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Previous Article | Next Article 
Molecular and Cellular Biology, November 2004, p. 9911-9919, Vol. 24, No. 22
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.22.9911-9919.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center,1 St. Louis University Cancer Center, St. Louis,4 Stowers Institute for Medical Research, Kansas City, Missouri,2 Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas3
Received 12 May 2004/ Returned for modification 21 July 2004/ Accepted 5 August 2004
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ABSTRACT |
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INTRODUCTION |
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Elongin was originally purified from rat liver nuclear extracts based on its ability to increase the catalytic rate of transcription by Pol II in an in vitro assay (4). Elongin is a heterotrimeric protein complex comprised of a 
110-kDa subunit (elongin A), an 18-kDa subunit (elongin B), and a 15-kDa subunit (elongin C) (2, 4, 12, 13). Elongin A is the transcriptionally active component of this complex, since addition of this protein alone to in vitro transcription assays is capable of stimulating elongation activity. The B and C subunits of the complex function as positive regulators and are unable to stimulate elongation in the absence of the A subunit (1, 4). To date, four elongin A family members have been identified, including a single elongin A homologue in Caenorhabditis elegans and the closely related elongins A2 and A3 in mammals (3, 4, 44). Each of these is able to interact with the elongin BC complex via a small sequence motif known as the BC box. Interestingly, the A2 and A3 homologues share the highest level of sequence homology, and structure-function analyses of fusion proteins containing elongin A3 and elongin A have revealed that the C-terminal domain of elongin A confers dependence on the BC complex for maximal transcription activity (44).
The conserved BC box motif is not exclusive to elongin A family members. This motif is also found in a large number of BC box proteins that are linked through elongins B and C to a protein module composed of a cullin family protein (Cul2 or Cul5) and the ring finger protein, Rbx1, to form elongin BC-based ubiquitin ligase complexes (13, 18, 19). Among the BC box proteins is the von Hippel-Lindau tumor suppressor protein, which functions as the substrate recognition subunit of an E3 ligase that targets the 
subunits of hypoxia-inducible transcription factors (1, 5, 7, 17, 20, 27).
In an effort to gain insight into the functional role(s) of elongin A in vivo, we have identified and characterized the unique Drosophila homologue of the elongin A family (dElongin A or dEloA). Appropriately, dEloA exhibits several in vitro and in vivo properties expected of a Pol II elongation factor. It is able to enhance Pol II elongation in an in vitro transcription assay, and it interacts with phosphorylated Pol II in Drosophila extracts. Further, dEloA colocalizes with Pol II at sites of active transcription on polytene chromosomes. These data support the hypothesis that elongin A is a Pol II elongation factor in vivo.
Developmental expression analysis reveals that dEloA mRNA has a broad pattern of expression, with its peak around early larval stage and continuing during early pupation. The protein levels for dEloA appear to also peak during the larval stages of development, indicating an important role for dEloA during the larval stage and early pupation. Interestingly, animals expressing reduced levels of dEloA due to RNA interference (RNAi) knockdown are unable to make the transition from the larval stages to adulthood and die as pupae, suggesting that dEloA is an essential factor that is required for proper metamorphosis. Our studies demonstrate the following: (i) an in vivo role for elongin A during transcriptional elongation; (ii) that not all elongation factors are created equal, i.e., they are not redundant, and each may have tissue/gene-specific roles during development; (iii) that the elongation factor elongin A is found on transcriptionally active developmental puff sites; and (iv) that elongin A is required for coordinated regulation of gene expression and development. The discovery of the role of elongin A as an RNA polymerase II elongation factor in vivo extends the important contribution played by Pol II elongation machinery in regulation of gene expression that is required for proper development.
