Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

Abstract

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the nondividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the ؊701 to ؊921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.

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

“…Human breast cancer cells MCF-7, MDA-MB-468, MDA-MB-231 and BT549 were obtained from ATCC (Manassas, VA) and were cultured in ATCC-recommended media [10,11]. FLAG M2 antibody was purchased from Sigma Chemical Co. (St. Louis, MO).…”

“…Human SLUG (hSLUG) gene ORF (NM_003068) was PCR amplified [10] from the cDNAs derived from MDA-MB-231 cells with ClaI and BamHI site-containing primers (5′-CAAGGTACCATGCCGCGCTCCTTCCTGC-3′, and 5′-CAAGGATCCG TGTGCTACACAGCAGCC-3′). The reverse primer did not have the endogenous stop codon.…”

“…The SLUG-3xFLAG ORF was then transferred to pLenti4/TO/V5-DEST vector (Invitrogen) by recombination using Gateway cloning reagents and protocols (Invitrogen). Transfection of 293 FT packaging cells with the plasmid constructs and human breast cells with the virus were done as described before [10]. The blasticidin (25 μg/ml)-resistant tetracycline repressorexpressing cells were further transduced by pLenti4/TO/V5-SLUG-3xFLAG-containing virus as described above, and the stable cell line was selected with 250 μg/ml zeocin.…”

“…RNA was extracted from cells using Trizol reagent (Invitrogen) and PCR amplifications were performed as described [10]. Primers used were: for SLUG, 5′-ATGCCGCGCTCCTTCCTGC-3′ and 5′-ATGGAGGAGGGGGACTCACTCG-3′, for VDR, 5′-CCAGTTCGT GTGAATGATGG-3′ and 5′-AGATTGGAGAAGCTGGACGA-3′, for β-actin, 5′-GCTCGTCGTCGACAACGGCTC-3′ and 5′-CAAACATGATCTG GGTCATCTTCTC-3′.…”

“…Cells were seeded on 24-well tissue culture plates and post 24 h, they were transfected with pGL3-Control and pRL-VDR promoter construct using Lipofectamine 2000 transfection reagent (Invitrogen). Luciferase activity was assayed 48 h later using a dual luciferase assay kit (Promega), as described before [10].…”