Characterization of the chicken β-globin insulator

Abstract

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...

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…Prior studies on cHS4 insulators showed that deletion of the CTCF binding sites was associated with loss of enhancer-blocking activity, but did not alter the barrier function of the element. 22,46 However, earlier work on the natural cHS4 locus could not readily assess a potential silencing effect of CTCF in addition to its enhancer-blocking activity. 27 Analysis of CTCF in several genomic contexts brought forward its key function in the organization of the chromatin architecture.…”

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

“…2,10,[15][16][17][18][19] Previous reports have shown that HS4 insulator protects transgenes from position effect variegation when introduced into MoMLV-based retroviral vectors. 20,21 Since the HS4 fragment is active in restricted cell types, 21 a quest for alternative insulators is necessary to propagate the use of the insulator in various cell types and tissues.…”

Modifications to the INSM1 promoter to preserve specificity and activity for use in adenoviral gene therapy of neuroendocrine carcinomas

“…15 The chicken bglobin HS4 insulator elements functionally block enhancer elements when positioned between the promoter and enhancer and have been shown to protect tissue-specific or -inducible promoters in adenovirus backbones. 13,15,[29][30][31] Therefore, we wanted to determine if inclusion of the chicken b-globin HS4 insulator element with our promoter construct could eliminate any potential interference encountered by the adenoviral vector. Extreme efforts were made to incorporate a 1.2 kbp HS4 element both upstream and downstream of the INSM1 promoter-luciferase 2 constructs.…”