Abstract
The Ets gene family has a complex evolutionary history with many family members known to regulate genetic programs essential for differentiation and development, and some known for their involvement in human tumorigenesis. To understand the biological properties associated with a recently described epithelium-restricted Ets factor ESX, an 11 kb fragment from the 1q32.2 genomically localized human gene was cloned and analysed. Upstream of the ESX promoter region in this genomic fragment lies the terminal exon of a newly identified gene that encodes a ubiquitin-conjugating enzyme variant, UEV-1. Tissues expressing ESX produce a primary 2.2 kb transcript along with a 4.1 kb secondary transcript arising by alternate poly(A) site selection and uniquely recognized by a genomic probe from the 3′ terminal region of the 11 kb clone. Endogenous expression of ESX results in a 42 kDa nuclear protein having fivefold greater affinity for the chromatin-nuclear matrix compartment as compared to other endogenous transcription factors like AP-2 and the homologous Ets factor, ELF-1. Exon mapping of the modular structure inferred from ESX cDNA and construction of GAL4(DBD)-ESX expression constructs were used to identify a transactivating domain encoded by exon 4 having comparable potency to the acidic transactivation domain of the viral transcription factor, VP16. This exon 4-encoded 31 amino acid domain in ESX was shown by mutation and deletion analysis to possess a 13 residue acidic transactivation core which, based on modeling and circular dichroism analysis, is predicted to form an amphipathic α-helical secondary structure. Using recombinant GST-ESX (exon 4) fusion proteins in an in vitro pull-down assay, this ESX transactivation domain was shown to bind specifically to one component of the general transcription machinery, TATA-binding protein (TBP). Transient transfection experiments confirmed the ability of this TBP-binding transactivation domain in ESX to squelch heterologous promoters independent of any promoter binding as efficiently as the transactivation domain from VP16.
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Acknowledgements
We thank Xiaohui Xiong and Yevgeniya Suzdaltseva for their excellent technical assistance. This work was supported in part by NIH sponsored multi-institutional program project (CA44768) and SPORE grants (CA58207), an NIH sponsored individual research grant (CA36773), as well as the Hazel P Munroe and Janet Landfear memorial funds. As 1997 – 1998 Visiting Scientist and Professor at the Friedrich Miescher Institute and University of Basel, CC Benz thanks N Hymes, M Burger, and R Herrmann for their assistance throughout that year and acknowledges funding support from the Krebsliga beider Basel.
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Chang, CH., Scott, G., Baldwin, M. et al. Exon 4-encoded acidic domain in the epithelium-restricted Ets factor, ESX, confers potent transactivating capacity and binds to TATA-binding protein (TBP). Oncogene 18, 3682–3695 (1999). https://doi.org/10.1038/sj.onc.1202674
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DOI: https://doi.org/10.1038/sj.onc.1202674
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