BIOLOGICAL OVERVIEW

Regulatory elements termed enhancers, or locus control regions are capable of exerting their influence over long distances in an orientation-independent manner to orchestrate the complex gene expression patterns required for embryonic development. How are the effects of enhancers confined to the genes they regulate? In recent years the concept of domain boundaries or insulator elements has developed, based on the genetic properties of several eukaryotic genes. These elements serve to isolate chromosomal regions, confining regulatory effects of enhancers to the genes they are meant to serve. For example, specialized chromatin structures (scs and scs') have been found at outside the coding sequence of the Drosophila hsp70 gene. These sequences are associated with chromatin structures and serve as boundaries that can prevent activation by enhancer elements. Similarly the chicken ßglobin insulator can inhibit activation fo the gamma-globin gene promoter by the ß-globin locus control region. The Drosophila gypsy insulator functions similarly, confering position-independent transcription to genes and preventing activation of promoters by enhancers separated from proximal promoters by insulator elements (Gdula, 1996 and references).

The best characterized system is the gypsy chromatin insulator of Drosophila. Gypsy is an infectious retrovirus consisting of two long terminal repeat regions that function in viral replication and integration and three retroviral genes: gag, pol and env, coding for nucleocapsid, reverse transcriptase and envelope proteins respectively (Song, 1994). The gypsy insulator is located in the 5' transcribed untranslated region of the gypsy element. The unlinked gene, suppressor of Hairy-wing plays an essential role in functioning of the gypsy insulator. SU(HW) binds to the insulator element, interacting directly with an octamer motif present in the gypsy insulator. SU(HW) binds to approximately 200 other sites on Drosophila polytene chromosomes, possibly corresponding to endogenous insulators that play a role in the organization of the genome into higher order chromatin domains that allow for the normal regulation of gene expression.

yellow is one of the genes that has been used in studying the insulator properties of gypsy. yellow is a non-essential gene, lending pigmentation to various embryonic, larval and adult cuticular structures. yellow is regulated by a series of tissue-specific transcriptional enhancers (located in the 5' region of the gene). These enhancers regulate expression in the wings, body cuticle, and larval pigmented structures; a second series of intronic enhancers regulate expression in bristles and tarsal claws (Gdula, 1996 and references).

Insertion of gypsy in the 5' region of yellow inhibits the interaction between the upstream wing and body cuticle enhancers but does not affect the larval enhancer located proximal to the promoter. Mutation of su(Hw) results in a non-functional insulator. In flies with mutated su(Hw), upstream enhancers function even in the presence of insulator sequences between the enhancer and the proximal region of the gene. This suggests strongly that su(Hw) is required for the insulating properties of gypsy.

modifier of mdg4 was discovered during an analysis of mutations that alter the ability of su(Hw) to interfere with enhancer-promoter interactions. Mutations in mod(mdg4) cause an enhancement of the yellow phenotype, resulting in flies in which every cuticular structure of the larva and adult is unpigmented. This phenotype can be interpreted as a consequence of the inactivation of the yellow promoter itself, or inactivation of every enhancer controling yellow expression.

Cloning of mod(mdg4) revealed the structure of the protein for which it codes. MOD(MDG4) protein contains a BTB domain in the amino terminal region, a motif found in GAGA protein (known as Trithorax-like), a protein that plays a role in chromatin remodeling. MOD(MDG4) and SU(HW) demonstrate a direct physical interaction. MOD(MDG4) protein lacks a recognizable DNA binding domain and is unable to bind to insulator sequences of gypsy. Nevertheless MOD(MDG)4 is present on polytene chromosomes, in 2-3 times the number of bands containing SU(HW), suggesting that MOD(MDG4) might also interact with other proteins. In fact, mutation of mod(mdg4) is lethal, suggesting thaat MOD(MDG4) has a broad function. In the absence of MOD(MDG4), gypsy loses its uni-directional effects on distal enhancers and instead silences both upstream and downstream enhancers. Thus the function of MOD(MDG4) appears to be establishment of uni-directionality on the gypsy insulator. A mutated mod(mdg4) does not act as simply as a null allele, but instead causes a variegated phenotype similar to those caused by juxtaposing active genes to heterochromatic sequences. The resemblence of mod(mdg4) mutations to position-effect variegation, suggests that the effects of SU(HW) and MOD(MDG4) are mediated by changes in chromatin and not simply by classical transcription factor mediated changes in gene activation (Gdula, 1996).

There is a paradox with respect to SU(HW) function that begs for a resolution. On one hand the SU(HW)-MOD(MDG4) interaction suggests a chromatin based mechanism of gene activation and silencing similar to that mediated by Trithorax - and Polycomb-group genes. On the other hand, analysis of enhancers insulated from distal promoters by the gypsy element reveals that these enhancers can still function to regulate genes not protected by the insulator (Cai, 1995 and Scott, 1995). Paradoxically, the enhancer regions themselves are still functional, even if they are insulated form certain promoters by the gypsy element. The continued ability of enhancers to function, suggests that enhancers are not subject to chromatin mediated silencing. The resolution of this paradox will come from a biochemical study of the effects of SU(HW) and MOD(MDG4) chromatin structure. The generality of the role of chromatin in boundary element functioning is illustrated by the characterization of another boundary element associated factor, BEAF-32, which binds and contributes to boundary function at the scs' element of the hsp70 gene (Zhao, 1995).

How does the suHw insulator function to block enhancer-promoter interactions? Using divergently transcribed reporter genes in transgenic Drosophila embryos, it has been shown that an enhancer blocked from the downstream promoter by suHw is fully competent to activate an upstream promoter. To probe the insulator mechanism, the effect of suHw copy number on its insulator strength was tested in Drosophila embryos. The zerknullt enhancer VRE (ventral repression element) has been shown to be partially blocked by suHw. 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. A single 340-bp suHw insulator element in the VS2 transgene partially blocks the upstream VRE enhancer. Two tandem suHw elements (arranged as direct repeats) were inserted between VRE and E2, resulting in VSS2. Instead of enhanced blockage, VSS2 embryos exhibit a loss of suHw insulator activity. This was observed in most VSS2 embryos 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 independent VS2 and VSS2 lines further verifies the structural integrity of the transgenes in vivo (Cai, 2001).

To determine whether the loss of insulator function in VSS2 embryos is enhancer-specific, transgenes were constructed using a rhomboid neuroectodermal enhancer (NEE) and a hairy stripe 1 enhancer (H1). The NLH embryos containing NEE and H1 enhancers separated by a 1.4-kb neutral spacer (L) exhibit composite lacZ pattern directed by both enhancers. A single suHw element in the NSH transgene blocks the upstream NEE enhancer, whereas two tandem suHw elements (NSSH) do not block the NEE enhancer. A second group of transgenes uses a twist mesoderm enhancer (PE) and an evenskipped stripe 3 enhancer (E3). Both enhancers are active when separated by the L spacer (PL3). Insertion of a suHw element in the PS3 transgene blocks the upstream PE enhancer, whereas two tandem suHw elements do not block the PE enhancer. Replacing one of the two suHw elements in PSS3 with a spacer of comparable size (A) restores the enhancer-blocking activity of the remaining suHw in PSA3 embryos, 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. These results suggest that the loss of insulator activity with tandemly arranged suHw is independent of the enhancer tested (Cai, 2001).

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, the SVS2 transgene was constructed 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 in VS2 embryos. 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, indicating that VRE is blocked from promoters on either side. suHw-mediated blockage of VRE is significantly reduced in SVS2/mod(mdg4)^u1 embryos, indicating that a MOD(MDG4)-mediated complex is required for the enhanced insulator activity. VSS2, NSSH, and PSS3 transgenes were also examined in a mod(mdg4)^u1 background, and no change in the staining patterns was seen (Cai, 2001).

Insulators block enhancer-promoter interactions only when positioned between them. How can this occur without inactivating the enhancer or promoter? It has been hypothesized that insulators may interact with each other to form chromatin loop domains, restricting interactions among neighboring regulatory elements. 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 abolish rather than enhance the insulator function. Instead, a single intervening suHw insulator may interact with other insulators or chromosomal/nuclear sites, 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. In contrast, suHw elements flanking an enhancer may readily interact as a result of their proximity, leading to better blockage of the enhancer. 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. 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. 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. 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 ß-globin 5' and 3' boundaries. 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 (Cai, 2001).

GENE STRUCTURE

Exons - 7

PROTEIN STRUCTURE

Amino Acids - 944

Structural Domains

SU(HW) contains 12 repeats of the C2H2 zinc finger motif. These zinc finger domains are located in the central portion of the protein. In addition, a highly acidic region, containing 48% Asp and Glu, is present between amino acids 154 and 202, immediately proceeding the first Zn finger motif (Parkhurst, 1988). A leucine zipper domain is found between the zinc finger domains and the C-terminal end of the protein (Harrison, 1993).

It is thought that su(Hw) forms discrete domains of gene activity by segregating promoters from enhancer elements through a change in chromatin organization. Functional domains of the su(Hw) protein were characterized that mediate the silencing effect of mod(mdg4) mutations. Two of three regions of su(Hw), regions B and C, that are located between the leucine zipper motif and the C-terminal acidic domain are conserved across Drosophila species and are necessary for both the unidirectional and bidirectional repression of transcription by su(Hw). These domains are implicated in an interaction with mod(mdg4) which is thought to mediate the unidirectional repression due to insulator function. In contrast, two acidic domains, the N-terminal acidic domain and the C-terminal acidic domain, dispensable for the unidirectional repression of enhancer elements, are critical for the bidirectional silencing of enhancer activity observed in mutants lacking functional mod(mdg4) protein. Bidirectional repression is thought to be due to changes in large blocks of chromatin structure (Gdula, 1997).

date revised: 10 May 98

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