The Elongation Factor ELL (Eleven-Nineteen Lysine-Rich Leukemia) Is a Selective Coregulator for Steroid Receptor Functions

Laurent Pascual-Le Tallec, Federico Simone, Say Viengchareun, Geri Meduri, Michael J. Thirman and Marc Lombès

Institut National de la Santé et de la Recherche Médicale (L.P.-L.T., S.V., M.L.), Unité 478, Institut Fédératif de Recherche Claude Bernard, Faculté de Médecine Xavier Bichat, 75870 Paris cedex 18, France; University of Chicago (F.S., M.J.T.), Section of Hematology/Oncology, Chicago, Illinois 60637-1470; and Laboratoire d’Hormonologie et de Génétique Moléculaire (G.M.), Hôpital du Kremlin Bicêtre, 94270 Bicêtre, France

Address all correspondence and requests for reprints to: Marc Lombès, Institut National de la Santé et de la Recherche Médicale, Unité 693, Faculté de Médecine Paris-Sud, 63 rue Gabriel Peri, 94276 Le Kremlin Bicetre cedex, France. E-mail: marc.lombes{at}kb.u-psud.fr.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The dynamic and coordinated recruitment of coregulators by steroid receptors is critical for specific gene transcriptional activation. To identify new cofactors of the human (h) mineralocorticoid receptor (MR), its highly specific N-terminal domain was used as bait in a yeast two-hybrid approach. We isolated ELL (eleven-nineteen lysine-rich leukemia), a RNA polymerase II elongation factor which, when fused to MLL (mixed lineage leukemia) contributes to the pathogenesis of acute leukemia. Specific interaction between hMR and ELL was confirmed by glutathione-S-transferase pull-down and coimmunoprecipitation experiments. Transient transfections demonstrated that ELL increased receptor transcriptional potency and hormonal efficacy, indicating that ELL behaves as a bona fide MR coactivator. Of major interest, ELL differentially modulates steroid receptor responses, with striking opposite effects on hMR and glucocorticoid receptor-mediated transactivation, without affecting that of androgen and progesterone receptors. Furthermore, the MLL-ELL fusion protein, as well as several ELL truncated mutants and the ELL L214V mutant, lost their ability to potentiate MR transcriptional activities, suggesting that both the elongation domain and the ELL-associated factor 1 interaction domains are required for ELL to fulfill its selector activity on steroid receptors. This study is the first direct demonstration of a functional interaction between a nuclear receptor and an elongation factor. These results provide further evidence that the selectivity of the mineralo vs. glucocorticoid signaling pathways also occurs at the transcriptional complex level and may have major pathophysiological implications, most notably in leukemogenesis and corticosteroid-induced apoptosis. These findings allow us to propose the concept of "transcriptional selector" for ELL on steroid receptor transcriptional functions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MINERALOCORTICOID AND GLUCOCORTICOID hormones exert similar, but nonoverlapping, effects through corticosteroid hormone signaling pathways. They share common features in terms of basic molecular mechanisms of action, mediated by two closely related nuclear receptors (1), the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Both receptors may be activated by mineralocorticoids and glucocorticoids, bind common response elements on promoters of target genes, and are able to interact with identical coregulators of the p160/steroid receptor coactivator 1 family, and the cAMP response element binding protein (CREB)-binding protein (CBP)/p300 leading to the recruitment of the general transcriptional machinery and turn-on gene expression (2, 3). Nevertheless, aldosterone and MR possess distinct and specific actions, differing from those of GR, as demonstrated by their well-established roles in the maintenance of sodium and potassium homeostasis (4, 5) and the regulation of blood pressure (6), as well as the modulation of neuronal activity in the central nervous system (7). With regard to these assertions, it is necessary to postulate that particular mechanisms account for MR vs. GR specificity of action.

