In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells - PubMed (original) (raw)

In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells

Mukul K Mittal et al. Biochem Biophys Res Commun. 2008.

Abstract

The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628bp fragment (-613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling.

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Figures

Fig. 1

Fig. 1

Inducible expression of FLAG-tagged SLUG in MCF-7 and MDA-MB-468 cells. (A) Domain structure of hSLUG protein. (B) Amino acid sequences of hSLUG protein showing different functional domains. The SNAG motif (residues 1–20), the P-motif (residues 91–97) and the five zinc finger motifs (residues 130–150, 161–181, 187–207, 215–235, and 243–259) are underscored. RT-PCR (C) and Western blotting (D) analyses data showing tetracycline-inducible expression of hSLUG mRNA and protein, respectively. β-Actin mRNA and protein were used as loading controls in these studies.

Fig. 2

Fig. 2

Down-regulation of VDR in SLUG-expressing human breast cells. RT-PCR (A) and Western blotting (B) analyses data showing the expressions of SLUG and VDR mRNA and protein, respectively, in different human breast cancer cells. (C) Western blotting analysis data showing tetracycline-inducible repression of VDR protein levels in the recombinant MCF-7 and MDA-MB-468 cells. (D) Immunofluorescence analysis showing tetracycline-inducible repression of VDR protein levels in the recombinant MDA-MB-468 cells. (Left panel) The nuclei of the cells were stained with DAPI (blue); (middle panel) VDR protein was tagged with red Alexafluor dye; and (right panel) the superimposed photograph. β-Actin mRNA and protein were used as loading controls in these studies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Fig. 3

Negative regulation of VDR gene promoter activity in SLUG-expressing human breast cells. (A) Nucleotide sequence of human VDR gene promoter showing (underscored) the SLUG-binding E2-box (CAGGTG/CACCTG) elements. The upstream sequences are shown in lower case letters. The 5′-end of the exon 1 sequences is in uppercase letters. (B) Dual luciferase assay data showing the repression of the function of VDR gene promoter in the recombinant MCF-7 and MDA-MB-468 cells. Results are means ± SE (n = 6). The differences in the luciferase activities between the control and the tetracycline-induced cells were statistically significant (p < 0.001).

Fig. 4

Fig. 4

In vivo binding of SLUG, CtBP1 and HDAC1 to the VDR gene promoter in human breast cells. (A) ChIP analysis data showing the in vivo binding of hSLUG, CtBP1, and HDAC1 at the native VDR gene promoter in the lentivirus-transformed MDA-MB-468 cells. The expression of hSLUG was induced with tetracycline (1 μg/ml) for 48 h before the ChIP analysis. Anti-FLAG antibody was used to pull down the SLUG complex. (B) Model showing the possible mode of action of hSLUG to repress VDR gene expression in human breast cells. For simplicity, hSLUG binding to only one of the three E2-boxes is shown. The hypothetical protein, denoted with ‘?’, is proposed to help recruit CtBP1 at the P-motif of hSLUG. ZF, zinc finger of hSLUG; P, the P-motif; and S, the SNAG motif of hSLUG.

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