CTCF binding sites promote transcription initiation and prevent DNA methylation on the maternal allele at the imprinted H19/Igf2 locus.
Imprinting at the H19/Igf2 locus depends on a differentially methylated domain (DMD) acting as a maternal-specific, methylation-sensitive insulator and a paternal-specific locus of hypermethylation. Four repeats in the DMD bind CTCF on the maternal allele and have been proposed to recruit methylation on the paternal allele. We deleted the four repeats and assayed the effects of the mutation at the endogenous locus. The H19DMD-DeltaR allele can successfully acquire methylation during spermatogenesis and silence paternal H19, indicating that these paternal-specific functions are independent of the CTCF binding sites. Maternal inheritance of the mutations leads to biallelic Igf2 expression, consistent with the loss of a functional insulator. Additionally, we uncovered two previously undescribed roles for the CTCF binding sites. On the mutant allele, H19 RNA is barely detectable in 6.5 d.p.c. embryos and 9.5 d.p.c. placenta, for the first time identifying the repeats as the elements responsible for initiating H19 transcription. Furthermore, methylation is abruptly acquired on the mutant maternal allele after implantation, a time when the embryo is undergoing genome-wide de novo methylation. Together, these experiments show that in addition to being essential for a functional insulator, the CTCF repeats facilitate initiation of H19 expression in the early embryo and are required to maintain the hypomethylated state of the entire DMD.
Engel
Nora
N
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Thorvaldsen
Joanne L
JL
Bartolomei
Marisa S
MS
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D000076246:CCCTC-Binding Factor; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D018392:Genomic Imprinting; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008954:Models, Biological; D011247:Pregnancy; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism*; D014158:Transcription, Genetic
D000076246:CCCTC-Binding Factor; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; C094335:H19 long non-coding RNA; C513121:IGF2 protein, mouse; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; D007335:Insulin-Like Growth Factor II; D004247:DNA