Such specificity-conferring mechanisms have been proposed at both prereceptor and receptor levels (8). First, it is generally admitted that the 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) enzymatic activity is essentially responsible for mineralocorticoid selectivity in vivo through prevention of illicit MR occupancy and activation by glucocorticoids (9, 10). Of importance, the expression of 11ßHSD2 seems to be restricted to epithelial aldosterone-target tissues questioning on the validity of the mechanisms of selectivity in nonepithelial tissues. At the receptor level, the ligand-binding domain (LBD) of MR plays a central role in hormone selectivity, most notably through its discrimination properties, i.e. affinity constants and dissociation kinetics (11, 12) allowing the proper exposure of the activating function 2 (AF2) and subsequent activation of transcription. However, the mechanisms above do not account for the overall selectivity of MR action at the transcriptional level. Accordingly, one can postulate that distinct actions of MR and GR may also depend on recruitment of specific coregulators that potentially mobilize different active transcriptional complexes. With regard to MR, few general coactivators, including glucocorticoid receptor-interacting protein 1, transcriptional intermediary factor 1{alpha}, steroid receptor coactivator 1, transcriptional intermediary factor 2, and RNA helicase A (RHA)/CBP, have been reported to interact with the receptor (13, 14, 15, 16). Yet, none of them described so far, distinguishes between MR and GR, suggesting that strict partners of MR or GR may exist and still remain to be identified. We and others (15, 16, 17, 18) have already postulated that the particular N-terminal domain (NTD) of MR could sustain specific action of the receptor because 1) it shares less than 15% sequence homology with the NTD of GR; 2) it is highly conserved among species; 3) it possesses two distinct activating functions referred to as AF1a and AF1b (15, 17), diverging from the unique one of GR (19).

To identify receptor-selective cofactors, we have previously reported a two-hybrid screening with the full-length NTD of human (h) MR, first describing the role of PIAS1 [protein inhibitor of activated STAT (signal transducer and activator of transcription) 1] interaction and sumoylation process in the specific repression of MR transactivation functions (17). In the present study, we have isolated, by the same approach using the amino acid residues 163–437 of NTD as bait, the human elongation factor ELL (eleven-nineteen lysine-rich leukemia), as a novel molecular partner of MR. ELL is mainly known to increase the RNA polymerase II (Pol II) processivity by suppressing polymerase pausing, premature arrest, and abortive elongation (20). Here, we demonstrated its discriminative role as a coactivator for MR and a corepressor for GR. In addition, the functional and physiological significance of the MR-ELL interaction has been investigated, describing the first example of a component of the transcriptional machinery that can act as a subtle selector modulating steroid receptor signaling pathways.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of ELL as a hMR Molecular Partner
With the aim to identify novel specific partners of hMR, we used its NTD to isolate such factors because it shares less than 15% of identity with other steroid receptors and thus is likely to confer part of the mineralocorticoid specificity. The subdomain spanning from amino acids 163–437 (Fig. 1AGo), which has been demonstrated to carry inhibitory functions and referred to as inhibitory domain (ID) (17), served as bait for yeast two-hybrid screening of a human kidney cDNA library. Of the 13 clones isolated, one of the strong interacting candidates was a 3.5-kb cDNA encoding for the human elongation factor ELL protein, which lacked the first 73 amino acids. To confirm biochemically this interaction, we performed glutathione-S-transferase (GST) pull-down assays using GST-NTD proteins and in vitro translated [35S]Met-labeled full-length ELL. Figure 1BGo (upper panel) shows that the GST-fused proteins containing the 163–437 fragment or the 1–602 fragment, which represents the entire NTD, interacted with ELL, whereas the luciferase protein, used as control, was unable to bind these hMR subdomains (lower panel). Due to the presence of only three methionines in the full-length ELL protein, the low specific activity of the radiolabeled protein led to very weak signals. Therefore, reciprocal pull-down experiments were performed with GST-ELL and in vitro translated hMR and confirmed this interaction (Fig. 1CGo). Both full-length hMR and 1–602 NTD were able to bind GST-ELL, demonstrating that ELL physically interacts with the NTD of hMR. Further delineations showed that the 445–602 fragment was also able to strongly bind ELL, whereas the fragments 1–167 and, most surprisingly, 163–437 were not retained by the protein (Fig. 1CGo, lower panel). To reconcile the apparent discrepancy concerning the interaction between ELL and the 163–437 domain, it should be noted that, both in yeast two-hybrid screening and pull-down experiments, the N-terminal extremity of the 163–437 fragment was fused to LexA and GST, respectively, probably allowing a more structured conformation and thus facilitating the interaction. Taken together, these data indicate that ELL interacts with hMR presumably through an overlapping region within the 163–437 and 445–602 junction. However, we could not exclude a bipartite interaction, which remains to be more precisely delineated.