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MATERIALS AND METHODS |
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For the purpose of performing RNA interference in vivo, a 593-bp fragment of dEloA (bp 400 to 993) was amplified by PCR from the pRSETa-dEloA construct using a 5' primer containing a nested BglII restriction site and a 3' primer containing a nested EcoRI restriction site. This product was then ligated to the BglII-EcoRI fragment of pSym-pUASTW (15), which was subsequently used for P-element-mediated transformation in Drosophila embryos.
To generate radiolabeled RNA from the dEloA cDNA for the developmental Northern analysis of dEloA expression, the SalI-EcoRI fragment was excised from the pRSETa construct, containing the 3' 930 bp of the dEloA cDNA sequence, with an EcoRI-restricted end at its 3' end and a SalI end at its 5' end. This fragment was then ligated to the SalI-EcoRI fragment of pGEM-3 (Promega), where it was placed under a T7 promoter at its 3' end. This was then used to transcribe antisense RNA in the reaction described below.
Northern blot analysis. Five micrograms of total RNA isolated from Drosophila melanogaster at each of the 10 developmental stages (gift of Dale Dorsett) was denatured in morpholinepropanesulfonic acid-formaldehyde-formamide solution and separated by electrophoresis on a 1% formaldehyde gel at low voltage. Resolved RNAs were transferred to a Biotrans membrane (ICN) and cross-linked to the membrane by UV irradiation. The membrane was incubated in hybridization solution (50% formamide, 10% PEG 8000, 3.5% sodium dodecyl sulfate [SDS], 150 mM sodium pyrophosphate, 250 mM NaCl, 1 mM EDTA, 2x Denhardt's) for at least 1 h at 65°C before the addition of radiolabeled probe. The antisense RNA probe was prepared by using the pGEM vector containing dEloA cDNA under the control of the T7 promoter at the 3' end. Antisense RNA transcription was performed using T7 RNA polymerase (Promega) under the following reaction conditions: 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, 10 mM dithiothreitol, 2 mM spermidine, 0.05% Tween 20, 0.5 mM (each) ATP, GTP, and UTP, 0.5 µCi of [32P]CTP (ICN), 0.1 U of RNasin (Promega), and 2 µg of template DNA. Reactions were carried out at 37°C for 60 min and then treated with RQ1 DNase (Promega) for 15 min to remove plasmid template. Antisense RNA was then purified using the RNeasy RNA purification kit (QIAGEN), added to the hybridization mixture, and incubated overnight at 65°C. The blot was then washed successively in buffers containing decreasing concentrations of SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and SDS until background radiation levels reached a minimum. Transcripts were visualized by autoradiography. To visualize the loading control, pGEM containing Drosophila rp49 cDNA (a gift of Dale Dorsett) under T7 was used to generate the antisense RNA radiolabeled probe.
Expression and purification of recombinant dEloA.
dEloA in pRSETa was used to transform BL21(DE3)/pLysE Escherichia coli. Positive transformants were selected by double selection with ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml) and were then used to produce overnight cultures in liquid broth containing each antibiotic. Cultures were diluted 1:50, allowed to grow to an optical density at 600 nm of 0.4, and then induced with isopropyl-ß-D-thiogalactopyranoside (Roche) at 0.5 to 1 mM concentrations for 4 h. Cells were harvested by centrifugation and lysed by repeated freeze-thaw cycles in 1x phosphate-buffered saline. Inclusion bodies were separated from soluble proteins by ultracentrifugation and then homogenized in buffer A (6 M guanidine-HCl, 40 mM Tris) to solubilize the recombinant protein. Homogenates were then ultracentrifuged again to separate soluble protein from cell debris. His-tagged recombinant protein was purified by using Probond nickel-chelating resin (Invitrogen). Briefly, proteins were bound to resin in buffer A. Resin-protein conjugates were washed with buffer B (buffer A plus 40 mM imidazole) three times to remove nonspecifically bound contaminants. Protein was eluted from resin with buffer C (buffer A plus 300 mM imidazole) and vortexing. This was followed by centrifugation to separate the resin from the supernatant containing the eluted protein. Load, flow-through, wash, and bound fractions were analyzed by Western blotting with anti-Express and anti-His6 antibodies (Invitrogen) to examine the efficiency of purification. Using this same strategy, 5 mg of recombinant protein was expressed and purified (the purity and identity of the expressed dEloA were determined by matrix-assisted laser desorption ionization-time of flight [MALDI-TOF] mass spectroscopy analysis) for the purpose of raising polyclonal antisera. Protein samples were sent to Research Genetics (Invitrogen, Huntsville, Ala.) for antibody production. For use in the in vitro transcription assay, purified recombinant dEloA was renatured by dialysis in buffer containing 250 mM KCl.
