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E1A-responsive elements for repression of rat fibronectin gene transcription
T Nakajima1, T Nakamura, S Tsunoda
1Nichirei Research Institute, Tokyo, Japan.
Molecular and Cellular Biology
|June 1, 1992
Summary
E1A protein expression down-regulates fibronectin (FN) gene transcription by interacting with specific GC-rich promoter elements. This repression mechanism involves the retinoblastoma susceptibility gene product (RB) in resting cells.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cellular Oncology
Background:
- Fibronectin (FN) gene transcription is high in resting 3Y1 cells but decreases upon growth stimulation.
- E1A expression also induces a steep decrease in FN gene transcription.
Purpose of the Study:
- To elucidate the mechanism of E1A-mediated down-regulation of the fibronectin gene.
- To identify the specific DNA elements and protein interactions involved in this repression.
Main Methods:
- Reporter gene assays using constructs with deleted/substituted 5' flanking regions of the FN gene fused to a CAT reporter.
- Introduction of these constructs into 3Y1 cells with E1A expression plasmids.
- Analysis of E1A deletion mutants to identify critical domains for repression.
Main Results:
- Specific G-rich sequences (G10 stretch and two GC boxes) in the FN promoter are identified as E1A-responsive elements.
- Mutations within these G-rich sequences abolish E1A-mediated repression.
- The E1A domain responsible for binding the retinoblastoma susceptibility gene product (RB) is crucial for repression.
Conclusions:
- E1A represses fibronectin gene transcription through specific GC-rich promoter elements.
- The retinoblastoma susceptibility gene product (RB) plays a role in repressing a negative regulatory factor in resting cells.
- E1A expression likely derepresses this factor, leading to reduced FN transcription.