title: Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance.
abstract: The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.
authors.affiliation: Department of Development and Genetics, Evolution Biology Centre, Uppsala University, S-752 36 Uppsala, Sweden.
authors.forename: Vinod
authors.initials: V
authors.lastname: Pant
authors.forename: Sreenivasulu
authors.initials: S
authors.lastname: Kurukuti
authors.forename: Elena
authors.initials: E
authors.lastname: Pugacheva
authors.forename: Shaharum
authors.initials: S
authors.lastname: Shamsuddin
authors.forename: Piero
authors.initials: P
authors.lastname: Mariano
authors.forename: Rainer
authors.initials: R
authors.lastname: Renkawitz
authors.forename: Elena
authors.initials: E
authors.lastname: Klenova
authors.forename: Victor
authors.initials: V
authors.lastname: Lobanenkov
authors.forename: Rolf
authors.initials: R
authors.lastname: Ohlsson
mesh_terms: D000818:Animals; D000076246:CCCTC-Binding Factor; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D018392:Genomic Imprinting; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008745:Methylation; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000076246:CCCTC-Binding Factor; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; D007335:Insulin-Like Growth Factor II