Physical Interaction and Functional Synergy between Glucocorticoid Receptor and Ets2 Proteins for Transcription Activation of the Rat Cytochrome P-450c27 Promoter

Jayati Mullick,Hindupur K Anandatheerthavarada,Govindasamy Amuthan,Shripad V Bhagwat,Gopa Biswas,Vijayasarathy Camasamudram,Narayan K Bhat,Shyam E.P Reddy,Veena Rao,Narayan G Avadhani

doi:10.1074/jbc.m100671200

Abstract

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak" Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.

Highlights

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor
In the present study we show that Dxmediated activation of Cytochrome P-450c27 (CYP27) gene expression involves GR binding to a variant GRE site and its synergistic interaction with factors binding to an adjacent “weak” consensus Ets site, referred to in this study as an Ets-like site
These results show that the transcription activation domain of Ets2 is highly essential for the synergistic activation of the promoter (Fig. 5), the transcription activation domain of GR may not be critical for the activity

Summary

Introduction

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets factor. Another study showed that mutations targeted to the Ets1-binding site, overlapping the GR binding motif of the tyrosine aminotransferase promoter, resulted in a 2-fold reduction in transcription activity, suggesting synergistic effects of the two factors [29]. In the present study we show that Dxmediated activation of CYP27 gene expression involves GR binding to a variant GRE site and its synergistic interaction with factors binding to an adjacent “weak” consensus Ets site, referred to in this study as an Ets-like site. The activation appears to require a novel functional and physical interaction between the DNA-binding domain of GR and the transcription activation domain of the ubiquitously expressed Ets transcription factor

Methods

Results

Conclusion