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Insulators Targets Pcg Proteins to a Downstream Promoter

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Insulators Targets Pcg Proteins to a Downstream Promoter Developmental Cell 11, 117–124, July, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.devcel.2006.05.009 PRE-Mediated Bypass of Two Short Article Su(Hw) Insulators Targets PcG Proteins to a Downstream Promoter Itys Comet,1 Ekaterina Savitskaya,2,3 dependent on the chromatin surrounding the transgene Bernd Schuettengruber,1 Nicolas Ne`gre,1,4 (barrier activity). Second, when an insulator is placed Sergey Lavrov,1,5 Aleksander Parshikov,2 between an enhancer and a promoter, it can prevent Franc¸ois Juge,1,6 Elena Gracheva,2,7 the enhancer from activating the promoter (enhancer Pavel Georgiev,2,* and Giacomo Cavalli1,* blocking activity). 1 Institute of Human Genetics The Drosophila Su(Hw) insulator, from the gypsy retro- Centre National de la Recherche Scientifique transposon, contains 12 binding sites for the Su(Hw) 141 rue de la Cardonille protein and can block enhancer-promoter interactions F-34396 Montpellier Cedex 5 in a Su(Hw)-dependent manner when inserted between France them (Cai and Levine, 1995; Geyer and Corces, 1992; 2 Department of the Control of Genetic Processes Scott et al., 1999). On the other hand, two intervening Institute of Gene Biology Su(Hw) insulators restore enhancer access to down- Russian Academy of Sciences stream promoters (Cai and Shen, 2001; Muravyova Moscow 119334 et al., 2001). This ability, which is shared by other insula- Russia 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 Summary vicinity of the downstream promoter. In addition to blocking enhancers, one copy of the Drosophila Polycomb group response elements (PRE) Su(Hw) insulator can also counteract the repressive ef- silence neighboring genes, but silencing can be fect of a Polycomb response element (PRE) (Mallin blocked by one copy of the Su(Hw) insulator element. et al., 1998; Sigrist and Pirrotta, 1997). PREs are bound We show here that Polycomb group (PcG) proteins can by proteins of the Polycomb group (PcG), inducing si- spread from a PRE in the flanking chromatin region lencing of both endogenous target genes and reporter and that PRE blocking depends on a physical barrier genes (Chan et al., 1994; Dejardin et al., 2005; Sigrist established by the insulator to PcG protein spreading. and Pirrotta, 1997). Here, we investigated molecular On the other hand, PRE-mediated silencing can by- mechanisms of insulator-dependent PRE blocking, pass two Su(Hw) insulators to repress a downstream and we analyzed the effect of the presence of two insu- reporter gene. Strikingly, insulator bypass involves lators on PRE-mediated silencing. Our data show that targeting of PcG proteins to the downstream pro- the spreading of PRE bound proteins into neighboring moter, while they are completely excluded from the in- chromatin domains is blocked by the Su(Hw) insulator. tervening insulated domain. This shows that PRE-de- In contrast, two copies of the insulator are bypassed pendent silencing is compatible with looping of the by the PRE, and this bypass involves the establishment PRE in order to bring PcG proteins in contact with of PcG binding at the repressed promoter. These data the promoter and does not require the coating of the strongly suggest that insulator pairing induces a contact whole chromatin domain between PRE and promoter. between PRE bound proteins and the downstream pro- moter, resulting in its silencing. Introduction Results Chromatin insulators are thought to regulate gene ex- Enhancer-Mediated Insulator Bypass pression by defining independent chromatin domains in the Presence of a PRE (Brasset and Vaury, 2005; Capelson and Corces, 2004; We first asked whether an enhancer can activate its pro- West et al., 2002). These elements are characterized moter across a chromosomal domain maintained in a si- mainly by two activities that have been identified in lenced state by a PRE and bordered by two Su(Hw) insu- transgenic assays. First, when an enhancer and its cog- lators. In the (E)S(P)YSW transposon (Figure 1A), the nate promoter are flanked by two insulators, they are white minigene and the yellow gene served as reporters isolated from activating or repressing position effects 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 *Correspondence: [email protected] (G.C.); georgiev_p@ from the bxd region of the homeotic gene Ultrabithorax mail.ru (P.G.) 3 Present address: Centre of Bioengineering, Russian Academy of (Ubx)(Sigrist and Pirrotta, 1997) (hereafter, called PRE). Sciences, Prosp. 