CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.
The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.
Hark
A T
AT
Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
Schoenherr
C J
CJ
Katz
D J
DJ
Ingram
R S
RS
Levorse
J M
JM
Tilghman
S M
SM
D000818:Animals; D000076246:CCCTC-Binding Factor; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D018392:Genomic Imprinting; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D009124:Muscle Proteins / Q000235:genetics*; D011485:Protein Binding; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D014157:Transcription Factors / Q000502:physiology*; D016335:Zinc Fingers
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D004268:DNA-Binding Proteins; C094335:H19 long non-coding RNA; D009124:Muscle Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; D014157:Transcription Factors; D007335:Insulin-Like Growth Factor II; D004247:DNA