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Fig. 1. ELL Is a Molecular Partner of hMR Interacting with Its NTD

A, Schematic representations of the functional domains of hMR and ELL. B, Biochemical interactions between hMR and ELL by GST pull-down assays. ELL (upper panel) and luciferase as control (lower panel) were labeled with [35S]Met by in vitro translation and incubated with glutathione sepharose-bound GST-NTDs. After extensive washes, bound proteins were eluted in sample buffer and separated onto 10% SDS-PAGE. C, Reverse GST pull-down experiments performed with in vitro translated full-length hMR or various subdomains of hMR and glutathione sepharose-bound GST-ELL, resolved onto 10% SDS-PAGE. DBD, DNA-binding domain.

 
ELL Acts as a Coactivator of MR
To further examine the functional significance of ELL-hMR interaction, we tested the ability of ELL to modulate hMR transactivation functions. Transient cotransfections of hMR and ELL were performed in rabbit renal RC.SV3 cells, which do not express endogenous functional MR, with glucocorticoid response element (GRE) promoter driving luciferase gene expression. Figure 2AGo (upper panel) shows that ELL increased MR transcriptional activity by 2.5-fold in a dose-dependent manner, without affecting total hMR protein levels as assayed by Western blot analysis performed with an anti-FLAG antibody (lower panel). Increasing amounts of hMR expression vector cotransfected with a constant amount of ELL plasmid also led to a major potentiation of hMR transactivation, which reaches a plateau. Under the same experimental conditions, hMR transactivation was linear with control vector (Fig. 2BGo). This result indicates that ELL directly affects hMR transcriptional activity and possibly constitutes a limiting cofactor in the cells. Dose-response experiments showed that the coactivating properties of ELL increase the maximal MR transactivation capacity (Fig. 2CGo), but, interestingly, the EC50 of aldosterone for MR was shifted from 5 x 10–10 M to 5 x 10–11 M, consistent with an increase in hormonal sensitivity. To examine the influence of the nature of the ligand on hMR responses to ELL, experiments performed with cortisol and spironolactone, for its partial mineralocorticoid agonist activity, led to the same potentiating effects (Fig. 2DGo). These results indicated that a particular LBD conformation induced by the nature of the bound agonistic ligand was not crucial for ELL action on MR and thus presumably independent of the AF2. We next investigated whether ELL was also effective on hMR stably expressed in the M cells derived from the parental RC.SV3 (21). Figure 2EGo shows that transient transfections, performed with increasing amounts of ELL expression vector, also increased the transcriptional activity of hMR expressed at physiological levels (21), thus confirming our previous results. Collectively, the elongation factor ELL fulfills many of the properties required for a nuclear receptor coactivator.



