Putting Boundaries on Silence

The functional tests we have used have identified different boundary elements with different functions; however, our testing is incomplete (see Table 1Table 1). Is there a fundamental difference between insulators and barriers, or might they operate by a common mechanism? Can all insulators serve as barriers, and vice versa? Barriers serve as the boundary between heterochromatin and euchromatin, while insulators operate within euchromatic domains. The insulator element by itself does not normally activate or inactivate either the promoter or the enhancer; both can interact with other regulatory elements that are on the same side of the insulator (tested with scs and the gypsy insulator). Thus while these elements insulate enhancer function, there is no indication that they mark the boundary between different modes of chromatin packaging. However, the gypsy insulator, made up of multiple copies of the SU(HW)-binding site, can function as a barrier, blocking the silencing activity of the PRE and protecting a transgene from silencing on insertion into heterochromatin (18xRoseman, R.R, Pirotta, V, and Geyer, P.K. EMBO J. 1993; 12: 435–442PubMedSee all References, 19xSigrist, C.J.A and Pirotta, V. Genetics. 1997; 147: 209–221PubMedSee all References). In contrast, the scs elements do not protect a mini-white transgene from exhibiting a variegating phenotype when resident in the pericentric heterochromatin (Kellum and Schedl 1991xKellum, R and Schedl, P. Cell. 1991; 64: 941–950Abstract | Full Text PDF | PubMedSee all ReferencesKellum and Schedl 1991). Indeed, previous observations showing the spreading of gene silencing over hundreds of kilobases and multiple genes (as seen in PEV) imply that many insulators capable of establishing domains of enhancer function are not able to serve as barriers to heterochromatin spreading. One would like to know whether the barriers to heterochromatin spreading characterized in yeast can serve as insulators, if separated from silencing elements.A distinction between insulators and the boundary elements of the BX-C complex is implied by a recent analysis in which the Fab-7 boundary element was replaced by either the scs or gypsy insulator. Both elements can substitute for the Fab-7 activity, preventing adventitious interaction between the iab-6 and iab-7 regulatory domains; however, the scs or gypsy insulators also blocked interaction of the distal cis-regulatory domains with the Abd-B promoter, with deleterious effect (Mihaly et al. 1998xMihaly, J, Hogga, I, Barges, S, Galloni, M, Mishra, R.K, Hagstrom, K, Muller, M, Schedl, P, Sipos, L, Gausz, J et al. Cell. Mol. Life Sci. 1998; 54: 60–70Crossref | PubMed | Scopus (105)See all ReferencesMihaly et al. 1998). Thus while Fab-7 can act as an insulator in a transgenic construct, it apparently does not have such an effect in its normal context, implying a distinction between these types of boundaries.How do elements that insulate enhancers work? Might they also act by disrupting chromatin structure? The various models fall into two general categories: those involving a local interaction between the proteins of the insulator element and the proteins of the enhancer (or other regulatory element), and those in which insulator activity is coupled to a structural role in higher-order chromatin/nuclear organization (10xKellum, R and Elgin, S.C.R. Curr. Biol. 1998; 8: R521–R524Abstract | Full Text | Full Text PDF | PubMedSee all References, 1xBell, A.C and Felsenfeld, G. Curr. Opin. Genet. Dev. 1999; 9: 191–198Crossref | PubMed | Scopus (186)See all References). At present, no single model satisfies all observations. Additional clues are being derived from genetic approaches. A recent analysis of the Drosophila genes Chip and Nipped-B has suggested that their protein products play a role in the interaction of remote enhancers with their target gene promoters; it has been suggested that SU(HW)-binding sites (gypsy insulator) block enhancer function by disrupting these interactions (Dorsett 1999xDorsett, D. Curr. Opin. Genet. Dev. 1999; 9: 505–514Crossref | PubMed | Scopus (149)See all ReferencesDorsett 1999). If insulators function by disrupting the cis interactions that bring an enhancer to a promoter, might barriers function by disrupting the cis interactions that bring together elements needed to maintain a silent state? Such a model could allow some insulators to function as barriers, and vice versa, without requiring that all do so, depending on the protein complement at a given boundary element. Given the current gaps in our knowledge, any such model will be highly speculative. No doubt further clues to the mechanism of both insulator and barrier activity will be obtained by identifying and characterizing the proteins that interact with these elements (see Table 1Table 1), and analyzing the complexes that they form. Only by establishing the requirements for each activity will we be able to identify the relationships between these different types of boundaries, and understand how the cell maintains its mosaic genome.