation reactions
To examine the effect of CG methylation on the promoter activity, Podxl promoter constructs -111,-181,-210 were mock methylated or methylated by Sss I methylase in the absence or presence of S-adenosylmethionine. The status of methylation was determined by digestion with the restriction enzyme Hpa II. Mock-methylated or Sss I methylated plasmids were isolated from 1% agarose gel using Gel Purification Kit from Qiagen.
Western blot analysis
For western blot analysis, cells were lysed in 100 μl of phosphate buffered saline containing 1% Triton X-100, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and 1% of protease inhibitors cocktail. Cellular extracts were resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with mAb against Sp1 (Sta Cruz sc-420) or Podxl (3D3) and subsequently with the horseradish peroxidase-conjugated secondary antibody (BioRad, Madrid, Spain). β-Actin was blotted in order to normalize the amount of lysate in the experiments. To develop the blots we use the ECL reagent.
Treatment of cells with 5-Aza-2 deoxycytidine
The cells were treated with 5-azaC at a final concentration of 1 μM or 5 μM for 7 days. The medium was changed every second day. Negative controls were treated in the same manner without 5-azaC. Then, cells were lysed for western blot analysis and blotted with the mAb anti-Podxl primary antibody (3D3).
Bisulfite modification of genomic DNA
To release small fragments of genomic DNA of Meg0l and Tera-1 cells we digested with restriction enzymes that don't cleave the CpG region of the Podxl-pr. Digested genomic DNAs were denatured, modified by bisulfite, purified, and desulphonated according to Herman et al . Degenerated primers for PCR were designed to amplify the region between -618 to +368 (Podxl-pr -618/-592 5'-GGTGAAGGAGTGAATTAGTC/TC/TAGC/TTTG-3' and Podxl-pr 368/343 5'-AGAATGGTGAGTGGC/TC/TGGC/TC/TTGGGTT-3). The PCR fragments were purified by the JetQuick Purification Kit (Genomed) and sequenced.
N. Butta is recipient of a tenure track grant Ramon y Cajal from the Spanish Ministry of Science. S. Larrucea was supported by a contract from the Comunidad de Madrid (08.4/0015.1/2001) and at present holds a research tenure track of the Spanish Council of Research. S. Alonso was recipient of a predoctoral fellowship from the Basque Autonomous Community (BF101-40). E G Arias-Salgado is recipient of a tenure research grant Juan de la Cierva. This work has been supported in part by grants from the Direccion General de Investigacion (SAP 2004-04345), Fondo de Investigaciones Sanitarias (FIS-PI021263 and PI050546) and Comunidad de Madrid (08.4/0029.1/2003). N. Butta was recipient of a grant from the Fundacion Rodríguez Pascual. We thank Dr. Kershaw for the generous gift of the mAb 3D3.
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