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Fig. 2. ELL Is a Coactivator of hMR Transcriptional Activity

Functional effects of ELL were evaluated by transient transfections in the rabbit kidney RC.SV3 cell line (A–D) and its derived M cell line (E). A (upper panel), RC.SV3 cells were cotransfected with hMR (0.1 µg pcDNA3.hMR) in the presence of increasing amounts of pCMV2-FLAG-ELL plasmid (0.1 and 0.5 µg), and treated with 10–8 M aldosterone for 24 h in a steroid-free medium. Relative transcriptional activities were expressed as a percentage of the control (same amount of pCMV2-FLAG plasmid). A (lower panel), COS1 cells were cotransfected with FLAG-hMR plasmid (0.1 µg pCMV2-FLAG-hMR) and increasing amounts of ELL plasmid (pcDNA3.ELL 0.1 and 0.5 µg). Western blot analysis of total proteins separated onto 7.5% SDS-PAGE was performed with mouse anti-FLAG antibody (M2-FLAG). B, Same experiments were performed with cells transfected with increasing amounts of hMR (from 1.5–150 ng of pcDNA3.hMR) in the presence of constant amounts (375 ng) of pCMV2-FLAG-ELL or pCMV2-FLAG plasmids. C, Cells were transfected with hMR (0.1 µg pcDNA3.hMR) and pCMV2-FLAG-ELL or pCMV2-FLAG plasmids (0.5 µg) and treated for 24 h with aldosterone (from 10–11 to 10–7 M). Results are expressed as percentage of control arbitrarily fixed at 100% for 10–7 M aldosterone. D, Agonist effects of 10–7 M cortisol and 10–6 M spironolactone were tested as described in panel C in the absence (black bars) and presence (white bars) of ELL. Results are expressed as percentage of transcriptional activity of hMR arbitrarily fixed at 100% in the absence of ELL for 10–8 M aldosterone. Note that the relative hMR transcriptional activities upon 10–7 M cortisol and 10–6 M spironolactone are 150% and 14%, respectively, compared with 10–8 M aldosterone treatment. Fold increases in transactivation with ELL are indicated on the top of each paired bars. E, M cells stably expressing hMR were transfected with increasing amounts of pCMV2-FLAG-ELL plasmid (0.5, 2, and 4 µg). All transfections were performed along with 0.5 µg of pSV.ß-Gal and 0.33 µg of pGL3-GRE2-TATA-Luc plasmids. Results are expressed as relative transcriptional activity (Luc/ß-gal activity) as a percentage of their respective controls. Results represent mean ± SD of at least three independent determinations. ***, P < 0.001. RLU, Relative light units.

 
To determine the relative contribution of hMR AFs, we investigated the effect of ELL on different hMR deletion mutants (Fig. 3AGo). Those mutants were first analyzed for their intrinsic transcriptional capacities. Figure 3BGo shows that the naturally occurring hMR mutant {Delta}5,6, lacking the entire LBD (22), exhibits 40% of wild-type hMR transcriptional activity, whereas the N454 hMR mutant lacking the first 453 N-terminal amino acids, as well as the {Delta}AB hMR mutant lacking the entire NTD, display about 20% of that of wild-type hMR. Interestingly, ELL was still able to potentiate the transcriptional activities of the {Delta}5,6 and N454 mutants by an approximately 5- and 3-fold factor, respectively (Fig. 3CGo); however, it had no significant effect on the transcriptional properties of the {Delta}AB mutant. These results indicated that both the AF2 located in the LBD, and the AF1a within the first 167 amino acids (see Fig. 3AGo), were dispensable for the potentiating effects of ELL on hMR and strongly suggested the implication of the AF1b. Furthermore, these results functionally confirmed the physical interaction of ELL with the 445–602 fragment (Fig. 1CGo and data not shown) because the N454 mutant transactivation was still increased to a similar extent compared with that of wild type. Taken together, ELL displays all the functional properties for an AF1b-specific transcriptional coactivator of MR in a ligand and/or AF2-independent manner.



