Abstract
We reported previously that the sarco(endo)plasmic reticulum Ca(2+)-ATPase type 3 (SERCA3) gene is expressed in many tissues and in a subset of cells such as endothelial, epithelial, and lymphoid lineages. Here we analyzed the mechanisms involved in the regulation of transcription of the SERCA3 gene in endothelial cells. The promoter of the murine SERCA3 gene was isolated, and a single transcription initiation site located 301 bp upstream of the translation initiation site was identified. Analysis of the transcriptional activity of fragments of the SERCA3 promoter showed the existence of a minimal promoter region located between bases -97 and +153 that contains one ETS-binding site (EBS) and two Sp1 elements that are essential for basal transcription. Mutation of the EBS or of the Sp1 sites abolished the basal activity of the promoter. We identified Ets-1 and Sp1 among endothelial nuclear factors that recognize the EBS and Sp1 sites on the promoter. Furthermore, transactivation of the -97/+301 promoter fragment by Ets-1 requires the presence of both the EBS and Sp1 sites, suggesting an interaction of the transcription factors on the gene promoter. Finally, overexpression of Ets-1 induced the expression of SERCA3 in endothelial cells and in fibroblasts.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF521044
An ϳ250-bp fragment was amplified when using primer 2 on all total RNA samples, suggesting that transcription of the sarco(endo)plasmic reticulum Ca2؉-ATPase type 3 (SERCA3) gene was initiated in the region between the binding sites for primer 1 and primer 2
Mutation of the ETS-binding site (EBS) resulted in a 70 and 85% loss of activity when transfected in EA.hy926 and MoBr 204 cells, respectively (Fig. 3B), and mutation of the Sp1 sites decreased the transcription activity to ϳ20% of control values in both cell lines, indicating that the EBS and Sp1 sites were necessary for the basal transcription of the SERCA3 gene
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF521044. We show here that the Ets-1 protein from nuclear extracts of endothelial cells binds to the SERCA3 promoter essential EBS and that Ets-1 is able to promote the transcription of reporter constructs of the gene in endothelial cells.
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