In vitro transcription elongation assay.
Vector containing an adenovirus major late promoter was cut with EcoRI and NdeI. The linear fragment was purified by gel electrophoresis and used in in vitro transcription assays. Preinitiation complexes were assembled at the adenovirus major late promoter with recombinant TBP, TFIIB, TFIIE, TFIIF, and purified rat TFIIH and RNA polymerase II as described previously (37). Transcription was initiated by the addition of 50 µM ATP, 50 µM GTP, 2 µM UTP, 10 µCi of [-32P]CTP (ICN) (specific activity, 3,000 Ci/mmol) and 7 mM MgCl2. After 10 min at 28°C, 100 µM nonradioactive CTP was added to the reaction mixture, and short transcripts were chased in the presence or absence of purified and renatured recombinant dEloA for the times indicated. Transcripts were analyzed by electrophoresis through a 6% acrylamide-7 M urea-0.5x Tris-borate-EDTA gel and developed using a Molecular Dynamics (Sunnyvale, Calif.) PhosphorImager. Mock-purified and renatured protein was used in the absence of dEloA as a control for the experiment.
Immunofluorescence staining. Salivary glands were dissected in gland buffer (8), fixed for 30 s in 2% formaldehyde in gland buffer, then in 45% acetic acid, 12% formaldehyde for 3 min, before storage in 67% glycerol-33% phosphate-buffered saline at –20°C. For optimal staining, dEloA polyclonal antibody (dilution, 1:300) was incubated with polytene chromosomes overnight at 4°C. Pol II antibodies (H5 and H14; Covance) were used at a 1:1,000 dilution and with overnight incubation. Appropriate secondary antibodies (Jackson Laboratory) were used at 1:1,000 dilution. Fluorescence detection was carried out with epifluorescence, using an Olympus BX60 fluorescence microscope with an NB barrier filter for fluorescein and Cy2 detection and a WG barrier filter for rhodamine and Cy3. Images were recorded with a SPOT charge-coupled device camera (Diagnostic Instruments, Inc.) using PAX-it imaging software (Midwest Information Systems, Inc.).
Coimmunoprecipitation of phospho-Pol II with dEloA. Wild-type (OregonR) Drosophila third-instar larvae were homogenized in ice-cold NUN buffer (1 M urea, 300 mM NaCl, 25 mM HEPES [pH 7.9]) (23). Extract was cleared by centrifugation and halved and incubated with dEloA antibody or preimmune serum at 4°C overnight with gentle agitation. Extract-antibody mixtures were then incubated with protein A Sepharose 4B fast flow resin, preincubated in NUN buffer plus 0.1% bovine serum albumin (pentax fraction V) and 0.1% NP-40 for 3 h at 4°C with gentle agitation. Resin-protein conjugates were washed three to four times with NUN buffer plus bovine serum albumin and NP-40, and bound proteins were released by boiling the resin in Laemmli buffer. Load, flow-through, wash, and bound fractions from each sample set (dEloA polyclonal or preimmune serum) were analyzed by Western blotting for the presence of Ser5-phospho-Pol II using H14 monoclonal antibody (Covance). Protein was visualized using the Western Lightning ECL kit (Perkin-Elmer Life Sciences).
