title: 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 non-dividing 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.
authors.affiliation: Division of Cancer Biology, Department of Biomedical Sciences, Meharry Medical College, Vanderbilt University Medical Center, Nashville, Tennessee 37208, USA.
authors.forename: Manish K
authors.initials: MK
authors.lastname: Tripathi
authors.forename: Smita
authors.initials: S
authors.lastname: Misra
authors.forename: Sheetal V
authors.initials: SV
authors.lastname: Khedkar
authors.forename: Nalo
authors.initials: N
authors.lastname: Hamilton
authors.forename: Charletha
authors.initials: C
authors.lastname: Irvin-Wilson
authors.forename: Chakradhari
authors.initials: C
authors.lastname: Sharan
authors.forename: Linda
authors.initials: L
authors.lastname: Sealy
authors.forename: Gautam
authors.initials: G
authors.lastname: Chaudhuri
mesh_terms: D000107:Acetylation; D000429:Alcohol Oxidoreductases; D024682:BRCA2 Protein / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000737:chemistry; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
chemical_list: D024682:BRCA2 Protein; D004268:DNA-Binding Proteins; D006657:Histones; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C000604407:SNAI1 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D004247:DNA; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D006655:Histone Deacetylases
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