A Dyad Oct-binding Sequence Functions as a Maintenance Sequence for the Unmethylated State within theH19/Igf2-imprinted Control Region

Abstract

DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages. DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages. imprinted control region CCCTC-binding factor dyad Oct-binding sequence dyad octamer motif-like sequence octamer motif-like sequence electrophoretic mobility shift assay octamer-wild type motif Mouse H19 and Igf2 (insulin-like growth factor 2) genes are ∼70 kb distant from each other and are a set of imprinted genes that are reciprocally expressed only from the maternal and paternal allele, respectively, in identical tissues with the same timing. Monoallelic expression of both genes depends on differential methylation of an imprinted control region (ICR),1 located 2–4-kb upstream of the H19 gene, that is hypermethylated in the paternal chromosome but unmethylated in the maternal chromosome. Genomic deletions of the ICR result in activation of the normally silent maternal Igf2 and paternal H19genes in liver, gut, kidney, heart, skeletal muscle, and lung (1Kaffer C.R. Srivastava M. Park K.Y. Ives E. Hsieh S. Batlle J. Grinberg A. Huang S.P. Pfeifer K. Genes Dev. 2000; 14: 1908-1919PubMed Google Scholar, 2Leighton P.A. Ingram R.S. Eggenschwiler J. Efstratiadis A. Tilghman S.M. Nature. 1995; 375: 34-39Crossref PubMed Scopus (683) Google Scholar). Additionally, deletion of endodermal enhancers downstream from theH19 gene results in loss of maternal expression ofH19 and paternal Igf2 in liver, gut, and kidney (3Leighton P.A. Saam J.R. Ingram R.S. Stewart C.L. Tilghman S.M. Genes Dev. 1995; 9: 2079-2089Crossref PubMed Scopus (358) Google Scholar). These results suggest that the paternal ICR repressesH19 expression and that the maternal ICR represses expression of the distant Igf2 through enhancer blocking activity at least in liver, gut and kidney. In fact, recent reports revealed that a 1.2-kb portion of the methylated ICR in the paternal chromosome functioned as a transcriptional silencer in endodermal tissue (4Drewell R.A. Brenton J.D. Ainscough J.F. Barton S.C. Hilton K.J. Arney K.L. Dandolo L. Surani M.A. Development. 2000; 127: 3419-3428PubMed Google Scholar) and that the unmethylated ICR functioned as a CTCF-dependent insulator (1Kaffer C.R. Srivastava M. Park K.Y. Ives E. Hsieh S. Batlle J. Grinberg A. Huang S.P. Pfeifer K. Genes Dev. 2000; 14: 1908-1919PubMed Google Scholar, 5Bell A.C. Felsenfeld G. Nature. 2000; 405: 482-485Crossref PubMed Scopus (1404) Google Scholar, 6Hark A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. Nature. 2000; 405: 486-489Crossref PubMed Scopus (1253) Google Scholar, 7Kanduri C. Pant V. Loukinov D. Pugacheva E., Qi, C.F. Wolffe A. Ohlsson R. Lobanenkov V.V. Curr. Biol. 2000; 10: 853-856Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Methylation of the CTCF-binding site inhibited binding of the CTCF protein (5Bell A.C. Felsenfeld G. Nature. 2000; 405: 482-485Crossref PubMed Scopus (1404) Google Scholar, 6Hark A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. Nature. 2000; 405: 486-489Crossref PubMed Scopus (1253) Google Scholar). Moreover, the binding of CTCF in mouse fetal liver was observed only on the maternal ICR (7Kanduri C. Pant V. Loukinov D. Pugacheva E., Qi, C.F. Wolffe A. Ohlsson R. Lobanenkov V.V. Curr. Biol. 2000; 10: 853-856Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). These observations suggest that the enhancer blocking activity is repressed on the hypermethylated paternal ICR, which enables association between downstream enhancers and theIgf2 promoter. Thus, the methylation status of the ICR is a crucial factor for monoallelic expression in both genes. The unmethylated state of the maternal ICR is established in the germ line and maintained during development (8Tremblay K.D. Saam J.R. Ingram R.S. Tilghman S.M. Bartolomei M.S. Nat. Genet. 1995; 9: 407-413Crossref PubMed Scopus (361) Google Scholar, 9Tremblay K.D. Duran K.L. Bartolomei M.S. Mol. Cell. Biol. 1997; 17: 4322-4329Crossref PubMed Scopus (298) Google Scholar, 10Ueda T. Abe K. Miura A. Yuzuriha M. Zubair M. Noguchi M. Niwa K. Kawase Y. Kono T. Matsuda Y. Fujimoto H. Shibata H. Hayashizaki Y. Sasaki H. Genes. Cells. 2000; 5: 649-659Crossref PubMed Scopus (180) Google Scholar), although de novo methylation is extensively induced in the 3′ portion of the maternal H19 structural gene and in various other genes in blastocysts and postimplantation embryos, respectively (11Brandeis M. Kafri T. Ariel M. Chaillet J.R. McCarrey J. Razin A. Cedar H. EMBO J. 1993; 12: 3669-3677Crossref PubMed Scopus (291) Google Scholar, 12Ferguson-Smith A.C. Sasaki H. Cattanach B.M. Surani M.A. Nature. 