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, Shirley M. Tilghman

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)3855-3863
Number of pages9
JournalMolecular and cellular biology
Volume25
Issue number10
DOIs
StatePublished - May 2005
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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