(A) Sites of mutations in Su(Tpl) alleles. Mutational lesions are indicated in red above the wild-type amino acid sequence of dELL at the site of the mutation. For frameshift alleles, the resulting out-of-frame peptide is also shown. (B) Effects of Su(Tpl) alleles on sev-Ras1^v12 (top) and GMR-yan^ACT (bottom). The dELL polypeptide is represented by a horizontal bar. Filled intervals represent conserved homology to the N-terminal ELL elongation domain and the C-terminal ZO-1 occludin binding domain. Alleles with an enhancer effect are shown in red, alleles with a suppressor effect are shown in blue, and alleles with no apparent effect are shown in black.

dELL(Δ1-189) is expressed and localized to the nucleus in Drosophila embryos. (A) Transgenic embryos expressing dELL(Δ1-189) or nontransgenic Oregon R embryos were fixed and stained with anti-six-His and DAPI (4′,6′-diamidino-2-phenylindole). Staining was visualized by fluorescent microscopy. (B) To confirm nuclear localization, embryos were fixed and immunostained with anti-six-His serum and anti-Pol IIo serum (H14; Covance). Merging of these images confirms that the dELL(Δ1-189) is a nuclear protein, since it colocalizes extensively with Pol IIo.

Transgene rescue of Su(Tpl) lethality. Rescue of flies carrying two different heteroallelic combinations of Su(Tpl) mutants was measured using dELL wild-type and mutant transgenes. Percent viability is computed as the percentage of expected Mendelian survival. Results for three independent transgene inserts are shown for the dELL(Δ1-189) construct. For each genotype, the number of flies scored ranged from 199 to 1,559. The upper 95% confidence interval is indicated above each bar.

The N-terminal domain of dELL is necessary and sufficient for localization to chromatin in vivo. (A) Schematic of dELL wild-type and deletion transgenes used in this study. Yellow indicates the conserved N-terminal domain, and red indicates the C-terminal domain. At top is a schematic of the mammalian ELL protein (Hs ELL), with blocks depicting the characterized domains in the protein sequence as follows: A, N-terminal Pol II binding and elongation activation domain; B, lysine-rich region; C, occludin homology domain. (B) Recombinant dELL mutants were expressed in E. coli and analyzed by Western blotting with anti-dELL polyclonal serum to confirm predicted molecular sizes: dELL(1-262), 42 kDa; dELL(Δ1-189), 95 kDa; dELL(Δ190-759), 56 kDa; and dELL(Δ760-1059), 80 kDa. (C) Immunolocalization pattern for dELL mutants on polytene chromosomes. dELL(Δ1-189) fails to localize to chromosomes in vivo (compare to nontransgenic OreR), while dELL(1-262) exhibits a localization similar to full-length dELL and both dELL(Δ760-1059) and dELL(Δ190-759). Landmark sites on the X (3C and 8EF), second(25BC), and third (68C) chromosomes are indicated with arrows to facilitate comparisons. (D) Distribution of dELL deletion mutant proteins is identical to full-length dELL on the X chromosome. Relative positions of the weakly staining landmark puff at 2B, the strongly staining bands at 3C, and 8EF are indicated in X chromosomes from each transgenic line showing chromosome targeting of dELL. (E) dELL(1-262) colocalizes with phosphorylated Pol II on polytene chromosomes. Chromosomes from transgenic flies expressing dELL(1-262) were fixed and stained with anti-six-His and anti-Pol IIo (H5; Covance). Images were merged to visualize extent of colocalization.

dELL(Δ190-759) is retained at developmental loci upon heat shock. (A) Both dELL(Δ760-1059) and dELL(Δ190-759) colocalize with phosphorylated Pol II under non-heat shock conditions. Chromosomes from transgenic flies expressing dELL(Δ760-1059) and dELL(Δ190-759) were fixed and stained with anti-six-His (left side) and anti-Pol IIo (H5; Covance) (right side). Images were merged to determine the extent of colocalization. (B) dELL(Δ190-759) is retained at developmental loci after heat shock. After a 20-min heat shock, chromosomes were preparedand immunostained as above. Unlike dELL(Δ760-1059), dELL(Δ190-759) retains its non-heat shock distribution after heat shock. Major heat shock puff sites are labeled with arrows. (C) Endogenous dELL is still recruited to heat shock puffs in transgenic flies expressing the dELL(Δ190-759) mutant. Chromosomes were prepared as described for panel B, except that anti-dELL polyclonal serum was used rather than anti-six-His serum. In these images, both the widely distributed pattern of localization for dELL(Δ190-759) and the heat shock puff staining observed for wild-type dELL are shown. Major heat shock puff sites are labeled with arrows.