Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells.
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.
Tripathi
Manish K
MK
Division of Cancer Biology, Department of Biomedical Sciences, Meharry Medical College, Vanderbilt University Medical Center, Nashville, Tennessee 37208, USA.
Misra
Smita
S
Khedkar
Sheetal V
SV
Hamilton
Nalo
N
Irvin-Wilson
Charletha
C
Sharan
Chakradhari
C
Sealy
Linda
L
Chaudhuri
Gautam
G
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
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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