ELL2, a new member of an ELL family of RNA polymerase II elongation factors

Abstract

We recently isolated an RNA polymerase II elongation factor from rat liver nuclei and found it to be homologous to the product of the human ELL gene, a frequent target for translocations in acute myeloid leukemia. To further our understanding of the possible role(s) of ELL in transcriptional regulation and human disease, we initiated a search for ELL-related proteins. In this report we describe molecular cloning, expression, and characterization of human ELL2, a novel RNA polymerase II elongation factor 49% identical and 66% similar to ELL. Mechanistic studies indicate that ELL2 and ELL possess similar transcriptional activities. Structure-function studies localize the ELL2 elongation activation domain to an ELL2 N-terminal region that is highly homologous to ELL. Finally, Northern blot analysis reveals that the ELL2 and ELL genes are transcribed in many of the same tissues, but that the ratio of their transcripts exhibits tissue-to-tissue variation, raising the possibility that ELL2 and ELL may not perform completely general functions, but, instead, may perform gene- or tissue-specific functions.

Figure 1

Comparison of the deduced amino acid sequences of human ELL2 and ELL. ELL2 residues 11–640 are from the sequence of a DNA fragment obtained by PCR amplification of a human fetal heart cDNA library. Residues 1–10 are from Human Genome Sciences ESTs HNFD055 and HNEAK22 and from an EST in the GenBank database (accession number W94585W94585). Identical amino acids are shown in white letters on a black background; similar amino acids (A, S, T, P; D, E, N, Q; H, R, K; I, L, M, V; F, Y, W) are shown in black letters on a gray background. Conserved regions 1, 2, and 3 are indicated by single, double, and triple underlines, respectively.

Figure 2

Tissue distribution of ELL2 and ELL mRNAs. A human multiple tissue northern blot (MTN1, CLONTECH) was probed sequentially with PCR-generated ELL2- and ELL-specific probes chosen from a region of sequence that was most divergent between the two genes. The ELL-specific probe contained sequences encoding amino acids 317–621, and the ELL2-specific probe contained sequences encoding amino acids 327–474. As a loading control, the same blot was probed with a probe specific for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. Probes were labeled with [α-³²P]dCTP by random priming performed according to the manufacturer’s instructions (Rediprime kit, Amersham). The blot was prehybridized in 10 ml of Hybrisol I solution (Oncor) for 3 h at 42°C. Probe DNA was denatured and added to hybridization solution at 10⁶ cpm/ml of solution. Hybridization was carried out at 42°C overnight. The blot was washed 10 min in 2× standard saline citrate (SSC)/0.1% SDS at room temperature, 15 min in 0.2× SSC/0.1% SDS at 45°C, 10 min in 0.1× SSC/0.1% SDS at 55°C, and then exposed to film (Hyperfilm-MP, Amersham) overnight at −80°C.

Figure 3

ELL2 and ELL have similar effects on elongation by RNA polymerase II during synthesis of promoter-independent and promoter-dependent transcripts. (A) Ten percent SDS/PAGE of recombinant ELL2 (rELL2) and ELL (rELL), purified by nickel chromatography and preparative SDS/PAGE. Proteins were visualized by silver staining. (B) Effects of ELL2 and ELL on the kinetics of promoter-dependent transcription. Preinitiation complexes were assembled at the AdML promoter with recombinant TBP, TFIIB, TFIIE, TFIIF, and purified rat TFIIH and RNA polymerase II as described (13). Transcription was initiated by addition of 50 μM ATP, 50 μM GTP, 2 μM UTP, 10 μCi of [α-³²P]CTP (>400 Ci/mmol, Amersham), and 7 mM MgCl₂. After 10 min at 28°C, 100 μM nonradioactive CTP was added to reaction mixture and short transcripts were chased in the absence or presence of ≈50 ng of SDS/PAGE-purified rELL2 or rELL for the times indicated. Transcripts were analyzed by electrophoresis through a 6% polyacrylamide/7.0 M urea gel. (C) Effects of ELL2 and ELL on the kinetics of promoter-independent transcription. SDS/PAGE-purified histidine-tagged ELL2 and ELL proteins were renatured and assayed in pulse–chase reactions as diagrammed in the figure using the oligo(dC)-tailed template pCpGR220 S/P/X. Reactions contained ≈0.01 unit of RNA polymerase II, 100 ng of pCpGR220S/P/X, and ≈50 ng of rELL2 or ≈50 ng of ELL and were performed essentially as described (13). The control reaction (mock) contained an identically prepared fraction from uninfected JM109(DE3) cells.

Figure 4

Localization of the ELL2 elongation activation domain. (A) Summary of ELL2 mutants and their activities in transcription. Wild-type ELL2 is diagrammed at the bottom of the panel. Conserved regions 1, 2, and 3 (R1, R2, and R3) are indicated by the shaded boxes. The alignment of region 3 with the C-terminal ZO-1 binding domain of occludin was generated with the bestfit program of the Genetics Computer Group package, using the symbol comparison table of Gribskov and Burgess (34). (B) Wild-type ELL2 and ELL2 mutants were expressed in E. coli and purified by nickel-affinity chromatography as described (13). Approximately 50 ng of each protein (in a maximum volume of 50 μl) was renatured and assayed as described (13) for its ability to stimulate synthesis of the 135-nucleotide transcript from the T-less cassette of oligo(dC)-tailed template pCpGR220 S/P/X. Reactions containing ≈0.01 unit of RNA polymerase II, 100 ng template, and the indicated ELL2 proteins were incubated at 28°C for 5 min in the presence of 50 μM ATP, 50 μM GTP, 1.8 μM CTP, and 10 μCi of [α-³²P]CTP. The control reaction (MOCK) contained an identically prepared fraction from uninfected JM109(DE3) cells.