Critical DNA binding interactions of the insulator protein CTCF: a small number of zinc fingers mediate strong binding, and a single finger-DNA interaction controls binding at imprinted loci.

The DNA-binding protein CTCF (CCCTC binding factor) mediates enhancer blocking insulation at sites throughout the genome and plays an important role in regulating allele-specific expression at the Igf2/H19 locus and at other imprinted loci. Evidence is also accumulating that CTCF is involved in large scale organization of genomic chromatin. Although CTCF has 11 zinc fingers, we show here that only 4 of these are essential to strong binding and that they recognize a core 12-bp DNA sequence common to most CTCF sites. By deleting individual fingers and mutating individual sites, we determined the orientation of binding. Furthermore, we were able to identify the specific finger and its point of DNA interaction that are responsible for the loss of CTCF binding when CpG residues are methylated in the imprinted Igf2/H19 locus. This single interaction appears to be critical for allele-specific binding and insulation by CTCF.

The insulation of genes from external enhancers and silencing chromatin.

Insulators are DNA sequence elements that can serve in some cases as barriers to protect a gene against the encroachment of adjacent inactive condensed chromatin. Some insulators also can act as blocking elements to protect against the activating influence of distal enhancers associated with other genes. Although most of the insulators identified so far derive from Drosophila, they also are found in vertebrates. An insulator at the 5' end of the chicken beta-globin locus marks a boundary between an open chromatin domain and a region of constitutively condensed chromatin. Detailed analysis of this element shows that it possesses both enhancer blocking activity and the ability to screen reporter genes against position effects. Enhancer blocking is associated with binding of the protein CTCF; sites that bind CTCF are found at other critical points in the genome. Protection against position effects involves other properties that appear to be associated with control of histone acetylation and methylation. Insulators thus are complex elements that can help to preserve the independent function of genes embedded in a genome in which they are surrounded by regulatory signals they must ignore.

Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities.

The 1.2-kb DNA sequence element (5'HS4) at the 5' end of the chicken beta-globin locus has the two defining properties of an insulator: it prevents an "external" enhancer from acting on a promoter when placed between them ("enhancer blocking") and acts as a barrier to chromosomal position effect (CPE) when it surrounds a stably integrated reporter. We previously reported that a single CTCF-binding site in 5'HS4 is necessary and sufficient for enhancer blocking. We show here that a 250-bp "core" element from within 5'HS4 is sufficient to confer protection against silencing of transgenes caused by CPE. Further dissection of the core reveals that 5'HS4 is a compound element in which it is possible to separate enhancer blocking and barrier activities. We demonstrate that full protection against CPE is conferred by mutant 5'HS4 sequences from which the CTCF-binding site has been deleted. In contrast, mutations of four other protein binding sites within 5'HS4 result in varying reductions in the ability to protect against CPE. We find that binding sites for CTCF are neither necessary nor sufficient for protection against CPE. Comparison of the properties of 5'HS4 with those of other CTCF-binding enhancer-blocking elements suggests that CPE protection is associated with maintenance of a high level of histone acetylation near the insulator, conferred by insulator binding-proteins other than CTCF.