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

Abstract

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.

ACKNOWLEDGMENTS

We thank A. Bowman, T. Caspary, M. Cleary, T. Bestor, J. Eggenschwiler, A. Hark, and R. Ingram for helpful discussions of this work. We also especially thank D. Unger for all of the biochemical advice.

S.M.T. was an investigator of Howard Hughes Medical Institute. This work was supported by a grant from the National Institute for General Medical Sciences.