12 pages[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 ~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.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.Peer reviewed