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.
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Authors | Manish K Tripathi, Smita Misra, Sheetal V Khedkar, Nalo Hamilton, Charletha Irvin-Wilson, Chakradhari Sharan, Linda Sealy, Gautam Chaudhuri |
Journal | The Journal of biological chemistry
(J Biol Chem)
Vol. 280
Issue 17
Pg. 17163-71
(Apr 29 2005)
ISSN: 0021-9258 [Print] United States |
PMID | 15734731
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- BRCA2 Protein
- DNA-Binding Proteins
- Histones
- Phosphoproteins
- RNA, Messenger
- RNA, Small Interfering
- Recombinant Proteins
- SNAI1 protein, human
- Snail Family Transcription Factors
- Transcription Factors
- DNA
- Alcohol Oxidoreductases
- C-terminal binding protein
- Histone Deacetylases
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Topics |
- Acetylation
- Alcohol Oxidoreductases
- BRCA2 Protein
(biosynthesis, genetics)
- Base Sequence
- Blotting, Northern
- Blotting, Western
- Breast Neoplasms
(genetics, metabolism, pathology)
- Cell Line, Tumor
- Cell Nucleus
(metabolism)
- Chromatin Immunoprecipitation
- DNA
(metabolism)
- DNA Mutational Analysis
- DNA-Binding Proteins
(metabolism)
- Gene Expression Regulation, Neoplastic
- Gene Silencing
- Histone Deacetylases
(metabolism)
- Histones
(chemistry)
- Humans
- Models, Genetic
- Molecular Sequence Data
- Mutagenesis, Site-Directed
- Phosphoproteins
(metabolism)
- Promoter Regions, Genetic
- Protein Binding
- Protein Conformation
- Protein Structure, Tertiary
- RNA, Messenger
(metabolism)
- RNA, Small Interfering
(metabolism)
- Recombinant Proteins
(chemistry)
- Reverse Transcriptase Polymerase Chain Reaction
- Snail Family Transcription Factors
- Transcription Factors
(metabolism)
- Transfection
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