Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region.
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 Mg(2+), 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.
Katz
David J
DJ
Department of Molecular Biology, One Nassau Hall, Princeton University, Princeton, NJ 08544-0015, USA.
Beer
Michael A
MA
Levorse
John M
JM
Tilghman
Shirley M
SM
D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D016678:Genome; D018392:Genomic Imprinting / Q000235:genetics*; D023281:Genomics; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D000072200:Ku Autoantigen; D008297:Male; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013379:Substrate Specificity
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C094335:H19 long non-coding RNA; D046912:Multiprotein Complexes; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D007335:Insulin-Like Growth Factor II; D004247:DNA; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen