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Effects of cis Arrangement of Chromatin Insulators on Enhancer-Blocking Activity

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Science  19 Jan 2001:
Vol. 291, Issue 5503, pp. 493-495
DOI: 10.1126/science.291.5503.493

Abstract

Chromatin boundary elements or insulators are believed to regulate gene activity in complex genetic loci by organizing specialized chromatin structures. Here, we report that the enhancer-blocking activity of the Drosophila suHw insulator is sensitive to insulator copy number and position. Two tandem copies of suHw were ineffective in blocking various enhancers from a downstream promoter. Moreover, an enhancer was blocked more effectively from a promoter by two flanking suHw insulators than by a single intervening one. Thus, insulators may modulate enhancer-promoter interactions by interacting with each other and facilitating the formation of chromatin loop domains.

Insulators regulate gene activity in diverse organisms (1–8). The defining feature of insulators as a class of regulatory elements is their ability to block enhancer-promoter interactions when positioned interveningly. One of the best characterized insulators is suHw, a 340–base pair (bp) element from the Drosophila gypsy retrotransposon. It protects transgenes from chromosomal position effects and blocks various enhancer-promoter interactions (9–13). SUHW, a zinc-finger DNA binding protein, and MOD(MDG4), a BTB domain protein, are essential for suHw function (13–16). Using divergently transcribed reporter genes in transgenic Drosophila embryos, we have shown that an enhancer blocked from the downstream promoter by suHw is fully competent to activate an upstream promoter (12).

To probe the insulator mechanism, we tested the effect of suHw copy number on its insulator strength in Drosophila embryos. The zerknullt enhancer VRE (ventral repression element) has been shown to be partially blocked by suHw (12).In blastoderm embryos, the V2 transgene containing VRE and E2, an evenskipped stripe 2 enhancer, directs reporter expression in a composite pattern of broad dorsal activation and dominant ventral repression of the E2 stripe (Fig. 1, A and D) (13, 17, 18). A single 340-bp suHw insulator element in the VS2 transgene partially blocked the upstream VRE enhancer (Fig. 1, B and D). Two tandem suHw elements (arranged as direct repeats) were inserted between VRE and E2, resulting in VSS2. Instead of enhanced blockage,VSS2 embryos exhibited a loss of suHw insulator activity (Fig. 1, C and D). This was observed in most VSS2 embryos (Fig. 1D) and in all 10 independent VSS2 lines, indicating that it is unlikely to be caused by chromosomal position effects. Genomic polymerase chain reaction (PCR) analysis of independentVS2 and VSS2 lines further verified the structural integrity of the transgenes in vivo (Fig. 1E) (19).

Figure 1

suHw-mediated blockage of the VRE enhancer depends on copy number of the insulator.eve-lacZ reporter expression was detected in transgenic embryos (anterior is left, dorsal is up) (19). In transgene diagrams (not drawn to scale), boxes represent enhancers, ovals represent suHw insulators, and arrows represent theeve-lacZ promoter. (A) A V2 embryo showing composite lacZ pattern consisting of VRE-directed broad dorsal stain and the anterior E2 stripe, repressed in the ventral region. (B) An intervening suHw in VS2greatly reduced the VRE-directed dorsal activation but did not affect ventral repression of E2. (C) Two tandem copies of suHw inVSS2 failed to block VRE-mediated activation, shown by the intense lacZ expression in the dorsal region. (D) Thirty transgenic embryos from three random insertion lines (10 embryos per line) were categorized for blockage of VRE-mediated activation by visual inspection. The most frequently observed staining patterns (asterisks) are those shown in (A) to (C). (E) IndependentVS2 and VSS2 lines (four each) were analyzed by genomic PCR using transgene-specific primers. Expected product sizes for intact transgenes are indicated.

To determine whether the loss of insulator function inVSS2 embryos is enhancer-specific, we constructed transgenes using a rhomboid neuroectodermal enhancer (NEE) and ahairy stripe 1 enhancer (H1) (13). TheNLH embryos containing NEE and H1 enhancers separated by a 1.4-kb neutral spacer (L) exhibited a composite lacZ pattern directed by both enhancers (Fig. 2, A and H). A single suHw element in the NSH transgene blocked the upstream NEE enhancer (Fig. 2, B and H), whereas two tandem suHw elements (NSSH) did not block the NEE enhancer (Fig. 2, C and H). A second group of transgenes uses a twist mesoderm enhancer (PE) and an evenskipped stripe 3 enhancer (E3) (13). Both enhancers are active when separated by the L spacer (PL3) (Fig. 2, D and H). Insertion of a suHw element in the PS3 transgene blocked the upstream PE enhancer (Fig. 2, E and H), whereas two tandem suHw elements (PSS3) did not block the PE enhancer (Fig. 2, F and H). Replacing one of the two suHw elements in PSS3 with a spacer of comparable size (A) restored the enhancer-blocking activity of the remaining suHw inPSA3 embryos (Fig. 2G), indicating that loss of insulator activity with two suHw elements is not due to the spacing change but to the presence of the additional insulator. Genomic PCR with individualNSH, NSSH, PS3, and PSS3lines indicated that the transgenes were structurally intact (Fig. 2I). These results suggest that the loss of insulator activity with tandemly arranged suHw is independent of the enhancer tested.

