The expression of the insulin-like growth factor 2 (Igf2) and H19 genes is imprinted. Although these neighbouring genes share an enhancer, H19 is expressed only from the maternal allele, and Igf2 only from the paternally inherited allele. A region of paternal-specific methylation upstream of H19 appears to be the site of an epigenetic mark that is required for the imprinting of these genes. A deletion within this region results in loss of imprinting of both H19 and Igf2 (ref. 5). Here we show that this methylated region contains an element that blocks enhancer activity. The activity of this element is dependent upon the vertebrate enhancer-blocking protein CTCF. Methylation of CpGs within the CTCF-binding sites eliminates binding of CTCF in vitro, and deletion of these sites results in loss of enhancer-blocking activity in vivo, thereby allowing gene expression. This CTCF-dependent enhancer-blocking element acts as an insulator. We suggest that it controls imprinting of Igf2. The activity of this insulator is restricted to the maternal allele by specific DNA methylation of the paternal allele. Our results reveal that DNA methylation can control gene expression by modulating enhancer access to the gene promoter through regulation of an enhancer boundary.

An insulator is a DNA sequence that can act as a barrier to the influences of neighboring cis-acting elements, preventing gene activation, for example, when located between an enhancer and a promoter. We have identified a 42 bp fragment of the chicken beta-globin insulator that is both necessary and sufficient for enhancer blocking activity in human cells. We show that this sequence is the binding site for CTCF, a previously identified eleven-zinc finger DNA-binding protein that is highly conserved in vertebrates. CTCF sites are present in all of the vertebrate enhancer-blocking elements we have examined. We suggest that directional enhancer blocking by CTCF is a conserved component of gene regulation in vertebrates.

Insulators, first identified in Drosophila, are DNA sequence elements that shield a promoter from nearby regulatory elements. We have previously reported that a DNA sequence at the 5 end of the chicken ␤-globin locus can function as an insulator. It is capable of shielding a reporter gene from the activating effects of a nearby mouse ␤-globin locus control region element in the human erythroleukemic cell line K562. In this report, we show that most of the insulating activity lies in a 250-bp CpG island (core element), which contains the constitutive DNase I-hypersensitive site (5HS4). DNA binding assays with the core sequence reveal a complex protein binding pattern. The insulating activity of the core element is multiplied when tandem copies are used. Although CpG islands are often associated with promoters of housekeeping genes, we find little evidence that the core element is a promoter. Furthermore, the insulator differs from a promoter in its ability to block the locus control region effect directionally.Insulators are DNA sequence elements that block activation of a promoter by an enhancer, but only when placed between them (1). Known insulators include the scs (2, 3) and gypsy (4) elements in Drosophila. We have previously reported that a DNA sequence at the 5Ј end of the chicken ␤-globin locus, containing a constitutive DNase I-hypersensitive site (5ЈHS4), can also function as an insulator (5). When two copies of this 1.2-kb fragment were introduced between a human A ␥-globin promoter and an upstream enhancer͞locus control region (LCR) element from the mouse ␤-globin locus (6-8), the expression of a reporter gene for neomycin resistance was greatly reduced. Other experiments showed that the insulator sequence could shield the white minigene from position effect in Drosophila.The hypersensitive site 5ЈHS4 marks the 5Ј end of the chicken ␤-globin domain (9, 10). Unlike the erythroid-specific hypersensitive sites further 3Ј in the domain, 5ЈHS4 is DNase I-hypersensitive in all tissue types (9). Upstream of 5ЈHS4, there is an abrupt change in higher order chromatin structure in chicken erythroid cells (10). The chromatin within the ␤-globin domain (i.e., downstream of 5ЈHS4) displays the uniform moderate sensitivity to DNase I characteristic of transcriptionally active chromatin domains, whereas the chromatin upstream of 5ЈHS4 is DNase I-resistant and may be thought of as a kind of ''heterochromatic'' region. Consistent with this view, the N-terminal tail of histone H4 within the ␤-globin domain is acetylated at elevated levels when compared with histone H4 in chromatin upstream of the 5ЈHS4 (10). Elevated histone acetylation has been found in transcriptionally active and decondensed regions, such as the active X chromosome, relative to acetylation in transcriptionally inactive and compacted regions, such as the inactive X chromosome (11).In this paper we describe in more detail the properties of the chicken insulator. We show by dissection of the 1.2-kb fragment that much of the activity is containe...