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BRCA1 regulates GADD45 through its interactions with the OCT-1 and CAAT motifs
Wenhong Fan1, Shunqian Jin, Tong Tong
1Department of Radiation Oncology, Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Abstract:
BRCA1, a breast and ovarian cancer susceptibility gene, has been implicated in gene regulation. Previous studies demonstrate that BRCA1 induces GADD45, a p53-regulated and stress-inducible gene that plays an important role in cellular response to DNA damage. However, the mechanism(s) by which BRCA1 regulates GADD45 remains unclear. In this report, we have shown that BRCA1 activation of the GADD45 promoter is mediated through the OCT-1 and CAAT motifs located at the GADD45 promoter region. Site-directed mutations of both OCT-1 and CAAT motifs abrogate induction of the GADD45 promoter by BRCA1. Both OCT-1 and CAAT motifs are able to confer BRCA1 inducibility in a non-related minimal promoter. Physical associations of BRCA1 protein with transcription factors Oct-1 and NF-YA, which directly bind to the OCT-1 and CAAT motifs, are established by biotin-streptavidin pull-down and coimmunoprecipitation assays. Such protein interactions are required for interaction of BRCA1 with the GADD45 promoter because either immunodepletion of Oct-1 and NF-YA proteins or mutations in the OCT-1 and CAAT motifs disrupt BRCA1 binding to the GADD45 promoter. These findings indicate that BRCA1 can up-regulate its targeted genes through protein-protein interactions and provide a novel mechanism by which BRCA1 participates in transcriptional regulation.
Insights
BRCA1 protein regulates GADD45 gene expression by interacting with transcription factors Oct-1 and NF-YA. This interaction binds BRCA1 to the GADD45 promoter, up-regulating gene activity and aiding DNA damage response.
Area of Science:
- Molecular Biology
- Cancer Genetics
- Gene Regulation
Background:
- BRCA1 is a tumor suppressor gene linked to breast and ovarian cancers.
- BRCA1 influences cellular responses to DNA damage.
- BRCA1 induces GADD45, a gene critical for DNA damage response, but its regulatory mechanism is unknown.
Purpose of the Study:
- To elucidate the mechanism by which BRCA1 regulates GADD45 gene expression.
- To identify the specific DNA elements and protein interactions involved in BRCA1-mediated GADD45 induction.
Main Methods:
- Site-directed mutagenesis of OCT-1 and CAAT motifs in the GADD45 promoter.
- Reporter assays to assess promoter activity.
- Biotin-streptavidin pull-down and coimmunoprecipitation assays to detect protein-protein interactions.
- Immunodepletion assays to evaluate the role of transcription factors.
Main Results:
- BRCA1 activation of the GADD45 promoter depends on OCT-1 and CAAT motifs.
- Mutations in these motifs abolish BRCA1-induced promoter activity.
- BRCA1 physically interacts with transcription factors Oct-1 and NF-YA.
- These protein interactions are essential for BRCA1 binding to the GADD45 promoter.
Conclusions:
- BRCA1 up-regulates GADD45 gene expression through physical interactions with Oct-1 and NF-YA.
- This study reveals a novel mechanism of transcriptional regulation by BRCA1 involving protein-protein interactions.
- Findings contribute to understanding BRCA1's role in cancer susceptibility and DNA repair pathways.