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First published online November 21, 2008
doi: 10.1242/10.1242/dev.024554

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The DNA-binding Polycomb-group protein Pleiohomeotic maintains both active and repressed transcriptional states through a single site

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Silencing activities of sequences from even skipped. (Top panel) Range of eye colors exhibited by heterozygous flies of different lines carrying each construct (indicated below both panels) is graphed in colors that approximate the eye color. The extent of each color bar represents the percentage of lines exhibiting that color. (Bottom panel) The percentage of lines showing pairing-sensitive silencing (PSS) is graphed. In both panels, the number of lines analyzed is indicated above each bar. The first two bars in each panel represent transgenes carrying mini-white without a PSS element. These showed no PSS activity, either with or without Glass-binding sites, and the presence of Glass activator binding sites strongly shifted the intensity of eye colors, indicative of increased expression of mini-white. PSS elements and derivatives were each assayed in the context of transgenes with Glass-binding sites (see Results). In addition, PRE300 caused PSS activity in 73% of lines and decreased the average eye color intensity; mutation of the Pho binding site (PRE300Pho) drastically decreased PSS activity (to 11%) and abolished the effect on eye color; mutation of either one, two or all three GAF-binding sites (PRE300gaga123) had a relatively weak effect on both PSS activity and eye color. The 3' region of PRE300 (PRE300-3') harbors most of its PSS activity (69% of lines for PRE300-3' compared to 5% for PRE300-5'), even though binding sites for several known PRE-binding proteins are removed (see Fig. 3). The eve promoter region (PSEpro) also confers PSS activity (53% of lines); combining it with PRE300 (PRE300 + PSEpro) did not significantly change this activity (50%), although it did increase the repression of heterozygous mini-white activity. Mutations in Pho- and GAF-binding sites, and deletions of PRE300, are as indicated in Fig. 3.

View larger version (80K): [in this window] [in a new window]   Fig. 2. The eve locus contains a Pho-dependent PRE. (A,B) Eyes of adult flies closely matched in age (based on body color) and within 14 hours of eclosion (during which time no discernable changes in eye color occurred), either heterozygous (A) or homozygous (B) for transgenic PRE300 in a white mutant background. Note the dramatically lighter color in B, owing to PSS of transgenic mini-white. (C,D) Eyes of pharate adults carrying a homozygous PRE300 transgene in a heterozygote (C; indistinguishable from wild-type eyes in this line) or homozygous (D) pho1 mutant background. Note the slightly darker eye color in D compared with C. (E,F) Eyes of pharate adults carrying a homozygous GFP-RR transgene in a heterozygous (E; indistinguishable from wild-type eyes in this line) or homozygous (F) pho1 mutant background. GFP-RR (described in the Materials and methods) carries the neuronal RP2+a/pCC enhancer and PRE300 (+7.9 to +9.2 kb) (Fujioka et al., 2003). As with PRE300 alone (Fig. 1), GFP-RR transgenic lines showed PSS (64%: 7 out of 11 lines). Note the darker eye color in F. (G-L) Anti-β-gal-stained stage 13 embryos carrying a bxd-Ubx-Z-derived transgene. (G-I) Transgenes (indicated below) inserted at attP-target site 95E5. (G) 500-bxd-Ubx-Z, a non-PRE-containing negative control. (H) PRE300-bxd-Ubx-Z. Note the lack of β-gal expression in the anterior region indicated by the black line, relative to G. (I) PRE300Pho-bxd-Ubx-Z. Note that mutation of the Pho-binding site caused de-repression of β-gal in many cells in the anterior regions, indicated by arrowheads (compare with H). (J-L) PRE300-bxd-Ubx-Z inserted at attP-target site 52D (Bateman et al., 2006). (J) Wild-type background. (K) Pc4 background. (L) ph503 background. Note de-repression of β-gal in the anterior region indicated by the black line in K and L, relative to J. (M-O) Anti-β-gal-stained CNS from stage 15 embryos carrying transgenes (indicated below) inserted at attP-target site 95E5. (M) 500-bxd-Ubx-Z. (N) PRE300-bxd-Ubx-Z. Note the lack of β-gal expression in the anterior region indicated by the black bracket, relative to M. (O) PRE300Pho-bxd-Ubx-Z. Note that mutation of the Pho-binding site caused de-repression of β-gal in many cells in the anterior region indicated by the black bracket, relative to N.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Binding site diagram and sequence of eve PRE300. The line diagram at the top and the sequence below show the locations of consensus binding sites for the known PRE-binding proteins Dsp1 (red), Zeste (blue), Sp1 (green) (Brown et al., 2005), Grh (purple) (Blastyak et al., 2006), and the functional Pho- (black, bold) and GAF-binding sites (black, solid underlines) based on our mutational analysis (see text). PRE300gaga1 (see Fig. 1) has the 5'-most GAF site mutated, whereas PRE300gaga12 has the 5'-most and the 3'-most sites mutated. The extents of the sub-elements PRE300-5' and PRE300-3' are shown as solid lines in the diagram. The sequences used to eliminate the GAF and Pho sites are shown under the main sequence. BsrGI and EcoRI restriction enzyme sites are marked with broken underlines.