Germ line transformation and fly culture.
yw67C23 embryos were injected with the pSympUASTW-dEloA 400-993 construct, together with the helper plasmid pvhs
2-3 "Turbo" (33) (kindly provided by Dale Dorsett, St. Louis University School of Medicine) essentially as described previously (40). G0 survivors were crossed to yw67C23 flies, and F1 adults were screened for the presence of w+. At least three independent transgenic lines were used for crosses with Gal4 lines (yw; daughterless Gal4 yw; actin5C Gal4/CyO y+) to activate RNAi.
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RESULTS |
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110 kDa, the size expected of full-length dEloA. Identity of the Coomassie-stained band was confirmed by tryptic digestion followed by MALDI-TOF analysis (data not shown). We then tested the ability of recombinant dEloA to stimulate Pol II elongation in an in vitro transcription assay. Promoter-specific transcription reactions were assembled with or without dEloA as described previously (14), and the accumulation of runoff transcripts after specific time points was examined by acrylamide gel electrophoresis and autoradiography. Figure 2C shows the dramatic stimulatory effect of dEloA on Pol II elongation. As with its mammalian counterparts, dEloA is capable of functionally interacting with Pol II in vitro, since it dramatically stimulates the rate of transcript accumulation in these reactions.
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dEloA is required for metamorphosis. While much has been learned about the functions of elongation factors in the regulation of transcribing Pol II in vitro, the in vivo roles of many of these factors remain unresolved. While it appears that some elongation factors have redundant functions, it is clear that some of these factors are essential. To determine whether dEloA is required for normal development in Drosophila, we used an RNAi-based strategy to deplete dEloA mRNA and protein levels in developing flies. We cloned an internal portion of the dEloA cDNA (approximately 600 bp) into pSym-pUASTW, placing it between two convergent Gal4 upstream activation sequence promoters (15). Transgenic flies bearing this construct were then crossed to flies carrying transgenes expressing yeast Gal4 under control of either an actin5C or a daughterless promoter. The effects of RNAi targeted against dEloA are shown in Fig. 6. Northern blot analysis of total and poly(A)+ RNA from control and dEloA-RNAi third-instar larvae revealed that dEloA mRNA levels are dramatically reduced when RNAi is activated (Fig. 6A). To confirm that dEloA protein levels were also reduced in these animals, we compared the levels of dEloA on polytene chromosomes under normal and RNAi-activated conditions. dEloA is virtually undetectable on the chromosomes when RNAi is activated, while control chromosomes exhibit normal levels and distribution of the dEloA protein (Fig. 6B). Our Western analysis studies also demonstrated that the dEloA level is decreased upon activation of RNAi (data not shown).
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Interestingly, we noticed that in all crosses, using four different pSym-pUASTW-dEloA transgene insertions, organisms in which dEloA levels were reduced by RNAi died invariantly during pupariation, with no observable effect on viability at other stages of development. The imaginal disks from dEloA-RNAi third-instar larvae exhibited normal size and morphology, indicating no general adverse effect on cell cycle progression in these tissues (data not shown). Close examination of the pupae generated in crosses where dEloA RNAi is activated revealed that these animals are unable to complete metamorphosis. There is no observable difference between the very early white prepupae (wpp) in dEloA RNAi and wild-type flies, but during the course of metamorphosis, significant defects in the dEloA-deficient pupae are evident. By the later stages of pupal development, control flies develop adult structures that are clearly visible through the pupal case (Fig. 6E, panels a to c), while the dEloA-deficient pupae lack any adult structures and have a shriveled appearance (panels d to f). We conclude that dEloA is an essential elongation factor that is required for development in Drosophila and may play a specific role in the developmental program regulating metamorphosis.
