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J. Biol. Chem., Vol. 277, Issue 4, 3047-3052, January 25, 2002

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Purification and Characterization of the Human Elongator Complex^*

Nicola A. Hawkes^§, Gabriel Otero^§, G. Sebastiaan Winkler, Nick Marshall^¶, Michael E. Dahmus^¶, Daniel Krappmann, Claus Scheidereit, Claire L. Thomas^**, Giampietro Schiavo^**, Hediye Erdjument-Bromage, Paul Tempst, and Jesper Q. Svejstrup^§§

From the  Mechanisms of Gene Transcription Laboratory, Imperial Cancer Research Fund Clare Hall Laboratories, Blanche Lane, South Mimms, Hertfordshire, EN6 3LD, United Kingdom, the ^¶ Section of Molecular and Cellular Biology, University of California Davis, Davis, California 95616, the  Max-Delbruck-Center for Molecular Medicine, Berlin 13122, Germany, the ^** Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom, and the  Molecular Biology Programme, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication, October 31, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts. The purified factor can exist in two forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic Elp3 subunit. Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly with RNA polymerase II in solution. Several human homologues of the yeast Elongator subunits were identified as subunits of the human Elongator complex, including StIP1 (STAT-interacting protein 1) and IKAP (IKK complex-associated protein). Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this disease might be due to compromised Elongator function and therefore could be a transcription disorder.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RNA polymerase II (RNAPII)¹ transcription can be reconstituted in vitro with a minimal set of general transcription factors, such as TBP, TFIIB, TFIIF, TFIIE, and TFIIH (1, 2). Each of these factors is absolutely required for promoter-specific initiation of transcription under most conditions. By contrast, transcript elongation by RNAPII can occur in the absence of co-factors in vitro. However, over the last few years a plethora of factors that either stimulate or repress transcript elongation have been isolated (3).

We previously took a biochemical approach to isolate factors that specifically associate with the elongating form of RNAPII (4). Purification of chromatin-associated, hyperphosphorylated RNAPII from yeast led to the isolation of the Elongator complex and identification of the genes (ELP1-ELP6) encoding subunits of this multisubunit factor (4-7). The functional entity of Elongator complex has recently been shown to be an unstable six-subunit complex, termed holo-Elongator, which can dissociate into two discrete three-subunit subcomplexes upon treatment with high salt and/or MonoQ chromatography (7). One of these subcomplexes is the Elp3-containing core Elongator complex initially identified (4), and the other is a complex of the Elp4, Elp5, and Elp6 proteins (7). Yeast cells lacking the ELP genes are viable but display a variety of phenotypes consistent with a role for the factor in transcript elongation in vivo. Significantly, the Elp3 subunit is a highly conserved histone acetyltransferase (HAT) (6). Mutations that debilitate the HAT activity of Elp3 in vitro also confer elp phenotypes in vivo, indicating that the HAT activity of Elongator is required for its function (7, 8).

Homologues of several Elongator subunits, such as the WD40 repeat protein Elp2 (5), the HAT Elp3 (6), and Elp1 and Elp4 can be found by data base searching in the genomes of higher eukaryotic cells. Intriguingly, the closest homologue of Elp1 in human cells is encoded by the IKAP gene (4, 9), while the closest mouse homologue of Elp2 is StIP1 (10). Mutations in the IKAP gene can result in the severe human hereditary disorder familial dysautonomia (11, 12), while StIP1 was identified through its interaction with the transcriptional activator signal transducer and activator of transcription 3 (STAT3) (10).

Here, we report the purification and characterization of the human Elongator complex. Our results reveal extensive structural and functional similarity between the yeast and the human complex. Most notably, human holo-Elongator is a histone acetyltransferase complex, which in addition to human ELP3 (hELP3) contains IKAP, human StIP1, human ELP4 (hELP4), and two additional proteins as subunits. The identification of IKAP as an Elongator subunit suggests a connection between Elongator and human disease.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Buffers and Solutions-- Buffers A, B, and C were as described previously (13), except that KCl was used as salt in buffers A and B. Buffer D was 40 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.05% Nonidet P-40, and the concentration of KCl indicated after the hyphen. Protease inhibitors were included in all buffers used (14).

