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Molecular and Cellular Biology, May 2005, p. 3855-3863, Vol. 25, No. 10
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.10.3855-3863.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Characterization of a Novel Ku70/80 Pause Site at the H19/Igf2 Imprinting Control Region

David J. Katz, Michael A. Beer, John M. Levorse, and Shirley M. Tilghman*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

Received 24 November 2004/ Returned for modification 15 December 2004/ Accepted 25 February 2005

The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg2+, implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

* Corresponding author. Mailing address: One Nassau Hall, Princeton University, Princeton, NJ 08544-0015. Phone: (609) 258-6100. Fax: (609) 258-1615. E-mail: smt{at}princeton.edu.

Molecular and Cellular Biology, May 2005, p. 3855-3863, Vol. 25, No. 10
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.10.3855-3863.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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