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Fig. 3. Potentiation of Transcriptional Activities of hMR Mutants by ELL

A, Schematic representations of hMR mutants. B, Transfections were performed as described in the legend of Fig. 2Go. Maximal intrinsic transcriptional activities of different hMR deletion mutants (0.1 µg of pcDNA3.hMR, {Delta}5,6, N454, {Delta}AB, and K12345) were evaluated after incubation with 10–8 M aldosterone and compared with wild-type hMR, arbitrarily set at 100%. C, Effects of ELL [0.5 µg of pCMV2-ELL (white bars) or empty pCMV2 (black bars)] were tested on the transcriptional activities of these mutants. Results represent mean ± SD of three independent experiments performed in triplicate. ***, P < 0.001. ns, Not significant (P > 0.05). DBD, DNA-binding domain.

 
We have previously showed that hMR possesses five functional sumoylation sites (Fig. 3AGo) responsible for the repression of MR transcriptional properties on a GRE2 promoter (17). We next addressed the possible relationship between these sites and ELL, using the K12345 mutant of hMR, which is unable to undergo sumoylation and possesses an intrinsic 3- to 5-fold increased transcriptional activity (Ref.17 and Fig. 3BGo). We showed that ELL still potentiates K12345 mutant transactivation by a 2-fold factor (Fig. 3CGo), indicating that ELL-mediated coactivation was independent of the sumoylation status of MR.

ELL Is a Specific Coregulator of Corticosteroid Receptors
We next tested the ability of ELL to modulate transcriptional activities of other steroid receptors [hGR, human progesterone receptor (hPR), and human androgen receptor (hAR)] on canonical GRE2 (Fig. 4Go). As expected, in RC.SV3 cells, ELL increased by 2.5-fold the hMR-mediated transactivation. Surprisingly, however, under the same experimental conditions, ELL drastically repressed the hGR-mediated transactivation up to 90% (Fig. 4AGo). Of interest, ELL had no effect on AR or PR-mediated transactivations. Similar discriminating properties of ELL were obtained with a natural GRE (–1177/–1147bp) from the promoter of human SGK1 gene (data not shown). These results were confirmed in COS1 cells (Fig. 4BGo), for which ELL strongly increased hMR transactivation by 12-fold and still repressed that of GR up to 75%, without affecting those of AR and PR, indicating that ELL effects were independent from the cellular context. We further demonstrated by Western blot that ELL did not affect the expression level of GR protein (Fig. 4CGo) or that of MR as previously shown (Fig. 2AGo). To explore the mechanisms by which ELL acts as a corepressor on GR activity, we also showed a direct interaction between GST-fused ELL and in vitro translated [35S]Met-labeled GR as demonstrated by GST pull-down assays (Fig. 4DGo).



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Fig. 4. ELL Is a Transcriptional Selector of Steroid Receptor Functions

Transfections were carried out in RC.SV3 (A) or COS1 (B) cells as described in the legend of Fig. 2Go. Cells were transfected with steroid receptor plasmids (0.1 µg of each pcDNA3-receptors) together with pCMV2-FLAG as control (0.5 µg, black bars), or pCMV2-FLAG-ELL (0.5 µg, white bars) and treated for 24 h with 10–8 M aldosterone for hMR, 10–7 M dexamethasone for hGR, 10–7 M progesterone for hPR, and 10–7 M dihydrotestosterone for hAR. Results represent mean ± SD of three independent experiments performed in triplicate. ***, P < 0.001. C, COS1 cells were cotransfected without (lane 1) or with hGR plasmid (0.1 µg pcDNA3-hGR) together with 0.5 µg pCMV2-FLAG control (lane 2) or pCMV2 FLAG-ELL plasmid (lane 3). Western blot analysis of total proteins separated onto 7.5% SDS-PAGE was performed with mouse anti-GR (N449) or anti-{alpha}-tubulin antibodies. D, GST pull-down experiments performed with in vitro translated [35S]Met-labeled hGR and glutathione sepharose-bound GST-ELL, resolved onto 10% SDS-PAGE. E, The sequence of the hELL1 region encompassing amino acids 206–250 and its alignment with other members of the ELL protein family from human (h), mouse (m), Xenopus (x) and Drosophila (d) are depicted. Putative nuclear receptor boxes are overlined. F, Leucine to valine ELL mutants were assayed for their ability to modulate MR and GR transactivation. Transfections were performed in RC.SV3 cells with 0.1 µg of hMR or hGR plasmid together with 0.5 µg of plasmids (in pCMV2-FLAG vector) encoding wild-type ELL or the different ELL mutants or empty pCMV2-FLAG as control. Results are expressed as percentage of transcriptional activities of control MR or GR arbitrarily fixed at 100% for 24-h treatment with 10–8M aldosterone and 10–7 M dexamethasone, respectively, and represent mean ± SE of at least four independent experiments performed in triplicate. ns, Not significant (P > 0.05).