Purification of Elongator from HeLa Cells-- Throughout purification, Elongator was followed by Western blotting using anti-IKAP (15) and anti-Elp3 (4) antibodies. HeLa cell extract was prepared essentially as described (16) until after the ultracentrifugation step. The soluble cell extract was then dialyzed into buffer B-50 (50 mM KCl), loaded onto heparin-Sepharose (Amersham Biosciences, Inc.), and eluted stepwise with buffer B-150, B-300, B-450, and B-700. Proteins from the B-300 eluate were loaded onto HTP hydroxyapatite (Bio-Rad), equilibrated in buffer C-10, and eluted with a gradient from 10 to 400 mM potassium phosphate over 10 column volumes (CV) in buffer C. Elongator-containing fractions were dialyzed into buffer A-50, loaded onto a MonoS HR 10/10 column (Amersham Biosciences), and eluted with a linear gradient from 70 to 800 mM salt in buffer A over 15 CV. Fractions containing Elongator were pooled, dialyzed into buffer B-50, loaded onto a Mono Q HR 5/5 column (Amersham Biosciences) equilibrated in buffer B-50, and eluted with a linear gradient from 70 to 500 mM salt in buffer B over 10 CV. Elongator fractions were loaded onto a Sephacryl S-300 (26/20) gel filtration column (Amersham Biosciences), equilibrated in buffer B-150, and 1-ml fractions were collected. The Elongator-containing fractions were diluted, applied to Progel-TSK DEAE-5PW (Supelco) equilibrated in buffer B-50, and eluted with a linear gradient from 70 to 800 mM salt in buffer B over 10 CV. Elongator fractions were loaded onto HTP hydroxyapatite (Bio-Rad), and the resin was resolved as described above. The purity of the fractions from hydroxyapatite was determined by silver staining.

In addition to some of the steps described, the procedure to purify holo-Elongator utilized DEAE-Sepharose fast flow (Amersham Biosciences, Inc.), with proteins eluted stepwise in buffer B containing 150, 300, 450, and 700 mM salt. Proteins in the B-300 eluate were precipitated by ammonium sulfate precipitation (40% saturation) before dialysis into buffer A-50 and MonoS HR5/5 chromatography as described above. Elongator fractions were incubated with IKAP antibody resin for several hours to overnight at 4 °C with gentle mixing. Following washing with B-100, antibody-associated proteins were released by batch incubation with 1 CV of 1 mg/ml IKAP peptide (15) in buffer D-500 at 4 °C for several hours and eluted from the resin by gravity flow.

Protein Identification-- Gel-fractionated proteins were digested with trypsin, and peptides were analyzed by matrix-assisted laser desorption/ionization reflectron time-of-flight MS and by electrospray ionization MS/MS as previously described (7). Selected mass values from the matrix-assisted laser desorption/ionization time-of-flight experiments were taken to search the protein nonredundant data base (NCBI, Bethesda, MD) using the PeptideSearch (17) algorithm. MS/MS spectra were inspected for y" ion series to compare with the computer-generated fragment ion series of the predicted tryptic peptides.

Histone Acetyltransferase Assay-- 15-µl reactions were carried out in HAT buffer (10 mM Hepes-KOH, pH 7.9, 10 mM MgCl₂, 50 mM NaCl, 5 mM dithiothreitol, 10 mM sodium butyrate, 0.25 mg/ml bovine serum albumin, 5% glycerol), using 0.25 µCi of [³H]acetyl-coenzyme A (4.6 Ci/mmol, 250 µCi/ml) per assay and 5 µg of core histones where indicated. Core histones were prepared from HeLa cells as described (18). Histone tail peptides, mimicking the last 25-30 amino acids of the respective tails (3 µg), were also used. HAT reactions were incubated for 45 min at 30 °C. Reactions were analyzed by scintillation counting or fluorography. For visualization by autoradiography, the reactions were terminated by the addition of SDS-sample buffer followed by SDS-PAGE using 16.5% Tris-HCl peptide gels (Bio-Rad). After staining of the histones by Coomassie Brilliant Blue, the gels were soaked in Amplify solution (Amersham Biosciences), dried, and subjected to fluorography for 2-14 days.

