The 1.2-kb DNA sequence element (5HS4) at the 5 end of the chicken ␤-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 5HS4 is necessary and sufficient for enhancer blocking. We show here that a 250-bp ''core'' element from within 5HS4 is sufficient to confer protection against silencing of transgenes caused by CPE. Further dissection of the core reveals that 5HS4 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 5HS4 sequences from which the CTCF-binding site has been deleted. In contrast, mutations of four other protein binding sites within 5HS4 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 5HS4 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.CTCF ͉ chromatin domain ͉ boundaries I nsulators are DNA sequence elements that protect transcribed regions from outside regulatory influences. They are present near chromatin domain boundaries or at sites where they prevent inappropriate activation of a promoter by a nearby heterologous enhancer. A growing number of insulators with varied binding sequences and associated proteins have been found in Drosophila, and a few have been found in vertebrates (1-3). Some time ago we identified, at the 5Ј end of the chicken ␤-globin locus, an element (5ЈHS4) with the characteristic properties of an insulator (4). We applied two defining tests for insulator activity. In the first, the ''enhancer-blocking'' assay, we placed a 1.2-kb DNA sequence element containing 5ЈHS4 between an enhancer and promoter and showed that the action of the enhancer was impeded (4-6). This impedance did not occur when the insulator was inserted elsewhere. In the second assay, we showed that when double copies of this 1.2-kb sequence were placed on either side of an erythroid-specific reporter and stably integrated into an erythroid cell line, the expression of the reporter was uniform from one line to another (7). Expression was maintained after 80 days of incubation in the absence of selection. In contrast, expression was variable among uninsulated lines, and in most cases expression of uninsulated lines was extinguished in far less than 80 days. We concluded that this variability of expression and rapid extinction were manifestations of chromosomal position effects arising from the influence on the reporter of dominant regulatory elements flanking the sites of inte...

The constitutive DNase I hypersensitive site at the 5 end of the chicken ␤-globin locus marks the boundary of the active chromatin domain in erythroid cells. The DNA sequence containing this site has the properties of an insulator, as shown by its ability in stable transformation experiments to block enhancer-promoter interaction when it lies between the two, but not when it lies outside, and to protect against position effects in Drosophila. We now show that the chicken insulator can protect a stably integrated gene, which is otherwise subject to great variability of expression, from chromatin-mediated repression in cell culture. When the integrated reporter gene is surrounded by insulator elements, stably transformed cell lines display consistent enhancer-dependent expression levels, in accord with the strength of the enhancer. In the absence of insulators, long-term nonselective propagation of cells carrying the integrated reporter gene results in gradual extinction of the reporter's expression, with expression patterns from tandemly repeated inserted genes suggesting that the extinction of adjacent genes is coupled. We show that the uninsulated reporter genes, in addition to becoming transcriptionally inactive, lose several epigenetic hallmarks of active chromatin, including nuclease accessibility, DNA hypomethylation, and histone hyperacetylation during time in culture. Treatment with inhibitors of histone deacetylase or DNA methylation reverses the extinction of the uninsulated genes. Extinction is completely prevented by flanking the reporter construct with insulators. Furthermore, in contrast to the uninsulated reporter genes, chromatin over the insulated genes retains nuclease accessibility and histone hyperacetylation. However, there is no clear correlation between the presence of the insulators and the level of DNA methylation. This leads us to propose a model for the insulator's ability to protect against extinction in the transformed cell lines and to function as a chromatin boundary for the chicken ␤-globin locus in normal erythroid cells.

We have studied developmentally regulated patterns of histone acetylation at high resolution across approximately 54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken beta-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5' insulator element separating the globin locus from the folate receptor region, which suggests that this insulator element may harbor a high concentration of histone acetylases.

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo.