Fig. 2. Drosophila ELL can increase the catalytic rate of transcription elongation catalyzed by RNA polymerase II in vitro. Pre-initiation complexes were formed in the presence of AdMLP DNA, TBP, TFIIB, TFIIF, TFIIE, TFIIH and RNA Pol II. Transcription reactions were initiated by the addition of rNTPs as described in Materials and methods. Nascent transcripts were chased after the addition of cold CTP in the presence or absence of recombinant dELL for the times indicated. Runoff transcripts were analyzed in a 6% acrylamide gel containing 7 M urea and 0.51× TBE, and visualized by PhosphorImager analysis.

Fig. 4. Drosophila ELL is associated with euchromatin on transcriptionally active sites. (A) Immunofluorescence localization of dELL on polytene chromosomes. Wild-type polytene chromosomes were prepared from third instar larvae salivary glands and probed with purified polyclonal antibodies raised against dELL. (B and C) To ensure that the purified dELL polyclonal antibodies were specific for dELL, we also generated a tagged dELL transgenic fly. Polytene chromosomes isolated from the transgenic dELL third instar larvae salivary glands were probed either with (B) monoclonal antibody directed against the His₆-tag on dELL or (C) purified polyclonal antibodies directed against dELL. (D) Polytene chromosomes were probed for dELL (red) and the heterochromatin-associated protein HP1 (green). The bracket indicates the heterochromatic chromocenter (cc), while a prominent region of euchromatic staining by HP1 in region 31 is indicated by a line (31).

Fig. 6. Heat shock induces a dramatic relocalization of dELL to heat shock loci. Immunofluorescence detection of wild-type third instar larvae salivary glands polytene chromosomes probed with polyclonal antibodies directed against dELL (A) before and (B) after heat shock induction. (C) Presence of dELL on heat shock loci 87A, 87C, 63C and 93D (left panel) before and (right panel) after heat shock induction. (D) Co-localization of Pol IIo and dELL at heat shock puffs 87A, 87C, and 93D in heat-shocked polytene chromosomes. Phase contrast (left panel), staining for Pol IIo (middle panel) and staining for dELL (right panel) on a heat-shocked polytene chromosome.

Fig. 1. Domain structure and conservation and expression pattern of D.melanogaster ELL. (A) Sequence alignment of dELL with human ELL2. Amino acid identities are highlighted in red. Gray boxes indicate the sequences homologous to the N-terminal elongation activation domain and the C-terminal immortalization domain of ELL, and a central region of high homology between dELL and ELL2. (B) Affinity chromatography and western analysis of recombinant dELL. His₆-tagged protein was expressed and one-step purified by nickel chromatography as described in Materials and methods. Chromatographic fractions were analyzed by western blotting with anti-Express monoclonal antibody (Invitrogen) to visualize recombinant dELL. As expected, a single band of ∼120 kDa appears in both the load and bound fractions. (C) Expression of dELL transcripts during development. (Top) An autoradiograph of a northern blot showing the two dELL mRNAs. (Bottom) An autoradiograph from the same northern blot probed for rp49 transcript as loading control. Each lane contained 5 µg of total RNA isolated from Oregon R wild-type flies at the developmental stage indicated: EE, 0–30 min after egg laying (AEL); LE, 30 min–16 h AEL; L1–L3, first to third instar larvae; P1–P4, first to fourth day of pupation; AM, 0- to 1-day-old male adults; AF, 0- to 1-day-old adult females. (D) Western analysis of dELL protein expression at different stages of fly development. Protein extracts were prepared from third instar larvae, salivary gland tissue from third instar larvae and adult Drosophila as described in Materials and methods. Extracts were subjected to western analysis with purified anti-dELL polyclonal antisera to examine relative levels of protein expression at the selected developmental stages. A single polypeptide band (∼120 kDa) was detected in the extracts from each stage, and this polypeptide co-migrates with recombinant dELL. Protein concentrations in each extract were determined prior to loading to ensure that total protein in each lane was similar.

Fig. 3. Whole-mount immunofluorescence detection of ELL in D.melanogaster embryo. Pre-cellularization Drosophila embryos were fixed as described in Materials and methods and probed either with (A) DAPI to visualize DNA, (B) monoclonal antibody directed against phosphorylated Pol II or (C) purified polyclonal anti-dELL serum. (D) Overlay of (B) and (C).

Fig. 5. Co-localization of dELL with hyperphosphorylated RNA polymerase II (Pol IIo) on polytene chromosomes. (A) Immunofluorescence detection of dELL. (B) Immunolocalization of Pol IIo. (C) Overlay of (A) and (B). Co-localization of red and green signals appears yellow.

Fig. 7. RNA polymerase II and dELL can physically interact. To determine biochemical interaction between dELL and Pol II, wild-type and His₆-tagged dELL transgenic fly extracts were fractionated by nickel chromatography. Load, flow-through, wash and bound fractions were subjected to 12% SDS–PAGE, and fractions were analyzed for the presence of Pol II by western blot analysis using a Pol II-specific monoclonal antibody as described in Materials and methods.