Expression of Human Elp3 in Insect Cells-- For expression in insect Sf9 cells, the hELP3 coding region was cloned into the KpnI-XbaI sites of pBlueBac4.5 (Invitrogen). Details of the clone are available on request. Virus production and protein expression in insect Sf9 cells were done according to instructions from the manufacturer. Extracts from cells containing hELP3 virus were centrifuged at 2000 rpm for 15 min. hELP3 protein was recovered from the insoluble fraction by redissolving in SDS gel loading buffer without reducing agent.

Preparation of Antibodies-- To produce anti-hELP4 antibodies, a peptide encompassing the last 20 amino acids of the hELP4 protein was synthesized. This peptide was coupled via glutaraldehyde cross-linking to keyhole limpet hemocyanin (Calbiochem) and used to immunize rabbits (Murex). For immunoblotting, the anti-hELP4 antibody was used at a final dilution of 1:1000, and the anti-IKAP, anti-Elp3, and anti-RNAPII (8WG16) antibodies were used at 1:2500 in 0.01% Tween, 5% milk phosphate-buffered saline.

To produce an IKAP antibody affinity column, IKAP antibodies (15) were coupled to protein A-agarose beads (Amersham Biosciences) using standard chemical coupling with sodium borate, pH 9.0, dimethylpimedilate, and ethanolamine (19). The IKAP beads were equilibrated in buffer D-100 prior to use. Proteins were eluted with a peptide mimicking the C terminus of IKAP (15).

Elongator-RNA Polymerase II Interaction Studies-- The presence of Elongator in elongation complexes was studied using anti-IKAP antibodies, and G11 early elongation complexes were generated as described (20, 21). For studies of Elongator-RNAPII interaction in solution, equal amounts of virtually homogenous calf thymus RNA polymerase II (22) and purified human Elongator (200-ng hydroxyapatite fraction) in 50 mM Tris-acetate, pH 7.8, 2% glycerol, 0.15 M NaCl, 0.01% Nonidet P-40, 1 mM dithiothreitol were mixed in a final volume of 50 µl on ice. The mixture was incubated for 1 h at 4 °C prior to loading onto Superose 6 PC3.2/30 (Amersham Biosciences). Human Elongator interacted with both RNAPII-A and RNAPII-0.

Immunostaining-- Experiments to determine the subnuclear localization of IKAP and hELP3 were carried out on normal culture HeLa cells using the method described by Osborne et al. (23) with primary antibody dilutions of 1:700 and the Alexa 488 secondary antibody (Molecular Probes, Inc., Eugene, OR) used at 1:200.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We identified, cloned, and sequenced the hELP3 gene. It also recently appeared in the human genome data base as hypothetical protein AAH01240 (g12654795). Like other metazoan Elp3 homologues (6), hELP3 is highly homologous to the yeast counterpart (77% identity, 82% similarity) over the entire coding region. Antibodies directed against yeast Elp3 recognized recombinant hELP3 protein isolated from Sf9 insect cells as well as a protein of the expected size in HeLa whole cell extracts (Fig. 1A and data not shown). Combined use of anti-Elp3 antibodies and antibodies against IKAP (15), the closest human homologue of Elp1, made it clear that IKAP and hELP3 protein co-purified during chromatography on a variety of resins, although IKAP could also be detected in fractions that did not contain significant amounts of hELP3 (Fig. 1A and data not shown). Immunoprecipitation using anti-IKAP antibodies co-depleted similar proportions of total IKAP and hELP3 protein (Fig. 1B, lanes 1 and 2), and both proteins could be eluted from the affinity resin with the IKAP peptide used to raise the antibody (Fig. 1B, lane 3, and Fig. 4A). Immunoprecipitation with beads alone did not bring down any of the proteins (data not shown). Based on these observations, we concluded that IKAP and hELP3 are associated in a human Elongator complex, and we next purified the complex to virtual homogeneity. Fig. 1C shows the most highly purified Elongator fraction from the final hydoxylapatite column. This procedure yielded a three-subunit complex, in which IKAP, hELP3, and a 95-kDa protein are indicated. We designate this complex human core Elongator.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Purification of human core-Elongator. A, IKAP and human ELP3 (hELP3) proteins co-purify during chromatography. Fractions eluting from DEAE-Sephacel were fractionated by SDS-PAGE, blotted, and probed with antibodies directed against the proteins indicated on the right. Lane 1 contains recombinant human Elp3 from insect cells. B, similar proportions of IKAP and hELP3 protein are immunoprecipitated by anti-IKAP antibody. The input and unbound fractions of protein incubated with protein A-bound anti-IKAP antibody were fractionated by SDS-PAGE together with 5% of the protein obtained by subsequent peptide elution of the beads. Separated proteins were blotted and probed with antibodies directed against the proteins indicated on the right. C, purification procedure (left panel) and the most purified core-Elongator fraction (right panel) from the final hydroxyapatite column. Migration of size markers is indicated on the left, and the proteins are designated on the right. The asterisk denotes a protein that was not consistently found in highly purified Elongator fractions and might be a contaminant or loosely associating protein. D, HAT activity and the hELP4 protein do not co-elute with the peak of core-Elongator during MonoQ chromatography. Fractions from MonoQ were resolved by SDS-PAGE, blotted, and probed with antibodies directed against the proteins indicated on the left (upper two panels) or assayed for HAT activity (lower panel).

Elongator-RNA Polymerase II Interaction-- In yeast, Elongator is the major component of native elongating RNAPII holoenzyme, and the Elongator-RNAPII interaction can be reconstituted with purified proteins (4). We tested if Elongator is a component of early elongation complexes (EECs) formed by incubating bead-immobilized DNA templates containing the adenovirus 2 major late promoter with HeLa nuclear extract under conditions that allow RNAPII to transcribe 11 nucleotides before pausing due to limiting amounts of CTP (20, 21). The beads containing the early elongation complexes were first washed under mild conditions and then stringently with 1% sarcosyl (Fig. 2A). Hyperphosphorylated RNAPII was detected on the beads after both washes (lanes 4 and 5), although the majority was found in the supernatant after the sarcosyl wash (compare lanes 1 and 4). Elongator could not be detected after sarcosyl treatment (Fig. 2A, compare lanes 4 and 5) but was detected on the beads after the salt wash, suggesting that it associates with EECs. The IKAP signal detected in EECs washed only with salt is stable up to and including rinse with 600 mM KCl.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Interaction of Elongator with RNA polymerase II. A, early elongation complexes contain Elongator. Proteins isolated with early elongation complexes (EECs) were fractionated by SDS-PAGE, blotted, and probed with antibodies directed against the proteins indicated on the left. The sarcosyl supernatant of G11 EECs is shown in lane 1. Lanes 2 and 3 show different amounts of RNAPII-A as reference. Lane 4 shows proteins remaining in EECs after the stringent sarcosyl wash, and lane 5 shows the proteins present after the mild washes. Note that RNAPII present in EECs as expected is hyperphosphorylated. B, highly purified Elongator can associate with sarcosyl-washed EECs. Sarcosyl-washed, bead-bound EECs (lane 1) or the control beads containing DNA only (lane 4) were incubated with or without core Elongator as indicated, and EEC-associated proteins were brought down by centrifugation in a pellet (P), separated from the nonassociated proteins of the supernatant (S). Proteins were resolved by SDS-PAGE, blotted, and probed with antibodies directed against the proteins indicated on the left. C, direct association of human Elongator with RNAPII in solution. Elongator and RNAPII were filtrated (as indicated on the left) through Superose 6, either as individual factors (upper panels) or after their mixing (lower panels). The eluted fractions were blotted and probed with antibodies against the proteins indicated on the right.

Sarkosyl treatment removes most proteins from DNA, and EECs treated with Sarcosyl contain equimolar amounts of transcript and RNAP II, indicating that Sarkosyl efficiently and selectively dissociates nontranscribing RNAP II (20). Significantly, we found that purified Elongator was able to associate with sarcosyl-washed EECs (Fig. 2B). Some association with DNA-beads alone was also observed although clearly to a lesser extent than with EECs (Fig. 2B, compare lanes 2 and 3 with lanes 5 and 6), indicating that Elongator may also have an intrinsic ability to bind DNA.

We finally investigated whether a mammalian RNAPII-Elongator holoenzyme could be reconstituted from RNAPII and Elongator in the absence of nucleic acids. Highly purified RNAPII and core Elongator were analyzed by gel filtration (Fig. 2C), either individually (two upper panels) or after mixing (lower panel). Mammalian RNAPII and Elongator formed a complex in vitro, as evidenced by the mobility shift of these proteins from the position they occupied when run as separate entities to that of a novel species. As previously observed with the yeast counterparts (4), a fraction of RNAPII eluted in earlier fractions, while Elongator was quantitatively shifted to elute in later fractions by the association. Collectively, these results indicate that the Elongator HAT complex has the intrinsic ability to associate directly with RNAPII in elongation complexes, suggesting a role for the complex during transcription in human cells.

Cellular Localization of Elongator Complex-- Elongator localization was determined by immunostaining (Fig. 3). As expected, both IKAP and hELP3 were found predominantly in the cell nucleus. Unexpectedly, however, significant fractions of the proteins localized to the nucleoli and both IKAP and hELP3 could also be detected in the cytoplasm.

View larger version (91K): [in this window] [in a new window]   Fig. 3.   Subcellular localization of Elongator. Immunostaining of HeLa cells with anti-IKAP and anti-Elp3 polyclonal antibodies as indicated. The lower panels are magnified views of two cells from the upper panel, highlighting the nucleolar and nucleoplasmic distribution of IKAP and hELP3. Some cytoplasmic staining is also seen. Confocal images corresponding to the projection of a selected series of 0.4-µm sections are shown. Bars, 10 µm.

Holo-Elongator, but Not Core Elongator, Has HAT Activity-- As previously observed in the case of yeast Elongator (7), immunoblotting of human Elongator fractions showed that hELP4, the human homologue of yeast Elp4 (7), did not co-elute precisely with the peak of IKAP and hELP3 during MonoQ chromatography (Fig. 1D). Moreover, when the HAT activity of the eluted fractions was measured (Fig. 1D, lower panel), little or no HAT activity co-eluted with the peak of Elongator in MonoQ fractions 30-32. By contrast, low but significant levels of H3 and H4 HAT activity were detected in fractions 27-29, the leading edge of the Elongator elution profile. This activity thus co-eluted with the hELP4 protein. These data might suggest that the human Elongator complex is unstable and that the hELP4-containing fractions represent an active form of human Elongator complex remaining after several columns of purification. Such a chromatographic behavior would be strikingly similar to that of yeast Elongator, which can dissociate into two three-subunit subcomplexes during purification (7). These findings prompted us to design an alternative procedure to purify the intact complex by taking advantage of an anti-IKAP immunoaffinity step (Fig. 4).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Purification of holo-Elongator and HAT activity of the highly purified factor. A, purification procedure (left panel) and the holo-Elongator fraction (right panel) after elution from the antibody resin. Migration of size markers is indicated on the left, and the proteins are designated on the right. B, HAT activity of holo-Elongator. The holo-Elongator complex and conventionally purified core Elongator were titrated into the HAT assay. The resulting fluorogram (upper panel) and the Coomassie-stained histone gel (middle panel) as well as a Western blot indicating the relative amounts of IKAP, hELP3, and hELP4 used in the assays (lower panel) are shown. Several independent experiments failed to detect HAT activity in core Elongator fractions from several independent purifications, even after very long fluorogram exposures. C, Elongator HAT activity is directed against the tails of histones H3 and H4. Peptides mimicking the indicated histone tails were used in HAT assays in the absence (white bars) or presence (filled bars) of holo-Elongator and quantitated by scintillation counting.

When the purification procedure that took advantage of affinity chromatography was employed, the subunit composition of human Elongator complex differed significantly from the core complex purified by conventional means (Fig. 4A). We designate this complex human holo-Elongator. The virtually homogenous, immunopurified complex was compared with core Elongator complex in HAT assays. Holo-Elongator had robust HAT activity directed against histone H3 and H4, whereas similar quantities of core Elongator had no activity (Fig. 4B), indicating that the additional subunits confer HAT activity to the holo-Elongator complex. As expected, the activity of holo-Elongator was directed against the tails of histone H3 and H4 (Fig. 4C). These results indicate that the three small holo-Elongator subunits, hELP4, p38, and p30, are required to activate the HAT activity of hELP3 or that one of these proteins has intrinsic HAT activity.

Identification of Elongator Subunits-- Four of the subunits of holo-Elongator were identified by a combination of peptide mass fingerprinting and mass spectrometric sequencing. In this way, the presence of IKAP, hELP3, and hELP4 in human holo-Elongator was confirmed. Moreover, the 95-kDa protein, which was also observed in core Elongator (p95; Fig. 1C), was identified as hypothetical protein BAB14193 (NCBI g10434263), a conserved WD40 repeat protein that is the human homologue of yeast Elp2 and mouse StIP1 (10) (Fig. 5). Interestingly, data base searching for protein motifs in hELP2 revealed the presence of a motif (signature 2 motif) found repeated several times in the regulator of chromosome condensation (RCC1) protein. RCC1 has recently been shown to bind to chromatin via association with histones (24), but whether this requires the signature 2 motif is not known.

View larger version (154K): [in this window] [in a new window]   Fig. 5.   Human ELP2 protein. Shown is the predicted amino acid sequence of the protein encoded by hELP2 and alignment with homologues from other species using ClustalX (26). H.s., Homo sapiens; M.m., Mus musculus; A.t., Arabidopsis thaliana; S.p., Schizosaccharomyces pompe; S.c., Sacchoromyces cerevisiae. The mouse Elp2 protein is identical with StIP1 (10). Conserved residues are shaded in light gray, and identical residues are shaded in dark gray. Human ELP2 is 30.3% identical/43.4% similar to S. cerevisiae Elp2 over the entire sequence. Otherwise, homology between these proteins ranges from 25.6% identical and 37.7% similar (S. cerevisiae and A. thaliana), to 77.9/83.6% (H. sapiens and M. musculus). The indicated region of homology to RCC1 signature 2 was found using PredictProtein (available on the World Wide Web at maple.bioc.columbia.edu/predictprotein/). WD40 repeats are numbered and underlined.

We conclude that the components of the Elongator complex are well conserved from yeast to humans.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this report, we describe the purification and characterization of human Elongator. We find that human Elongator is remarkably similar to the yeast complex on several levels. First, it consists of homologues of the yeast Elp proteins. We have so far identified human homologues of yeast Elp1, Elp2, Elp3, and Elp4 in the holocomplex, which, like the yeast counterpart, appears to consist of six subunits. The p38 and p30 subunit of the human complex might be encoded by homologues of yeast Elp5 and Elp6. Second, as in yeast, human Elongator complex appears to exist as a core, three-subunit complex (hELP1/IKAP, hELP2, and hELP3) or as a larger, six-subunit holo-Elongator complex. In all likelihood, the human holo-Elongator complex dissociates during purification, at least partly as a consequence of treatment with high salt and MonoQ chromatography, as has previously been demonstrated for the yeast complex (7). Third, human holo-Elongator complex has histone H3 and H4 HAT activity, which has also been observed for the yeast holocomplex.² Finally, human Elongator complex has the ability to associate with RNAPII both in solution and in elongation complexes. Based on these results, we suggest that human Elongator complex serves a role in RNAPII-associated chromatin remodeling during transcript elongation in higher cells.

Significantly, we found that only holo-Elongator complex has detectable HAT activity, indicating that the additional components (hELP4, p38, and p30) confer activity to the hELP3 catalytic subunit present in the catalytically inactive core Elongator complex or that one of the additional components itself has HAT activity. We favor the former possibility, because similar results have been found for yeast Elongator complex. Neither yeast Elp4, Elp5, nor Elp6 contain motifs with homology to known HATs, and the complex of these proteins has no HAT activity in itself but confers activity to Elp3 in the core Elongator complex.²

The largest subunit of human Elongator is encoded by the IKAP gene. This protein was originally isolated biochemically as a subunit of a large complex containing IB kinases and proposed to be a scaffold protein for the assembly of these proteins (9). However, IKAP is the closest human homologue of yeast Elp1 with regions of high similarity spread over the entire coding region (4, 9), and recent data seriously question the proposed role of IKAP in IB kinase signaling (15). Thus, IKAP exists in a complex of the size expected for Elongator and immunoprecipitation of the protein from crude extracts yields a 5-7-subunit complex (15) of a composition similar to the holo-Elongator complex presented here. Importantly, this immunoprecipitated IKAP complex does not include IB kinases, and the IB kinase complex still forms and is activated normally in cells where IKAP mRNA and protein levels are reduced to very low levels by antisense oligonucleotides (15). Finally, overexpression of IKAP blocks not only induction of a NF-B-dependent reporter gene but also transcription from several NF-B-independent promoters (15). Taken together, the data thus suggest that IKAP is not involved in IB kinase signaling but rather plays a role in RNAPII transcription as a component of Elongator.

A splicing site mutation in the gene encoding IKAP causes the severe human recessive disorder, familial dysautonomia (11, 12). This mutation leads to the tissue-dependent expression of a truncated version of the IKAP protein, which results in the poor development, survival, and progressive degeneration of the sensory and autonomic nervous system. The disorder is invariably fatal, with only 50% of patients reaching age 30 years (see Ref. 12 and references therein). The identification of IKAP as a component of the human Elongator complex opens the possibility that the disease might be caused by reduced tissue-specific expression of genes and that it thus could be a transcription disorder. A further intriguing connection between Elongator and human disease stems from the recent finding that amino acid substitutions in the IKAP gene product can significantly increase the risk for bronchial asthma in children (25). Taken together, these connections suggest that cell lines with perturbed Elongator function might be used in genomics approaches to identify genes whose altered expression are causing these diseases.

Intriguingly, the hELP2 subunit of human Elongator is encoded by the homologue of mouse StIP1, which was recently isolated in a two-hybrid screen for proteins that interact with the conserved coiled-coil domain of the STAT3 protein (10). StIP1 exhibits an affinity for several STATs and also Janus kinases, and overexpression of the STAT3-binding domain of StIP1 blocks STAT3 activation, nuclear localization, and STAT3-dependent induction of a reporter gene (10). StIP1 was thus proposed to be a scaffold protein for the assembly of factors in the STAT signaling cascade in much the same way as IKAP was initially proposed to be a scaffold protein for the IB signaling cascade (9). These data are surprising in light of our finding of human StIP1 (hELP2) as a component of the RNAPII-interacting Elongator complex and because signaling pathways such as the NF-B- and the Janus kinase/STAT pathway are not conserved in lower eukaryotes, while StIP1/Elp2 clearly is. Possible explanations to this conundrum include that the reported interactions between the WD40 repeats of StIP1 (Elp2) and proteins of the Janus kinase/STAT pathway are nonspecific. Alternatively, Elongator might shuttle between the cytoplasm and the nucleus, or it might serve multiple distinct roles, or proteins that are subunits of the complex might play multiple roles. Our finding that IKAP is present both in Elongator and in fractions lacking detectable hELP3, and the fact that IKAP (and to a lesser degree hELP3) can be detected in the nucleoplasm, nucleoli, and cytoplasm of HeLa cells would be consistent with multiple roles for Elongator proteins in distinct cellular compartments.

	ACKNOWLEDGEMENTS

We acknowledge Grace Dahmus for providing purified RNAPII and the Imperial Cancer Research Fund service facilities, especially cell production services, for help. We thank Danny Reinberg for communicating results prior to publication, and we thank Peter Verrijzer and members of the Svejstrup laboratory for comments on the manuscript.

	FOOTNOTES

^* This work was supported by the Imperial Cancer Research Fund; Human Frontier Science Project Grant RG-193/97; an EMBO Long Term Fellowship (to G. S. W.); NCI, National Institutes of Health (NIH), Core Grant P30 CA08748 (to P. T.); and NIH Grant GM-33300 (to M. E. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) g8922735, g15292349, g15222770, and g7492054.

^§ These authors contributed equally to this work.

^§§ To whom correspondence should be addressed. Tel.: 44-207-269- 3960; Fax: 44-207-269-3801; E-mail: j.svejstrup@icrf.icnet.uk.

Published, JBC Papers in Press, November 19, 2001, DOI 10.1074/jbc.M110445200

² G. S. Winkler, and J. Q. Svejstrup, unpublished results.

	ABBREVIATIONS

The abbreviations used are: RNAPII, RNA polymerase II; HAT, histone acetyltransferase; IKAP, IKK complex-associated protein; STAT, signal transducer and activator of transcription; hELP1, -2, -3, and -4, human ELP1, -2, -3, and -4, respectively; CV, column volumes; MS, mass spectrometry; EEC, early elongation complex.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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