Related Experiment Videos
Net, a negative Ras-switchable TCF, contains a second inhibition domain, the CID, that mediates repression through
P Criqui-Filipe1, C Ducret, S M Maira
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, ECNRS/INSERM/ULP, 1 Rue Laurent Fries, BP 163, 67404 Illkirch cedex, France.
Abstract:
Signalling cascades are integrated at the transcriptional level by the interplay between factors such as the ternary complex factors (TCFs) that interact with serum response factor (SRF) and the serum response element (SRE) of the fos promoter. Net is a negative TCF that is switched to a positive regulator by the Ras signal. To understand the mechanisms of repression by Net, we used a yeast two-hybrid screen to identify factors that interact with its inhibitory domain. We isolated mCtBP1, the murine homologue of huCtBP1, a factor implicated in negative regulation of transformation by E1A plus Ras. We show that mCtBP1 interacts strongly with Net both in vitro and in vivo. The CtBP interaction domain of Net, the CID, mediates repression independently of the previously identified negative element, the NID. The CID inhibits by recruiting the co-repressor mCtBP1. The CID and mCtBP1 need to use de-acetylase activity for repression, whereas the NID apparently represses by other mechanisms. Finally, we provide evidence that CtBP and de-acetylation repress the c-fos SRE in low serum when it is inactive, but not in high serum when it is active. These results provide insights into the cross-talk between pathways that inhibit and stimulate transformation at the level of Net, a regulator of gene expression.
Insights
Net, a transcriptional regulator, uses its inhibitory domain to recruit mCtBP1, a co-repressor. This interaction, dependent on deacetylase activity, represses the c-fos serum response element (SRE) under low serum conditions, revealing cross-talk in transformation pathways.
Area of Science:
- Molecular Biology
- Gene Regulation
- Signal Transduction
Background:
- Signaling pathways converge at the transcriptional level, involving ternary complex factors (TCFs) interacting with serum response factor (SRF) and serum response elements (SREs).
- Net acts as a negative TCF, becoming a positive regulator upon Ras signaling, and its repressive mechanisms are not fully understood.
Purpose of the Study:
- To elucidate the molecular mechanisms by which Net exerts transcriptional repression.
- To identify factors interacting with Net's inhibitory domain and their role in gene regulation.
Main Methods:
- Yeast two-hybrid screening to identify interacting partners of Net's inhibitory domain.
- In vitro and in vivo interaction assays to confirm binding between Net and mCtBP1.
- Functional assays to assess the role of CtBP interaction domain (CID) and NID in repression, including deacetylase activity dependence.
Main Results:
- Murine CtBP1 (mCtBP1), a homolog of human CtBP1, was identified as a Net-interacting protein.
- mCtBP1 binds strongly to Net both in vitro and in vivo.
- Net's CtBP interaction domain (CID) mediates repression by recruiting mCtBP1, requiring deacetylase activity, independently of the NID.
- CtBP-mediated deacetylation represses the c-fos SRE in low serum but not in high serum.
Conclusions:
- Net represses gene expression by recruiting the co-repressor mCtBP1 via its CID, utilizing deacetylase activity.
- This mechanism highlights the cross-talk between pathways regulating transformation and gene expression.
- The findings provide insights into the dynamic regulation of the c-fos promoter by Net and CtBP in response to serum levels.