Binding and functional characteristics of two E‐box motifs within the S100A6 (calcyclin) gene promoter

Abstract

S100A6 (calcyclin) is a small calcium-binding protein of the S100 family often associated with cancer and metastasis. We have previously shown that the E-box sequence at position -283/-278 of the S100A6 gene promoter interacts with USF transcription factor and contributes to promoter transcriptional activity. We now present evidence that a second E-box motif at position -593/-588 of the promoter also binds USF and that the USF1/USF2 heterodimer is the prevailing dimeric form of the transcription factor bound. Using the chromatin immunoprecipitation assay (ChIP), we show that USF is bound in vivo to the E-box regulatory element(s). Depletion of the endogenous USF pool by means of a decoy oligonucleotide evokes a severe inhibition of S100A6 gene promoter activity. Furthermore, we show that S100A6 gene promoter activity can be stimulated by palmitate and that mutation of the -283/-278 E-box sequence completely blocks this stimulation.

Expression of S100A6 in Cardiac Myocytes Limits Apoptosis Induced by Tumor Necrosis Factor-α

“…Therefore, the peptide growth factor and cytokine-responsive sequences must be located upstream of the basic promoter fragment. Reported sequences upstream of the Ϫ167/ϩ134 include an NF-B-binding site at position Ϫ460/Ϫ451 (13), an antioxidant response element located at position Ϫ290/Ϫ281 (16) and two canonical E-boxes located at position Ϫ283/Ϫ278 that bind the ubiquitous upstream stimulatory factors 1 and 2 (38). Of interest, the NF-B-binding site has been implicated in S100A6 transcriptional regulation by TNF-␣ in a liver cell line (13).…”

Epigenetic Control of the S100A6 (Calcyclin) Gene Expression

“…A question therefore arose whether this would affect the binding of transcription factors. We showed that indeed the in vivo binding of USF, a ubiquitous transcription factor, which activates the S100A6 promoter (Les ´niak and Kuz ´nicki, 2006) is much less effective in HEK293 cells than in Hep-2 cells. This may be due both to the limited DNA accessibility suggested by the type of histone modifications and to the diminished ability of this transcription factor to bind to methylated DNA, as has been shown recently (Fujii et al, 2006).…”

“…We have then checked whether differences in DNA methylation and modifications of histone H3 between the two cell types examined could affect the in vivo function of the USF transcription factor, which binds to and activates the S100A6 gene promoter (Les ´niak and Kuz ´nicki, 2006). We therefore used the ChIP method and an antibody against USF1, which can bind DNA either as a homodimer or as a dimer with USF2 (Sirito et al, 1992).…”

“…The ChIP assay was performed essentially as described earlier (Les ´niak and Kuz ´nicki, 2006) using salmon sperm DNA/Protein A agarose (Upstate Biotech, Lake Placid, NY) and the following polyclonal rabbit antibodies: anti-USF1 from Santa Cruz Biotechnology (Santa Cruz, CA), anti-acetyl-histone H3, anti-trimethyl-histone H3 (Lys 9), and anti-trimethyl-histone H3 (Lys 27) from Upstate Biotech. Antirabbit IgG antibody was used as a control.…”

“…Furthermore, using the chromatin immunoprecipitation (ChIP) method, we have compared the extent of acetylation and Lys 9 and Lys 27 methylation of histone H3 bound to the S100A6 gene promoter. Additionally, we also compared the in vivo binding of upstream regulatory factor (USF), a transcription factor known to activate S100A6 expression (Les ´niak and Kuz ´nicki, 2006), to the transcriptionally active and inactive gene promoter. Our results point to the role of epigenetic factors in the control of S100A6 expression.…”