Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive.
In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1] and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin-like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles [1] [2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4] [5]. A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated [6] [7]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8] [9] and references therein) to the H19 DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19 DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.
Kanduri
C
C
Department of Development and Genetics, Uppsala University, Uppsala, S-75236, Sweden.
Pant
V
V
Loukinov
D
D
Pugacheva
E
E
Qi
C F
CF
Wolffe
A
A
Ohlsson
R
R
Lobanenkov
V V
VV
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D000076246:CCCTC-Binding Factor; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005260:Female; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D011485:Protein Binding; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D000076246:CCCTC-Binding Factor; 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