1993; 362: 751-755Crossref PubMed Scopus (368) Google Scholar, 13Kafri T. Ariel M. Brandeis M. Shemer R. Urven L. McCarrey J. Cedar H. Razin A. Genes Dev. 1992; 6: 705-714Crossref PubMed Scopus (610) Google Scholar, 14Shemer R. Birger Y. Dean W.L. Reik W. Riggs A.D. Razin A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6371-6376Crossref PubMed Scopus (72) Google Scholar, 15Yoder J.A. Walsh C.P. Bestor T.H. Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1548) Google Scholar). Previous reports showed that a cis-acting sequence within the ICR directs parent-of-origin-specific methylation in the ICR. Maternal transmission of a chromosome containing a 1.6-kb deletion (dDMD) within the ICR results in moderate methylation of the remnant ICR (16Thorvaldsen J.L. Duran K.L. Bartolomei M.S. Genes Dev. 1998; 12: 3693-3702Crossref PubMed Scopus (573) Google Scholar). However, when a 1.2-kb region (dSilencer) that comprises the hypermethylation-dependent silencer within the ICR is deleted, the unmethylated status of the ICR is maintained on the maternal allele (4Drewell R.A. Brenton J.D. Ainscough J.F. Barton S.C. Hilton K.J. Arney K.L. Dandolo L. Surani M.A. Development. 2000; 127: 3419-3428PubMed Google Scholar). These results indicate that the unmethylated state of the maternal ICR is actively maintained during development by a cis-acting sequence existing within an 0.75-kb portion (putative differential methylation control region) that is in the dDMD region but not in the dSilencer region (Fig. 1 a). In this study, to address the cis-acting sequence that is involved in the maintenance of the unmethylated state within the ICR, particularly within the dDMD region, we used a transfection assay system in the mouse embryonal carcinoma (EC) cell line, P19. Because the P19 line was derived from a teratocarcinoma formed by transplantation of a 7.5-day embryo into the testis and exhibits pluripotency of differentiation into neuroectodermal, endodermal, and mesodermal derivatives (17McBurney M.W. Int. J. Dev. Biol. 1993; 37: 135-140PubMed Google Scholar), we used this line as a model for the epiblasts, early postimplantation embryonic cells that undergo genome-wide de novo methylation (reviewed in Ref. 18Li E. Reik W. Surani A. Genomic Imprinting. Oxford University Press, New York1997: 1-20Google Scholar). Using stable transformants with the H19 upstream region, we revealed that a dyad Oct-binding sequence (DOS) in the putative differential methylation control region is involved in demethylation of low-density methylated DNA and that DOS is required to maintain the unmethylated state in both the CTCF-binding site and CpG sequences widely distributed within the ICR. All constructs were cloned into pBluescript II SK(−) (Stratagene). Plasmid pEE was cloned from a mouse genomic library constructed from testis DNA and has a 3.8-kb insert flanked by EcoRI sites. Construct pEEd260 was generated by deletion of a 260-bp AvaII fragment from pEE. Construct pEEm1m2 was generated by ligation of DNA fragments derived from pEE and synthetic oligonucleotides that were used for construction of the bm1m2 fragment (described below). Five fragments within the dDMD region were defined as: fragment a, 360-bpHinfI-HinfI; fragment b, 159-bpHinfI-HinfI; fragment c, 213-bpHinfI-NspI; fragment d, 509-bpNspI-AvaI; fragment e, 338-bpAvaI-HindIII. These fragments were excised from pEE and cloned into the SmaI site of pBluescript II SK(−). The cloned fragments were excised with HindIII andBamHI for further plasmid construction. The mutant series of fragment b was generated by ligation of the mutatedHhaI-HapII fragment, generated by initialized synthetic oligonucleotides, and the flanking fragments on either side. The resulting plasmids, bm1, bm2, and bm1m2, have mutations only at site 1 (m1 in Fig. 3 a), only at site 2 (m2), and at both sites, respectively. Human DOS fragments, hD1 and hD1m, were generated by annealing of synthetic oligonucleotides to form a double-stranded fragment that was cloned. For demethylation assays, five fragments (a–e) from the dDMD region and the mutant series of fragment b were inserted into the blunted HindIII site within the non-CpG island region of H19 (pEdBSl, Fig.1 d, left panel). The H19 fragment was derived from an EcoRI-SalI fragment encompassing the H19 structural gene with a deletion of a 1-kbBamHI fragment. All test constructs were linearized with NaeI and used for transfection or further modification. Test constructs used for demethylation assays were methylated in vitro with the appropriate DNA methylase according to the manufacture's instruction. Constructs pEE and pEEm1m2 were methylated with