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Molecular and Cellular Biology, December 1998, p. 7397-7409, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Coactivator OBF-1 Makes Selective Contacts with Both the POU-Specific Domain and the POU Homeodomain and Acts as a Molecular Clamp on DNA

Patrick Sauter and Patrick Matthias*

Friedrich Miescher-Institute, CH-4058 Basel, Switzerland

Received 4 August 1998/Accepted 10 September 1998

The lymphoid-specific transcriptional coactivator OBF-1 (also known as OCA-B or Bob-1) is recruited to octamer site-containing promoters by interacting with Oct-1 or Oct-2 and thereby enhances the transactivation potential of these two Oct factors. For this interaction the POU domain is sufficient. By contrast, OBF-1 does not interact with the POU domains of other POU proteins, such as Oct-4, Oct-6, or Pit-1, even though these factors bind efficiently to the octamer motif. Here we examined the structural requirements for selective interaction between the POU domain and OBF-1. Previous data have shown that formation of a ternary complex among OBF-1, the POU domain, and the DNA is critically dependent on residues within the octamer site. By methylation interference analysis we identified bases that react differently in the presence of OBF-1 compared to the POU domain alone, and using phosphothioate backbone-modified probes in electrophoretic mobility shift assays, we identified several positions influencing ternary complex formation. We then used Oct-1/Pit-1 POU domain chimeras to analyze the selectivity of the interaction between OBF-1 and the POU domain. This analysis indicated that both the POU specific domain (POUS) and the POU homeodomain (POUH) contribute to complex formation. Amino acids that are different in the Pit-1 and Oct-1 POU domains and are considered to be solvent accessible based on the Oct-1 POU domain/DNA cocrystal structure were replaced with alanine residues and analyzed for their influence on complex formation. Thereby, we identified residues L6 and E7 in the POUS and residues K155 and I159 in the POUH to be critical in vitro and in vivo for selective interaction with OBF-1. Furthermore, in an in vivo assay we could show that OBF-1 is able to functionally recruit two artificially separated halves of the POU domain to the promoter DNA, thereby leading to transactivation. These data allow us to propose a model of the interaction between OBF-1 and the POU domain, whereby OBF-1 acts as a molecular clamp holding together the two moieties of the POU domain and the DNA.

* Corresponding author. Mailing address: Friedrich Miescher-Institute, Maulbeerstr. 66, P.O. Box 2543, CH-4058 Basel, Switzerland. Phone: 41-61-697 66 61. Fax: 41-61-697 39 76. E-mail: matthias{at}fmi.ch.

Molecular and Cellular Biology, December 1998, p. 7397-7409, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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