Developmental Cell 11, 117–124, July, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.devcel.2006.05.009 Short Article PRE-Mediated Bypass of Two Su(Hw) Insulators Targets PcG Proteins to a Downstream Promoter Itys Comet, 1 Ekaterina Savitskaya, 2,3 Bernd Schuettengruber, 1 Nicolas Ne ` gre, 1,4 Sergey Lavrov, 1,5 Aleksander Parshikov, 2 Franc ¸ois Juge, 1,6 Elena Gracheva, 2,7 Pavel Georgiev, 2, * and Giacomo Cavalli 1, * 1 Institute of Human Genetics Centre National de la Recherche Scientifique 141 rue de la Cardonille F-34396 Montpellier Cedex 5 France 2 Department of the Control of Genetic Processes Institute of Gene Biology Russian Academy of Sciences Moscow 119334 Russia Summary Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. We show here that Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and that PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. On the other hand, PRE-mediated silencing can by- pass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream pro- moter, while they are completely excluded from the in- tervening insulated domain. This shows that PRE-de- pendent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter. Introduction Chromatin insulators are thought to regulate gene ex- pression by defining independent chromatin domains (Brasset and Vaury, 2005; Capelson and Corces, 2004; West et al., 2002). These elements are characterized mainly by two activities that have been identified in transgenic assays. First, when an enhancer and its cog- nate promoter are flanked by two insulators, they are isolated from activating or repressing position effects dependent on the chromatin surrounding the transgene (barrier activity). Second, when an insulator is placed between an enhancer and a promoter, it can prevent the enhancer from activating the promoter (enhancer blocking activity). The Drosophila Su(Hw) insulator, from the gypsy retro- transposon, contains 12 binding sites for the Su(Hw) protein and can block enhancer-promoter interactions in a Su(Hw)-dependent manner when inserted between them (Cai and Levine, 1995; Geyer and Corces, 1992; Scott et al., 1999). On the other hand, two intervening Su(Hw) insulators restore enhancer access to down- stream promoters (Cai and Shen, 2001; Muravyova et al., 2001). This ability, which is shared by other insula- tor elements (Gruzdeva et al., 2005; Melnikova et al., 2004), was suggested to depend on pairing of the two in- sulators that might bring the upstream enhancer in the vicinity of the downstream promoter. In addition to blocking enhancers, one copy of the Su(Hw) insulator can also counteract the repressive ef- fect of a Polycomb response element (PRE) (Mallin et al., 1998; Sigrist and Pirrotta, 1997). PREs are bound by proteins of the Polycomb group (PcG), inducing si- lencing of both endogenous target genes and reporter genes (Chan et al., 1994; Dejardin et al., 2005; Sigrist and Pirrotta, 1997). Here, we investigated molecular mechanisms of insulator-dependent PRE blocking, and we analyzed the effect of the presence of two insu- lators on PRE-mediated silencing. Our data show that the spreading of PRE bound proteins into neighboring chromatin domains is blocked by the Su(Hw) insulator. In contrast, two copies of the insulator are bypassed by the PRE, and this bypass involves the establishment of PcG binding at the repressed promoter. These data strongly suggest that insulator pairing induces a contact between PRE bound proteins and the downstream pro- moter, resulting in its silencing. Results Enhancer-Mediated Insulator Bypass in the Presence of a PRE We first asked whether an enhancer can activate its pro- moter across a chromosomal domain maintained in a si- lenced state by a PRE and bordered by two Su(Hw) insu- lators. In the (E)S(P)YSW transposon (Figure 1A), the white minigene and the yellow gene served as reporters responsible, respectively, for red eye pigmentation and for dark pigmentation of the larval and adult cuticle and its derivatives. Silencing was dependent on a PRE from the bxd region of the homeotic gene Ultrabithorax (Ubx)(Sigrist and Pirrotta, 1997) (hereafter, called PRE). The upstream region of the transposon carried the eye- specific enhancer, separated from the white reporter by an intervening domain containing the PRE and the yel- low gene bordered by two Su(Hw) insulators. The eye- specific enhancer was flanked by loxP sites (lox), and the PRE was flanked by frt sites, allowing excision by crossing transgenic flies with flies expressing Cre- or Flp-recombinase (Golic et al., 1997; Siegal and Hartl, *Correspondence: giacomo.cavalli@igh.cnrs.fr (G.C.); georgiev_p@ mail.ru (P.G.) 3 Present address: Centre of Bioengineering, Russian Academy of Sciences, Prosp. 60-let Oktiabria, bld.7-1, Moscow 117312, Russia. 4 Present address: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520. 5 Present address: DMGC, Institute of Molecular Genetics, RAS, 2, Kurchatov sq., Moscow, 123182, Russia. 6 Present address: Institute of Molecular Genetics, CNRS-UMR 5535. 1919, route de Mende, F-34293 Montpellier Cedex 5, France. 7 Present address: Department of Biology, Washington University, St. Louis, Missouri 63130.