Modulation of a Transcription Factor Counteracts Heterochromatic Gene Silencing in Drosophila  Kami Ahmad, Steven Henikoff  Cell  Volume 104, Issue 6, Pages 839-847 (March 2001) DOI: 10.1016/S0092-8674(01)00281-1

Figure 1 Rationale for Probing Heterochromatin with GAL4 Activator The target transposon carries two reporter genes (green and red lines), and shows their respective transcripts (thin lines). GAL4 is produced from a separate transposon source. The control target insertion is located in euchromatin, and can be activated by GAL4. Variegator derivatives of this insertion are juxtaposed to heterochromatin (red wavy line). (B) Relative timing of activation by GAL4 driver lines. The top line indicates the developmental stages of Drosophila development (e: embryos, L1-L3: larval stages, and P: pupal stages), and the lower lines the expression profile of each driver Cell 2001 104, 839-847DOI: (10.1016/S0092-8674(01)00281-1)

Figure 2 Derepression of UASGFP Variegation in the x21 Variegator Lines Depends on the Timing of the GAL4 Driver Eye-antennal discs from L3 larvae are oriented with the anterior and mitotically active region on the right, and the posterior differentiating region on the left. Arrowheads mark the position of the morphogenetic furrow. GFP fluorescence is in green, and the counterstained tissue is in gray Cell 2001 104, 839-847DOI: (10.1016/S0092-8674(01)00281-1)

Figure 3 A Weak GAL4 Driver Fails to Derepress UASGFP Variegation Each target line (euchromatic control or variegator) was crossed to the strong A5CGAL or to the weak armGAL driver, and eye discs were examined from L3 larvae Cell 2001 104, 839-847DOI: (10.1016/S0092-8674(01)00281-1)

Figure 4 New Derepressed Cells Appear in Cultured Discs Eye imaginal discs from larvae carrying the GawBT80 driver and the x21 variegator were photographed immediately after dissection (A) and 24 hr later (B). A merge of phase contrast images (gray) and GFP fluorescence (green) of the disc at each time point is shown. (C and D) Magnifications of the bracketed regions in (A) and (B). Only the GFP channel is shown. New GFP+spots have appeared in the later time point (D) Cell 2001 104, 839-847DOI: (10.1016/S0092-8674(01)00281-1)

Figure 5 GAL4 Binding at a UAS Counteracts Silencing at a Nearby mini-white Gene Each set of heads carries the indicated target chromosome (control or a variegator). Flies in each row are male siblings from a cross that either carry the indicated GAL4 source (“+” = A5CGAL or GMRGAL) or a dominantly marked homolog (“−”). The last pair of heads from the x18.4.1 variegator line also carry a dominant suppressor of silencing, Su(var)20502 Cell 2001 104, 839-847DOI: (10.1016/S0092-8674(01)00281-1)

Figure 6 Uncoupled Expression of UASGFP and mini-white in Variegator Lines Images of an eye from an adult fly carrying (A) x21 and A5CGAL (B) x18.4.1 and GMRGAL. Light microscope images of the eyes (left), and epifluorescent images (middle and right) are shown. In the epifluorescent images, pigment of the eye is in red and in the merged images, GFP is in green. The red pigments of the eye obscure GFP fluorescence where they coincide. The lines mark patches where mini-white is silenced but UASGFP is expressed Cell 2001 104, 839-847DOI: (10.1016/S0092-8674(01)00281-1)

Figure 7 The Site Exposure Model for Variegated Silencing (adapted from Widom, 1999) When DNA containing a factor binding site (black) is wrapped around a nucleosome (gray), a single factor site is inaccessible (the N conformation). Transient unwrapping of the DNA exposes the site (conformation N′), which can then be bound by its cognate factor (F) (giving conformation N′F). N and N′F correspond to the silent and active states, respectively. The production of the N and N′F conformations is determined by the relative reaction rates (arrows) involving the common substrate N′. In euchromatin, k1 weakly favors the N conformation, but k2 is large, and the production of N′F is strongly favored. We postulate that in heterochromatin, k2 remains large, but k1 has increased. Thus, both N and N′F conformations will be frequently produced, resulting in mosaic expression Cell 2001 104, 839-847DOI: (10.1016/S0092-8674(01)00281-1)