Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive

Abstract

In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([ 1 Ohlsson R. Genomic imprinting, an interdisciplinary approach.in: Hennig W. Nover L. Scheer U. Results and Problems in Cell Differentiation, vol 25. Heidelberg, Springer, Berlin, New York 1999 Google Scholar ] 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 Ohlsson R. Genomic imprinting, an interdisciplinary approach.in: Hennig W. Nover L. Scheer U. Results and Problems in Cell Differentiation, vol 25. Heidelberg, Springer, Berlin, New York 1999 Google Scholar , 2 Bartolomei M.S. Tilghman S.M. Genomic imprinting in mammals. Annu Rev Genet. 1997; 31 : 493-525 Crossref PubMed Scopus (478) Google Scholar ]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [ 3 Leighton P. Saam J. Ingram R. Stewart C. Tilghman S. An enhancer deletion affects both H19 and Igf2 expression. Genes Dev. 1995; 9 : 2079-2089 Crossref PubMed Scopus (323) Google Scholar ] from accessing the Igf2 promoters on the maternal chromosome [ 4 Thorvaldsen J.L. Duran K.L. Bartolomei M.S. Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes Dev. 1998; 12 : 3693-3702 Crossref PubMed Scopus (515) Google Scholar , 5 Kanduri C. Holmgren C. Pilartz M. Franklin G. Ullerås E. Kanduri M. et al. The 5′-flank of the murine H19 gene in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication. Curr Biol. 2000; 10 : 449-457 Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar ]. A differentially methylated domain (DMD) in the 5′ flank of H19 is maintained paternally methylated and maternally unmethylated [ 6 Olek A. Walter J. The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet. 1997; 17 : 275-276 Crossref PubMed Scopus (172) Google Scholar , 7 Tremblay K. Duran K. Bartolomei M.S. A 5′ 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development. Mol Cell Biol. 1997; 17 : 4322-4329 Crossref PubMed Scopus (285) Google Scholar ]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([ 8 Filippova G.N. Lindblom A. Meincke L.J. Klenova E.M. Neiman P.E. Collins S.J. et al. A widely expressed transcription factor with multiple DNA sequence specificity, CTCF, is localized at chromosome segment 16q22.1 within one of the smallest regions of overlap for common deletions in breast and prostate cancers. Genes Chromosomes Cancer. 1998; 22 : 26-36 Crossref PubMed Scopus (113) Google Scholar , 9 Awad T.A. Bigler J. Ulmer J.E. Hu Y.J. Moore J.M. Lutz M. et al. Negative transcriptional regulation mediated by thyroid hormone response element 144 requires binding of the multivalent factor CTCF to a novel target DNA sequence. J Biol Chem. 1999; 274 : 27092-27098 Crossref PubMed Scopus (58) Google Scholar ] 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.