dELL is an essential RNA polymerase II elongation factor with a general role in development - PubMed (original) (raw)

dELL is an essential RNA polymerase II elongation factor with a general role in development

Joel C Eissenberg et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Several eukaryotic proteins increase RNA polymerase II (Pol II) transcription rates in vitro. The relative contributions of these factors to gene expression in vivo is unknown. The ELL family of proteins promote Pol II elongation in vitro, and the Drosophila ELL homolog (dELL) is associated with Pol II at sites of transcription in vivo. The purpose of this study was to test whether an ELL family protein is required for gene expression in vivo. We show that dELL is encoded by the Suppressor of Triplo-lethal locus [Su(Tpl)]. We have characterized seven distinct mutant alleles of Su(Tpl) and show that a dELL transgene rescues recessive lethality of Su(Tpl). Su(Tpl) mutations cause abnormal embryonic segmentation and dominantly modify expression of diverse genes during development. These data show that an ELL family elongation factor is essential, acts broadly in development, and is not functionally redundant to other elongation factors in vivo.

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Figures

Figure 1

Figure 1

Molecular organization of the dELL locus. dELL is encoded by sequences lying within the large intron of dMi-2. The relative position of the P-element associated with the Su(Tpl) JE3 allele is shown at the apex of the triangle below the map.

Figure 2

Figure 2

dELL mutations are present in six alleles of Su(Tpl). (A) The complete amino acid sequence of dELL is shown; the N-terminal shaded region indicates sequence homologous to the transcription elongation activity domain/p53 interaction domain (9, 35), and the C-terminal shaded region indicates sequence homologous to mammalian ELL sequences required for the transformational activity of MLL–ELL fusion protein in myeloid precursor cells. Above the sequence are indicated the sites of mutations in Su(Tpl) mutant alleles. (B) Sequence alignment of the conserved C-terminal domain of mammalian and Drosophila ELL proteins. Residues that are identical in all four proteins are in red; residues representing conservative substitutions in all four proteins are in blue. Sites of C-terminal domain mutations found in the Su(Tpl) S-192 allele are indicated on the last line.

Figure 3

Figure 3

A dELL transgene complements the recessive lethality of Su(Tpl). (A) Immunofluorescence colocalization of the transgenically expressed 6xHis-dELL protein and phosphorylated RNA Pol II on transgenic third instar larval salivary gland polytene chromosomes. The 6xHis-dELL is detected by using 6xHis-Gly Ab (Invitrogen), and phosphorylated Pol II is detected with a mAb specific for the phosphorylated form of the Pol II large subunit C-terminal domain (H14; Covance). (B) A transgenic copy of dELL complements the recessive lethality of heteroallelic Su(Tpl) mutant flies.

Figure 4

Figure 4

Su(Tpl) mutations cause recessive embryonic segmentation defects. (A) Tabulation of cuticular defects observed in various heteroallelic combinations of Su(Tpl) mutations. (B) Examples of the most commonly observed cuticle defects in heteroallelic mutant Su(Tpl) embryos. F, fusion of adjacent denticle belts; H, failure of head involution; I, incomplete denticle belt; and M, missing denticle belt.

Figure 5

Figure 5

Su(Tpl) mutations are dominant enhancers of N nd-1 and ct 53d. (A) Wings of adult males that are Nnd-1 and either Su(Tpl) + or mutant for one of five different dELL mutations. (B) Tabulation of wing nicks in adult males that are ct 53d and either Su(Tpl) + or mutant for one of five different dELL mutations.

Figure 6

Figure 6

Models of ELL function in vivo. Several factors, including the ELL family proteins and TFIIF, operate in vitro to increase the overall rate of transcription elongation by altering the _K_m and/or _V_max of elongating polymerase. Three models may account for the functional nonredundancy of ELL elongation factors and other members of its kinetic class, which includes Elongins, CSB, and DRB sensitivity-inducing factor (represented here by TFIIF). (A) ELL may act specifically on a distinct subset of genes from those requiring other elongation factors. (B) ELL may act at a distinct kinetic phase of Pol II elongation. (C) ELL may act additively or cooperatively with other elongation factors to achieve optimal Pol II elongation rates in vivo.

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