60-let Oktiabria, bld.7-1, Moscow 117312, Russia. The upstream region of the transposon carried the eye- 4 Present address: Department of Genetics, Yale University School specific enhancer, separated from the white reporter by of Medicine, New Haven, Connecticut 06520. an intervening domain containing the PRE and the yel- 5 Present address: DMGC, Institute of Molecular Genetics, RAS, 2, low gene bordered by two Su(Hw) insulators. The eye- Kurchatov sq., Moscow, 123182, Russia. specific enhancer was flanked by loxP sites (lox), and 6 Present address: Institute of Molecular Genetics, CNRS-UMR 5535. 1919, route de Mende, F-34293 Montpellier Cedex 5, France. the PRE was flanked by frt sites, allowing excision by 7 Present address: Department of Biology, Washington University, crossing transgenic flies with flies expressing Cre- or St. Louis, Missouri 63130. Flp-recombinase (Golic et al., 1997; Siegal and Hartl, Developmental Cell 118 that could be excised are indicated between brackets in all constructs throughout the manuscript). In five lines with single copy insertions at independent genomic sites, the PRE silenced yellow as excision of the PRE induced yellow derepression (Figure 1A). In contrast, white was not repressed in four of these lines since the eye color ranged from brown to red and was generally not influenced by excision of the PRE, al- though eye pigmentation was reduced after excision of the eye enhancer. These data show that the eye-specific enhancer can activate white, bypassing a PRE-contain- ing domain bordered by two Su(Hw) insulators. We note here a slight increase in eye pigmentation upon PRE ex- cision in the I-3 and the III-1 lines. This might suggest a partial ability of the PRE to overcome the Su(Hw) insu- lator in these lines, consistent with previous reports of partial enhancer blocking by chromatin insulators (Hag- strom et al., 1996; Savitskaya et al., 2006). In the four lines showing insulator bypass, strong re- duction in eye pigment levels was observed and eye color variegation appeared in a su(Hw) mutant back- ground (data not shown). These characteristic features of PRE-mediated silencing indicated that the PRE gained the ability to repress white when the insulator was neutralized. Taken together, these results suggest that an insulated PRE is unable to affect insulator by- pass by capturing an enhancer on its way to the down- stream promoter. PRE-Mediated Insulator Bypass We next tested whether PRE-mediated silencing can by- pass a domain bordered by two Su(Hw) insulators. The transposons PSYSW, (P)(S)YSW, and (P)SY(S)W (Fig- ures 1B–1D) carry in their upstream regions the yellow body- and wing-specific enhancers and the PRE. These elements are followed by a domain in which two Su(Hw) insulators border the bristle-specific enhancer and the yellow coding sequence and, further downstream, by the white reporter. The phenotypes of PSYSW, (P)(S)YSW, and (P)SY(S)W were similar, suggesting that the frt or loxP sequences (absent in the PSYSW transposon) did not affect any functional element. In all transgenic lines, the yellow gene was expressed at Figure 1. Transposons Used in This Study and Phenotypic Analysis high levels by the bristle enhancer (Figures 1B–1D) of Transgenic Lines and, as expected, was only weakly expressed in the (A–D) (A) (E)S(P)YSW, (B) PSYSW, (C) (P)(S)YSW, (D) (P)SY(S)W. body and the wing, as the proximal insulator separates Brackets indicate elements that were excised by using Flp- or Cre- the yellow coding sequence from the body and wing en- recombinase. The transgene maps, not drawn to scale, indicate the yellow promoter and coding region (y), the yellow body (Eb), hancers. In contrast, the eye pigmentation was strongly wing (Ew) and bristle enhancer (Ebr), the white minigene (w), the reduced, ranging from complete white to pale yellow in eye-specific enhancer (Ee), the PRE, and the Su(Hw) insulators (S). most lines (Figures 1B–1D), suggesting that the PRE The P element ends, the frt, and lox sites are indicated by empty tri- strongly repressed white across the two Su(Hw) insula- angles, gray triangles, and gray lozenges, respectively. The trans- tors. Some of these lines showed variegation and in- genic line name and the derivative lines obtained after excision of creased silencing in the homozygous state, typical fea- PRE, enhancer, or insulator (DPRE, DEe, DS, respectively) are indi- cated in (A), (C), and (D). Phenotypic data are schematically repre- tures of PRE-mediated silencing (Kassis, 2002). Other sented above each construct. The number of lines showing the lines escaped silencing, but the frequency of strongly re- same phenotype is indicated in (B). For each line, pigmentation pressed lines was over 50%, similar to previous reports levels reflecting expression of the yellow (in the bristles) or the white of PRE-mediated silencing of white-containing trans- (eye color) reporters are indicated by boxes with a five level scale. genes (Chan et al., 1994; Sigrist and Pirrotta, 1997). White indicates no expression, and black indicates maximal expres- The excision of the PRE strongly derepressed white sion.
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