bacterialHapII methylase (M.HapII) and HhaI methylase (M.HhaI) or SssI methylase (M.SssI) in vitro. In pEE, there are 99 CpG dinucleotides, 8 HhaI sites (GCGC), and 6 HapII sites (CCGG) in the inserted EcoRI fragment derived from theH19 gene (Fig.1 a) and 296 CpG dinucleotides, 32HhaI sites, and 19 HapII sites on the entire plasmid. Construct pEEd260 was methylated with M.HapII and M.HhaI (M.HapII + M.HhaI). Construct pEdBSl and its derivatives were methylated with M.HapII (TaKaRa or New England BioLabs). Methylation reactions were tested with (8–10 unit/μg) HapII and HhaI restriction endonucleases. Plasmids that were methylated completely at the appropriate sites were used for transfection experiments. The mouse embryonal carcinoma cell line P19 was maintained in alpha of with fetal and P19 cells were with test constructs and the plasmid containing the gene under the control of the early at a of by the were with we and the methylation status on constructs in A. R. Cedar H. T. Y. Nat. Genet. 1996; 13: PubMed Scopus Google Scholar). All were for the methylation of we used the HapII of and England England and of Genomic DNA was with appropriate to and by to were with the and to in and DNA at were in at for and in at for were as by E. W. 17: PubMed Scopus Google Scholar). site site site site and were generated by annealing the synthetic sites and sites and sites and sites and and and respectively. a genomic region flanked and sites. a region of the insulator that was generated by annealing the oligonucleotides and The an sequence. For of the CTCF binding we used which binding sites in a genomic region that was excised from the cloned mouse gene with D. J. J. Genes Dev. PubMed Scopus Google and site The binding reactions of and of P19 were in 1 1 of and of fragment b, site 1 and site 2 or 1 1 of and of the at for at or 2 of were to the protein and and were from and respectively. was as R. M.W. 1998; PubMed Scopus Google Scholar). The DNA was DNA and used as a for The DNA region encompassing one of CTCF-binding sites on the pEE was by a on the and the or the to maintain the unmethylated state within the ICR, we a 3.8-kb Fig.1 a) in various of methylation into the embryonal carcinoma cell line P19. pEE of a region of H19 and a portion of the ICR that the entire dDMD region and CTCF-binding sites (Fig. 1 a). which was methylated with in in a methylated state after of (Fig.1 b, transfection of unmethylated pEE, or pEE methylated to a with M.HapII and in in the ICR (Fig. 1 b, the other the region of the when either methylated at or methylated through this (Fig. 1 that the demethylation activity is to low-density methylation within the ICR on but not we that a cis-acting sequence demethylation of low-density methylation within the ICR and the of methylation. the cis-acting we the demethylation of five fragments derived from the dDMD region as in Fig.1 a). fragment was inserted into a non-CpG island sequence (Fig. 1 The were methylated in vitro with M. HapII and used for fragment b, located within the putative differential methylation control region, demethylation of HapII sites the In the other fragments not show demethylation activity (Fig. 1 The demethylation activity of fragment b was with the deletion mutant a 260-bp AvaII fragment, fragment b and a flanking region on each was of this 260-bp sequence in a loss of demethylation activity in the ICR (Fig. 1 These results that the cis-acting in fragment b was required for demethylation of low-density methylation within the ICR. the sequence that directs demethylation within the ICR, we for protein binding sites within the sequence of fragment b and octamer motif-like sequences site and with a sequence 2 a). dyad of with various of sequences was with in the H19 gene and in the H19 gene (Fig. we on dyad octamer motif-like sequences for further the of the in demethylation of the ICR, we mutations into both or one of in fragment b a) and tested the between protein binding to the and demethylation activity of the protein in P19 cells to the was by b formed with in P19 b, 2 and b with a in one or of the and but fragment b with a not the of we demethylation activity of the fragment b derivatives when inserted into non-CpG island DNA as in Fig.1 in Fig. 3 c, the the in a methylated that the demethylation activity by type fragment b was by the and by the mutations and These results revealed that the is the sequence demethylation and that the dyad of binding sites and the binding of were for the the we used against and for R. EMBO J. PubMed Scopus Google Scholar), and which are known to expressed in P19 cells G. A. K. M. K. Development. 