 
Most coactivators possess nuclear receptor boxes defined by the peptide sequence LXXLL, a motif that acts as the physical interface between the coactivator and the steroid receptor (23). Thus, inspection of ELL primary sequence reveals that its N-teminal domain possesses three potential direct or inverted nuclear receptor boxes, which could account for its coregulatory activity (Fig. 4EGo). To test the respective contribution of these motifs, we mutated leucines to valines at positions 214, 226, and 245 and examined the ability of these ELL mutants to modulate MR and GR transcriptional activities. Figure 4FGo shows that, whereas all ELL mutants maintained their corepressive activity on GR-mediated transactivation, changing leucine 214 to valine (L214V) almost totally abolished ELL-stimulatory effects on MR function. In contrast, the two other mutants (L226V and L245V) displayed the same coactivating properties as wild-type ELL. This strongly suggests that the sequence encompassing L214 plays a crucial role in potentiating MR activity.

Taken together, our findings demonstrated that ELL is a specific coregulator of corticosteroid receptors (MR and GR) with no effect on other steroid receptors. Furthermore, because ELL is able to discriminate among steroid receptors by directly acting as a MR coactivator and as a GR corepressor, we propose a novel concept of subtle transcriptional selector for ELL, which governs positively or negatively corticosteroid receptor-mediated transactivation.

Functional and Physiological Significance of ELL-hMR Interaction
We analyzed the in vivo binding between MR and ELL using cells cotransfected with epitope-tagged versions of ELL and MR or negative [p300/CREB-binding protein-associated factor (PCAF)] and positive [ELL-associated factor 1 (EAF1)] protein controls. Comparable expression of PCAF, MR, and EAF1 proteins was first verified by Western blot with anti-FLAG antibody (Fig. 5Go, left panel). In the same cotransfected cell lysates, comparable protein levels of ELL were also observed using anti-hemagglutinin (HA) antibody (Fig. 5Go, middle panel). Thus, we demonstrated by immunoprecipitation of the FLAG-MR protein complexes, followed by Western blot with anti-HA antibody, that ELL and MR physically interact in vivo (Fig. 5Go, right panel). The binding of transfected MR to ELL was less pronounced than that of transfected EAF1 to ELL, suggesting either a lower binding affinity of MR for ELL, supported by the fact that we failed to coimmunoprecipitate transfected MR with endogenous ELL using the ELL antiserum, or that only a limited amount of MR is capable of interaction with ELL.



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Fig. 5. Physical in Vivo Interaction between ELL and hMR

HEK 293 cells were transiently cotransfected with HA-tagged ELL and FLAG-tagged MR. As a positive control for ELL binding, HEK 293 cells were cotransfected with HA-ELL and FLAG-EAF1. As a negative control for ELL binding, HEK 293 cells were cotransfected with HA-ELL and FLAG-PCAF. Expression of the transfected proteins was detected with {alpha}-FLAG antibody for PCAF, MR, and EAF1 proteins (left panel) and with {alpha}-HA antibody for ELL protein (middle panel). Cell lysates were first immunoprecipitated with the {alpha}-FLAG antibody and then immunoblotted with {alpha}-HA antibody (right panel).