Figure 2

Loss of insulator activity with tandem suHw is independent of the enhancer tested. (A)NLH embryos exhibit ventrolateral expression directed by NEE and an anterior transverse stripe directed by H1. (B) NEE-directed ventrolateral expression is blocked by suHw in theNSH embryos, whereas H1 expression is unaffected. (C) NEE-directed expression is not blocked by two tandem suHw elements in NSSH embryos. (D) PL3embryos showing PE-directed lacZ expression in the ventral region and an E3-directed mid-embryo stripe. PE expression is more intense in the anterior, possibly as a result of repressors bound to E3. (E) PS3 embryos exhibit greatly reduced PE-directed ventral expression. The E3 stripe was unaffected. (F) Two tandem copies of suHw failed to block the upstream PE enhancer in PSS3 embryos, as shown by the stronglacZ staining in the ventral region. (G) Replacing one of the tandem suHw elements with a 320-bp neutral spacer in PSA3 restored the suHw insulator function, resulting in the blockage of the PE-directed ventral expression, whereas the E3 stripe was unaffected. (H) suHw-mediated blockage of the upstream enhancer was categorized in 30 embryos from three random lines (10 embryos per line) for each transgene. The most frequently observed staining patterns (asterisks) are those shown in (A) to (F). (I) Genomic PCR of NSH, NSSH,PS3, and PSS3 lines (two each) yielded products of the expected size for intact transgenes (sizes indicated).

The enhancer-blocking activity of suHw may require its interaction with other sites (or insulators) within the nucleus. A second suHw nearby may compete dominantly for the existing suHw and affect the neighboring enhancer-promoter interactions, depending on the cis arrangement of these elements. To test this hypothesis, we constructed the SVS2 transgene in which the VRE enhancer is flanked by two suHw elements. In contrast to the loss of insulator function seen in VSS2 embryos, the VRE enhancer is more effectively blocked in SVS2 embryos than inVS2 embryos (Fig. 3, A, B, and D). Thus, it is the tandem arrangement rather than physical proximity that causes the loss of insulator activity. VRE-mediated dorsal activation of the divergently transcribed miniwhite is also diminished in SVS2 embryos (19), indicating that VRE is blocked from promoters on either side. suHw-mediated blockage of VRE is significantly reduced inSVS2/mod(mdg4)u1 embryos (Fig. 3C), indicating that a MOD(MDG4)-mediated complex is required for the enhanced insulator activity (13, 16, 20). VSS2,NSSH, and PSS3 transgenes were also examined in amod(mdg4)u1 background, and no change in the staining patterns was seen (19). The structural integrity of the SVS2 transgenes was confirmed by genomic PCR (Fig. 3E).

Figure 3

Two flanking suHw elements can block VRE more effectively. VRE-mediated dorsal staining is blocked more effectively in SVS2 embryos (A) than inVS2 embryos (B). E2 expression and VRE-mediated repression of E2 are unaffected. (C) suHw insulator activity was reduced in embryos hypomorphic formod(mdg4) u1, shown by the intense dorsal stain. (D) suHw-mediated blockage of VRE was categorized in three random lines of VS2, SVS2, and SVS2/mod(mdg4)u1 embryos. The most frequently observed staining patterns (asterisks) are those shown in (A) to (C). (E) Genomic PCR of threeSVS2 lines yielded products of the expected size for intact transgenes (sizes indicated).

Insulators block enhancer-promoter interactions only when positioned between them. How can this occur without inactivating the enhancer or promoter (12)? It has been hypothesized that insulators may interact with each other to form chromatin loop domains, restricting interactions among neighboring regulatory elements (5, 21, 22). We showed that the enhancer-blocking activity mediated by suHw is abolished when insulators are in tandem, and is enhanced when they flank the enhancer. Thus, suHw does not seem to block distal enhancers by locally capturing the enhancer complex or its associated proteins, because two tandem elements abolished rather than enhanced the insulator function. Instead, a single intervening suHw insulator may interact with other insulators or chromosomal/nuclear sites (Fig. 4A) (21), separating the enhancer and the promoter into topologically distinct chromatin domains. Two tandem suHw elements may preferentially interact with each other, excluding other interactions necessary to sequester the enhancer from the promoter, and may even augment the enhancer-promoter interaction by “looping out” the intervening DNA (Fig. 4B) (23). In contrast, suHw elements flanking an enhancer may readily interact as a result of their proximity, leading to better blockage of the enhancer (Fig. 4C). Loss of insulator function was seen when the distance between the two tandem suHw elements is 50, 150, and 170 bp (VSS2, PSS3, and NSSH, respectively). It has also been observed with spacers ranging from 200 bp to 5 kb in length (23). Therefore, it is unlikely to be caused by nonspecific steric hindrance due to the close juxtaposition of the insulators. DNA looping has been observed between interacting regulatory elements as close as 100 bp apart (24). Insulator assembly may induce alternative chromatin structure, resulting in DNA bending or nuclease-hypersensitive sites, which often indicate nucleosome-free DNA, to facilitate loop formation (4, 25,26). Insulators or chromatin boundaries are frequently found in multiple copies, flanking enhancers or the genetic locus they regulate, such as the scs and scs′ elements, the Mcp-1 and Fab boundaries, and the chicken β-globin 5′ and 3′ boundaries (22, 27–29). Selective interactions between neighboring insulators may regulate the access of tissue-specific enhancers to target promoters by forming alternative chromatin loop domains. It is conceivable that these domains not only block inappropriate enhancers but also facilitate interaction between distant enhancers and the target promoter.

Figure 4

Insulator-mediated loop formation. (A) A suHw insulator (S) may interact with other nuclear sites/insulators (I), separating the enhancer (E) and the promoter (P) into distinct domains and blocking their interaction. (B) Interactions between two tandem suHw insulators fail to sequester the enhancer and may even facilitate enhancer-promoter interaction by “looping out” the intervening DNA. (C) Enhancer blocking may be strengthened by the preferred interactions between two suHw insulators flanking the enhancer.

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