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Interference of Myb transactivation activity by a conditional dominant negative protein: functional interference in a
1Ludwig Institute for Cancer Research, Imperial College School of Medicine at St.Mary's, London, W2 1PG, UK.
Abstract:
The ability to ablate the activity of specific transcription factors in vivo is a potentially important tool to study their roles in cellular processes such as the cell cycle. Previously, production of a dominant interfering c-Myb protein (comprising a fusion of the c-Myb DNA binding domain with the Drosophila Engrailed transrepressor) was found to inhibit the proliferation of immature thymocytes in the developing thymus of transgenic mice. We report here the further development of this stratagem by rendering the c-Myb/Engrailed protein conditionally active by fusion to a modified estrogen receptor hormone binding domain, ER. Co-transfection experiments in NIH 3T3 fibroblasts showed that the resulting chimeric protein, Myb/En/ER, repressed transactivation of a c-Myb-responsive reporter only in the presence of the synthetic steroid, 4-hydroxytamoxifen (OHT). Additionally, we found that Myb/En/ER could counteract transactivation by C/EBP-beta of the mim-1 promoter, which contains juxtaposed Myb and C/EBP binding sites. Cytotoxic T-cells stably producing the inactive Myb/En/ER protein were readily obtained by gene transfection. The addition of OHT to these cells resulted in inhibition of proliferation and arrest in G1. The utility of this experimental system to study Myb and other transcription factors is discussed.
Insights
Scientists developed a conditional c-Myb/Engrailed protein (Myb/En/ER) for targeted gene regulation. This tool allows in vivo studies of transcription factors, inhibiting cell proliferation when activated by 4-hydroxytamoxifen (OHT).
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Transcription factors play crucial roles in cellular processes like the cell cycle.
- Previous studies utilized dominant interfering c-Myb proteins to inhibit thymocyte proliferation in transgenic mice.
Purpose of the Study:
- To develop a conditionally active c-Myb/Engrailed fusion protein (Myb/En/ER) for studying transcription factor roles in vivo.
- To enable precise temporal and spatial control over gene activity using a hormone-inducible system.
Main Methods:
- Constructed a chimeric protein Myb/En/ER by fusing c-Myb DNA binding domain, Engrailed transrepressor, and a modified estrogen receptor hormone binding domain.
- Performed co-transfection experiments in NIH 3T3 fibroblasts to assess protein activity in the presence of 4-hydroxytamoxifen (OHT).
- Generated gene-transfected cytotoxic T-cells stably producing Myb/En/ER and analyzed their proliferation and cell cycle arrest upon OHT addition.
Main Results:
- The Myb/En/ER protein repressed c-Myb-responsive reporter activity specifically in the presence of OHT.
- Myb/En/ER counteracted C/EBP-beta transactivation of the mim-1 promoter.
- OHT addition to T-cells expressing Myb/En/ER resulted in proliferation inhibition and G1 cell cycle arrest.
Conclusions:
- The developed Myb/En/ER system provides a powerful tool for conditional ablation of transcription factor activity in vivo.
- This inducible system allows for detailed investigation of transcription factor functions, such as c-Myb, in various cellular processes and disease models.