The insulation of genes from external enhancers and silencing chromatin

Abstract

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.

Figure 1

Two kinds of insulator functions. (A) Some insulators may function as barriers against the encroachment of adjacent genomic condensed chromatin. (B) Some insulators may serve as positional enhancer-blocking elements that prevent enhancer action when placed between enhancer and promoter, but not otherwise.

Figure 2

The chicken β-globin locus (Upper) showing the four genes, the strong enhancer between the adult β gene (β^A) and the ɛ gene, and the constitutive HS, 5′HS4. The boundary between the open chromatin domain and the condensed chromatin domain further 5′, as determined by Hebbes et al. (4), is shown (Lower).

Figure 3

(A) Construction used in an assay for enhancer (ENH) blocking activity (2, 3). Expression of a gene conferring G418 resistance (neo) is driven by an erythroid-specific enhancer and promoter. This plasmid is stably transfected into K562 human erythroleukemia cells, and G418-resistant colonies are counted. Typically transfection produces tandem integrants (Control). The test for the putative insulator (I) is to insert it so that it can block enhancer action, and again to count colonies. (B) Results of inserting a 1.2-kb fragment (see Fig. 2) containing 5′HS4 on colony number in the above assay. The control has an approximately equal length of λ phage DNA on either side of the reporter to keep distances constant. The 5′HS4 element strongly reduces enhancer (ENH) activity. Other experiments show that it has a much smaller effect when placed on the other side of the enhancer, confirming the positional enhancer blocking activity (2, 3).

Figure 4

The ends of the open β-globin domain. (A) 3′end. A second constitutive HS that binds CTCF and has enhancer blocking activity is found upstream of the beginning of a condensed chromatin region containing a gene for an odorant receptor (8). (B) 5′ end. A condensed chromatin region extends for about 16 kb upstream of 5′HS4, and beyond that is an erythroid-specific FR gene (9).

Figure 5

The mouse Igf2/H19 imprinted locus. In the maternally transmitted allele, Igf2 is silent, but in the paternal allele it is expressed, and the ICR is methylated. The ICR has been shown to contain four CTCF binding sites, which have strong enhancer blocking properties; enhancer blocking is abolished by methylation of cytosines at CpG sites within the ICR. These and other results lead to a model in which the maternal ICR blocks the action of downstream endodermal enhancers (E) on the Igf2 promoter. Methylation of the paternal ICR abolishes enhancer blocking and permits Igf2 activation (–12).

Figure 6

Constructions to measure protection against position effects (barrier function). A reporter containing a fragment of the IL-2R driven by the chicken adult β-globin promoter and the downstream β/ɛ enhancer (see Fig. 2) is stably transformed into the avian erythroid line 6C2. Expression of IL-2R on the cell surface is monitored by FACS analysis. (Upper) The control construction. (Lower) The control reporter is surrounded by two copies of the 1.2-kb chicken 5′HS4 insulator on each side. In most lines transformed with the control construction, IL-2R expression was extinguished after 80–100 days in culture. Almost all lines carrying the insulated construction still expressed IL-2R after 80–100 days (14).

Figure 7

Chromatin immunoprecipitation of modified histones across the β-globin locus. (Top) Diacetylated histone H3 and tetraacetylated histone H4 in 6C2 cells, arrested at the CFU-E stage of chicken erythroid development. (Middle) Map of the locus showing positions of HSs above the line and of probes used in PCR detection below the line. (Bottom) Patterns of histone H3 methylation at lysines 4 and 9 and diacetylated (lysines 9 and 14) histone H3 across the locus in 10-day embryonic chicken erythrocytes. (Adapted from refs. and .)