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Molecular and Cellular Biology, October 2002, p. 7337-7350, Vol. 22, No. 20
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.20.7337-7350.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mechanism of E47-Pip Interaction on DNA Resulting in Transcriptional Synergy and Activation of Immunoglobulin Germ Line Sterile Transcripts

Sujatha Nagulapalli,1 Aisha Goheer,1 Leslie Pitt,1 Lawrence P. McIntosh,2 and Michael L. Atchison1*

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,1 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z32

Received 7 February 2002/ Returned for modification 25 March 2002/ Accepted 16 July 2002

E47 and Pip are proteins crucial for proper B-cell development. E47 and Pip cooperatively bind to adjacent sites in the immunoglobulin kappa chain 3' enhancer and generate a potent transcriptional synergy. We generated protein-DNA computer models to visualize E47 and Pip bound to DNA. These models predict precise interactions between the two proteins. We tested predictions deduced from these models by mutagenesis studies and found evidence for novel direct interactions between the E47 helix-loop-helix domain (Arg 357 or Asp 358) and the Pip N terminus (Leu 24). We also found that precise spatial alignment of the binding sites was necessary for transcriptional synergy and cooperative DNA binding. A Pip dominant negative mutant that cannot synergize with E47 inhibited enhancer activity in plasmacytoma cells and could not activate transcription in pre-B cells. Using electrophoretic mobility shift assays, we found that Pip can bind to the heavy-chain intron enhancer region. In addition, we found that in fibroblasts Pip greatly increased E47 induction of germ line Iµ transcripts associated with somatic rearrangement and isotype class switching. However, a Pip dominant negative mutant inhibited germ line Iµ transcripts. The importance of these results for late B-cell functions is discussed.

* Corresponding author. Mailing address: University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce St., Philadelphia, PA 19104. Phone: (215) 898-6428. Fax: (215) 573-5189. E-mail: atchison{at}vet.upenn.edu.

Molecular and Cellular Biology, October 2002, p. 7337-7350, Vol. 22, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.20.7337-7350.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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