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DISCUSSION |
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Functional redundancy of elongation factors has been suggested by the fact that several fall within the same biochemical class as elongin A (i.e., factors that increase the overall Km for nucleotides and/or Vmax of Pol II during transcription in vitro). Previously, we demonstrated that one of the proteins in this class, dELL, is essential in Drosophila and is not functionally redundant with other members (10). A recent report by Yamazaki et al. indicates that mammalian elongin A is not essential for viability of mouse ES cells but is important for proper cell cycle progression and expression of a specific subset of genes (43). Whether elongin A is required for normal mouse development is not known. However, there are three known elongin A homologues in mammalian cells, while only a single elongin A homologue exists in Drosophila. Thus, there may be functional redundancy among the mammalian elongin A homologues in vivo, although it has been shown that elongin A and elongin A3 do not have complementary properties in vitro (44). Another possibility is that multiple homologues in mammalian systems have tissue-specific expression and/or functions, some of which may be nonessential. A number of elongation factors appear to have evolved specialized functions in metazoans and are limiting for the expression of specific genes, with mutations that result in very specific phenotypes (10, 16, 21). Consistent with these observations, Northern blots for elongin A, elongin A2, and elongin A3 reveal differential expression at the tissue level (3, 44).
Understanding the role(s) of the different elongation factors with respect to specific gene expression and the regulation of developmental processes is of major interest. While the widespread distribution of dEloA on polytene chromosomes is consistent with a general role for this factor in transcription, our data also support a more specific role for dEloA in Drosophila development. As with dELL, dEloA is required for viability, although it is important to note that dELL is maternally loaded and that dELL-null flies die during late embryogenesis (10, 14). Animals lacking wild-type levels of dEloA die during the pupal stages of development, at or shortly after the stage in which dEloA mRNA and protein expression levels peak. Thus, dEloA may play an important role in metamorphosis, and a reduction in dEloA levels at the larval stage of development could affect the levels and/or timing of gene expression, resulting in failure to complete this process. It is important to recognize, however, that RNAi does not result in complete elimination of the dEloA protein. While levels of the protein are dramatically reduced, it is therefore possible that sufficient dEloA remains to permit viability during larval stages, whereas a true knockout may result in earlier death.
We have identified the first targets of elongin A regulation during animal development. We have shown that a loss of dEloA correlates with reduced steady-state levels of larval cuticle proteins (data not shown). The genes encoding these proteins are found within a coordinately regulated gene cluster that is activated specifically during late larval development (11, 39). Further, these genes are downstream targets of the hormone ecdysone. We have observed localization of dEloA to ecdysone-induced puff sites, also suggesting a role for dEloA in expression of ecdysone-induced genes. The shriveled appearance of pupae lacking wild-type levels of dEloA is interesting, given that the role of the larval cuticle proteins is to establish a barrier to prevent desiccation of the developing pupae. Future experiments should identify additional targets of dEloA in the Drosophila developmental program.
Given our observations of the pattern of expression and requirement of dEloA, it is an attractive possibility that dEloA is required for the proper regulation of specific genes involved in metamorphosis. Our evidence is consistent with the presumption that elongin A is an elongation factor in vivo, although the relationship between elongation activity and viability remains unclear. Overall, our study presented here demonstrates the following: (i) that elongin A associates with the elongating form of RNA polymerase II during transcriptional elongation; (ii) that not all elongation factors are created equal, i.e., they are not redundant, and each may have a tissue/gene-specific role during development; (iii) the elongation factor elongin A associates with the transcriptionally active developmental puff sites; and (iv) elongin A is required for coordinated regulation of gene expression and development. These novel in vivo studies on the role of elongin A as an RNA polymerase II elongation factor extend the important contribution played by Pol II elongation machinery in regulation of gene expression that is required for proper development.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants from the National Science Foundation, MCB 0131414 (to J.C.E.), the American Cancer Society (RP69921801), and the National Institutes of Health (1R01CA089455) and a Mallinckrodt Foundation Award to A.S. A.S. is a scholar of the Leukemia and Lymphoma Society.
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FOOTNOTES |
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