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

Abstract

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...

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

“…However, wellknown insulator-binding protein CTCF is not a likely candidate since this protein is reported to be strongly associated with enhancer-blocking activity, rather than anti-silencing effect in studies using cHS4. 18 One can also attribute the cell type selectivity to the physiological role of insulators, that is, the regulation of stage-specific gene expression by regulating higher chromatin structure. 16,17 Recently, double-stranded DNA-binding protein, SATB1, was reported to play a key role in forming thymocytespecific nuclear architecture.…”

“…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.…”

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis‐regulatory regions in zebrafish

“…The gateway cassette (Invitrogen, Cassette B from Gateway Vector Conversion System) was cloned in blunt into the KpnI restriction site generating the insulator vector pminiTol2-Z48-Gw-CARGFP. Two copies of the ␤-globin 5Ј HS4 250-pb insulator (Recillas-Targa et al, 2002), previously cloned in pBluescript, were amplified with the T3 and T7 primers. The PCR fragment was cloned in TOPO vector (Invitrogen, pCR®8/GW/TOPO® TA cloning KIT) and transferred to the insulator test vector by in vitro recombination (Invitrogen, Gateway LR clonase II Enzyme mix).…”

“…Third, the enhancer reporter cassette in ZED is flanked by insulator sequences that we show decrease position effects, thereby increasing the specificity of the signal. We have used two different insulators, the 5ЈHS4 insulator from the chicken beta-globin gene (Chung et al, 1993;Recillas-Targa et al, 2002) and the GAB insulator element from the mouse tyrosinase gene (Montoliu et al, 1996), which we show here to maintain their insulator function in zebrafish. Also, ZED contains a cardiac actin promoter driving the expression of a red fluorescent protein (DsRed) that serves as control for transgenesis efficiency in vivo in the F0 and the F1 embryos.…”

Integration-free and stable expression of FVIII using a human artificial chromosome

“…Construction of the FVIII-HAC using CHO cells A single copy of the FVIII expression cassette was 9.1 kb and composed of the following DNA elements: the HS4 insulator sequences derived from pCJ5-4 (gifted by Dr G Felsenfeld), 11,12 the cytomegalovirus immediate early enhancer-chicken b-actin hybrid promoter 13 (gifted by Dr M Okabe), a human B-domain deleted factor VIII (FVIII) 14 and a bovine growth hormone poly A signal. FVIII-expression cassettes (1 to 16 copies) were inserted into the modified pPAC4 vector (see Supplementary Figure 1).…”