1996; PubMed Google Scholar). site 1 and site 2 to both the and Moreover, site 1 and site 2 also formed with other and although we not Because both sites to the mouse dyad octamer motif-like was dyad Oct-binding sequence or DOS. hD1 and are located in the DNA region the CTCF-binding sites upstream of the H19 hD1 has an upstream from the and also the demethylation activity of in a fragment in Fig. 3 a) and with mutations in 3 sites in a). mouse the hD1 region demethylation activity in (Fig. 3 d, and activity in one of d, showed demethylation activity at that hD1 has but demethylation activity in P19 is of the DOS is involved in the demethylation of low-density methylated CpG sequences within the ICR. address this we the Oct-binding sequence mutations into the 3.8-kb pEE (Fig. and transfection assays to demethylation the in a methylated state within the ICR, a of the demethylation activity (Fig. These results that the DOS demethylation of low-density methylation within the ICR. Furthermore, is a the DOS has the to against de novo methylation and for maintenance of the unmethylated state of the ICR. the DOS we unmethylated pEE and pEEm1m2 into P19 cells and experiments. The ICR in pEE type DOS de novo methylation in the in the DOS in de novo methylation at sites within the ICR (Fig. result that the demethylation activity by the DOS is for maintenance of the unmethylated state within the ICR. CTCF-binding sites in the ICR have to an in enhancer blocking activity in an unmethylated (5Bell A.C. Felsenfeld G. Nature. 2000; 405: 482-485Crossref PubMed Scopus (1404) Google Scholar, 6Hark A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. Nature. 2000; 405: 486-489Crossref PubMed Scopus (1253) Google Scholar, 7Kanduri C. Pant V. Loukinov D. Pugacheva E., Qi, C.F. Wolffe A. Ohlsson R. Lobanenkov V.V. Curr. Biol. 2000; 10: 853-856Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). the DOS maintain the unmethylated state of a CTCF-binding site within the ICR, we the methylation status at a CTCF-binding site and the region pEE and Because on the of the binding but not on the CTCF binding A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. Nature. 2000; 405: 486-489Crossref PubMed Scopus (1253) Google Scholar), the methylation status of the in the CTCF binding region was the (Fig. 6 a). were and were methylated at on pEE methylated sites and on pEEm1m2 in from (Fig. 6 there was a that the of CTCF in P19 cells de novo methylation of the CTCF-binding site and that binding of CTCF is for maintenance of the unmethylated state in the CTCF binding address this we the of CTCF protein that to the site by These results revealed that the binding of CTCF protein not maintain the unmethylated state of the site within ICR a DOS. These that the DOS was to maintain the unmethylated state of the CTCF-binding site and that DOS is an cis-acting sequence for the ICR to as an observed demethylation activity that low-density methylation but not hypermethylation within the ICR on in P19 cells (Fig. 1 we not the demethylation activity was or this activity appropriate to an unmethylated state maintenance activity in methylation of the ICR is established in the germ line and maintained in cells throughout embryonic demethylation activity the ICR maintains the unmethylated state of the maternal allele during postimplantation this activity not the hypermethylated paternal ICR. sites are located and upstream of theH19 transcriptional site and to the sites in this These sites various methylation on both during early embryonic development in (8Tremblay K.D. Saam J.R. Ingram R.S. Tilghman S.M. Bartolomei M.S. Nat. Genet. 1995; 9: 407-413Crossref PubMed Scopus (361) Google Scholar, 9Tremblay K.D. Duran K.L. Bartolomei M.S. Mol. Cell. Biol. 1997; 17: 4322-4329Crossref PubMed Scopus (298) Google Scholar, 12Ferguson-Smith A.C. Sasaki H. Cattanach B.M. Surani M.A. Nature. 1993; 362: 751-755Crossref PubMed Scopus (368) Google Scholar, H. A.C. Barton S.C. Surani M.A. Development. 1995; Google Scholar), in to the ICR. The demethylation activity in maintain the state of the These were with the observed demethylation activity in P19 that DOS is required for the demethylation mouse the hD1 region showed demethylation activity for one (Fig. 3 of the of the mouse P19 with the sequence. is that each of the the demethylation activity in in the P19 and maintains the unmethylated state of CTCF-binding sites. expression of in and is with methylation of theH19 upstream region on the maternal allele M.A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, H. C. Y. de A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). However, the of in the methylation is the have not for and of the demethylation activity of on the entire region, and the to the molecular of the maternal methylation. of a CTCF-binding site and the region revealed that DOS maintains the unmethylated state in a (Fig. 6 However, as in Fig. c, the de novo methylation was at sites that were within the to the of cis-acting that the region of ICR we a demethylation activity within the ICR on pEEm1m2 (Fig. reports have that cis-acting with protein binding regulate the unmethylated state of DNA The site D. J. J. Genes Dev. PubMed Scopus Google Scholar, M. D. M. A. V. Razin A. Cedar H. Nature. PubMed Scopus Google Scholar), the enhancer of the A. R. Cedar H. T. Y. Nat. Genet. 