View larger version (87K): [in this window] [in a new window]   Fig. 4. Pho is directly involved in maintaining eve CNS expression. (A) GFP expression driven by the eve RR regulatory region in the wandering third instar larval CNS, in a wild-type background. When a region including PRE300 was removed from the RR transgene construct (to generate RN, see text), GFP expression was not maintained to the third larval instar (not shown). (B) Mutation of a single Pho-binding site decreased the intensity and penetrance of GFP expression (sequences shown in Fig. 3). This line is representative of five out of the eight lines with normal embryonic expression; the other three showed essentially complete loss of expression at this stage, similar to F. (C,D) The same GFP-RR transgenic line shown in A in either a doubly homozygous phol81A, pho1 (C) or a homozygous pho1 (D) mutant background. (A-D) Arrows/arrowheads indicate eve-positive aCC/RP2 neurons in two adjacent hemisegments. (B-D) Note that GFP expression is decreased in both aCC and RP2 neurons, and the effect tends to be more pronounced in RP2 neurons. (E,F) Two GFP-RR lines inserted at the same chromosomal location (site 25C) (Bateman et al., 2006) using the RMCE system, one with an unaltered PRE300 (E), the other with the single Pho site mutated (F). Mutation of the Pho site at this chromosomal location abolishes specific expression, as well as increasing background expression throughout the CNS.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Pho binds to the eve locus. (A) Map of the eve locus showing the location of primer sets (numbered black bars) used to analyze Pho binding. The location of the 3' PRE, PRE300, is shown as a yellow box. (B-G) Analysis of Pho binding in wild type (yw) 2-6 hours AED (after egg deposition, B-D) and 7-13 hours AED (E-G) embryos. For 2-6 hours AED embryos, eggs were collected for 4 hours and developed for 2 hours at room temperature; these embryos spanned stages 4-11. For 7-13 hours AED embryos, eggs were collected for 6 hours at room temperature and developed overnight at 17°C; these embryos spanned stages late 11 to early 16. (B,E) ChIP with Pho-specific antiserum (raised in rabbit). (C,F) Negative control: parallel analysis using normal rabbit serum. (D,G) Positive control: parallel PCR reactions using 1/300 of input material. Note that, in addition to positive signals from elsewhere in the locus, some of which change with time, Pho binding is specifically detected at the 3' PRE in both early and later-stage embryos.

View larger version (86K): [in this window] [in a new window]   Fig. 6. The Pho consensus site is required for Pho binding in vivo. ChIP analysis of Pho binding (as in Fig. 5) to transgenes carrying PRE300 (GFP-RR, Fig. 2), either with a normal (A-C) or a mutated (D-F) Pho site, in 0-15 hours AED embryos (eggs were collected for 15 hours at temperature; embryos spanned stages 1 to late 16). The signal from the transgene (`wt t'gene' or `mut t'gen', using a transgene-specific primer set) is compared in each case with that from the endogenous eve 3' PRE (`endo eve', primer set 13 of Fig. 5), as well as to a gene (actin) that Pho is not expected to bind. Note that the Pho signal is reduced when the Pho site is mutated (transgene signal in D versus that in A), relative both to the endogenous eve signal and to the (presumably background) signal from the actin gene.

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