Gene Regulation
DNA Binding by the ETS Protein TEL (ETV6) Is Regulated by Autoinhibition and Self-association*

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The ETS protein TEL, a transcriptional repressor, contains a PNT domain that, as an isolated fragment in vitro, self-associates to form a head-to-tail polymer. How such polymerization might affect the DNA-binding properties of full-length TEL is unclear. Here we report that monomeric TEL binds to a consensus ETS site with unusually low affinity (Kd = 2.8 × 10−8 m). A deletion analysis demonstrated that the low affinity was caused by a C-terminal inhibitory domain (CID) that attenuates DNA binding by ∼10-fold. An NMR spectroscopically derived structure of a TEL fragment, deposited in the Protein Data Bank, revealed that the CID consists of two α-helices, one of which appears to block the DNA binding surface of the TEL ETS domain. Based on this structure, we substituted two conserved glutamic acids (Glu-431 and Glu-434) with alanines and found that this activated DNA binding and enhanced trypsin sensitivity in the CID. We propose that TEL displays a conformational equilibrium between inhibited and activated states and that electrostatic interactions involving these negatively charged residues play a role in stabilizing the inhibited conformation. Using a TEL dimer as a model polymer, we show that self-association facilitates cooperative binding to DNA. Cooperativity was observed on DNA duplexes containing tandem consensus ETS sites at variable spacing and orientations, suggesting flexibility in the region of TEL linking its self-associating PNT domain and DNA-binding ETS domain. We speculate that TEL compensates for the low affinity, which is caused by autoinhibition, by binding to DNA as a cooperative polymer.

DNA-binding Protein
Ets Family Transcription Factor
Protein-DNA Interaction
Transcription Factors
Transcription Repressor
Autoinhibition
Polymerization

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*

This work was supported, in whole or in part, by National Institutes of Health Grants R01GM38663 (to B. J. G.) and P50CA42014 (to the Huntsman Cancer Institute for support of shared resources). This work was also supported by funds from the Canadian Cancer Society Research Institute (to L. P. M.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Tables 1 and 2.

1

Present address: Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024.

2

Received instrument support from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, the British Columbia Knowledge Development Fund, the UBC Blusson Fund, and the Michael Smith Foundation for Health Research.