1996; 13: PubMed Scopus Google Scholar), and the site C.L. Mol. Cell. Biol. PubMed Scopus Google Scholar) as cis-acting for the maintenance of the unmethylated state within a CpG specific and demethylation of the of the respectively. is the we are of in which the cis-acting sequence is required for low-density the known and sites methylated DNA C.L. Mol. Cell. Biol. PubMed Scopus Google Scholar, M. G. Cedar H. Y. Cell. Full Text PDF PubMed Scopus Google Scholar, J. W. K. 1995; PubMed Scopus Google Scholar). However, we suggest that both the CpG a cis-acting sequence and the binding of the factor to its methylated binding site a cis-acting sequence the methylated Methylation of CpG on pEE completely the ICR from although methylation of the HapII and HhaI sites, to and the was not to the demethylation of the DOS 1 b The Oct-binding sequences not CpG but were by five CpG within 3 a). we that a that the binding of the protein to the DOS and that of demethylation (Fig. cis-acting sequence is also to parent-of-origin-specific differential which directs imprinted at the methylated region 2 of the imprinted insulin-like growth factor 2 The cis-acting the allele novo methylation only on the paternal allele and differential methylation in Y. Shemer R. J. Razin A. Nature. PubMed Scopus Google Scholar). have that DOS is in and is located within the putative differential methylation control region by in on the low-density methylated but not hypermethylated ICR, is required for maintenance of the unmethylated ICR, and through binding of in cells that These results suggest that the DOS on the mouse maternal allele the ICR from de novo to maintain the methylated ICR at least in early postimplantation (Fig. However, de novo methylation in the 3′ portion of the H19 gene in the (11Brandeis M. Kafri T. Ariel M. Chaillet J.R. McCarrey J. Razin A. Cedar H. EMBO J. 1993; 12: 3669-3677Crossref PubMed Scopus (291) Google Scholar, 14Shemer R. Birger Y. Dean W.L. Reik W. Riggs A.D. Razin A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6371-6376Crossref PubMed Scopus (72) Google Scholar), although the maternal ICR de novo methylation activity and unmethylated at this Additionally, after demethylation of the paternal allele in germ the unmethylated state of the ICR is maintained throughout the growth as genome-wide de Hence, the of DOS in both and Thus, is that DOS maintain the state of the ICR against a de novo methylation at stages. The 1.6-kb deletion in study (16Thorvaldsen J.L. Duran K.L. Bartolomei M.S. Genes Dev. 1998; 12: 3693-3702Crossref PubMed Scopus (573) Google Scholar) in methylation of the remnant ICR on the maternal allele and methylation on the paternal allele, the methylation status of the maternal and paternal are DOS. Because the of DOS revealed by the loss of hypermethylation maintenance we that mechanisms of the unmethylated state maintenance mechanisms by DOS. the 1.6-kb deletion is not for the loss of the hypermethylated state maintenance mechanisms hypermethylation of a DNA region a cis-acting for the maintenance of the hypermethylation M.W. Chaillet J.R. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). have also that DOS is required for maintenance of the unmethylated state at one of CTCF-binding sites in the ICR and maintain the unmethylated state of widely distributed within the ICR. These that DOS directs the unmethylated state of CTCF-binding sites within the ICR and that the ICR, as a CTCF-dependent insulator, requires the unmethylated state maintenance sequence in to CTCF-binding sites (Fig. other CTCF-dependent and 1 A.C. Felsenfeld G. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, C.P. J.M. Lobanenkov V.V. Nat. Genet. PubMed Scopus Google Scholar), have to a site that is known as a cis-acting of the unmethylated state within a CpG island D. J. J. Genes Dev. PubMed Scopus Google Scholar, M. D. M. A. V. Razin A. Cedar H. Nature. PubMed Scopus Google Scholar), although the of the site is we suggest that the CTCF-binding site but not for enhancer blocking activity of the CTCF-dependent insulator that on the or by de