 
We also examined the effect on MR and GR transcriptional activities of the mixed lineage leukemia (MLL)-ELL protein, recently reported to play a critical role in the pathogenesis of acute leukemia. ELL, in this context of fusion protein with MLL, completely lost its capacity to stimulate MR function (Fig. 6Go) but instead caused a drastic inhibition of MR transcriptional activity such as that observed for GR either with ELL or MLL-ELL. We were also interested to determine which domain of ELL was required for the potentiation of MR functional activity by using various N- or C-terminal truncated forms of ELL as depicted in Fig. 6Go. Because ELL was found to contain two domains that bind EAF1, one in the N terminus and one in the C terminus, we tested the effects of several deletion mutants including ELL 1–210, ELL 207–411, and ELL 400–621. All ELL deletion mutants efficiently inhibited hMR transcriptional activity. Similarly, ELL, as well as all ELL mutants, was equally potent in inhibiting GR transcriptional activity. Collectively, our results firmly establish a strikingly different behavior of full-length ELL and ELL mutants or MLL-ELL on MR and GR functions. This also suggests the necessary requirement of the integrity of the two EAF1 interacting domains as well as the importance of leucine 214 for ELL-mediating MR potentiation.



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Fig. 6. Effects of MLL-ELL Fusion Protein and ELL Deletion Mutants on hMR- and hGR-Mediated Transactivation

MLL-ELL and full-length and truncated ELL proteins are schematically depicted together with the putative functional domains and their amino acid positioning onto the protein. Transfections were performed in RC.SV3 cells as described in the legend of Fig. 2Go, with 0.5 µg of plasmids (in pCMV2-FLAG vector) encoding the different ELL mutants or MLL-ELL proteins or pCMV2-FLAG as control. Results are expressed as percentage of transcriptional activities of control MR or GR arbitrarily fixed at 100% in the absence of ELL for 24-h treatment with 10–8 M aldosterone and 10–7 M dexamethasone, respectively, and represent mean ± SE of at least four independent experiments performed in triplicate.

 
Finally, to examine whether the physical interaction between ELL and hMR had a potentially physiological significance, we investigated whether hMR and ELL were indeed colocalized within the same aldosterone-sensitive tissues. As shown in Fig. 7AGo, using specific antibodies, we were able to demonstrate that hMR, together with ELL, was detected in the nuclei of cortical collecting duct cells of human kidney. We also addressed the question of whether ELL gene expression could be submitted to mineralocorticoid regulation using the recently established aldosterone-sensitive renal cell line KC3AC1 (24). As illustrated in Fig. 7BGo, quantitative real-time PCR revealed that the ELL mRNA level was up-regulated by at least 4-fold upon 10–8 M aldosterone treatment for 1 h and remained significantly higher after 24 h stimulation in KC3AC1 cells. This is the first demonstration that ELL is an early inducible mineralocorticoid target gene under physiological aldosterone concentrations. Altogether, our findings provide evidence for a direct relationship between ELL and the mineralocorticoid signaling pathway.



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Fig. 7. Colocalization of ELL-hMR and Functional Significance

A, Serial sections of human kidney were used to immunodetect ELL and hMR in the distal parts of the distal nephron. Right panel, ELL was clearly identified in the nuclei of the tubular cells using anti-ELL antibodies (43 ). Left panel, The same positively stained cells also expressed hMR as revealed by immunostaining with the anti-hMR polyclonal antibody (A4) (45 ). B, Real-time PCR analysis of ELL mRNA expression in renal cells after aldosterone treatment. Highly differentiated renal cortical collecting duct KC3AC1 cells were grown on filters and treated for 1 h or 24 h with 10–8 M aldosterone. Results normalized by amplification of the 18S ribosomal RNA are expressed as relative fold induction compared with control conditions set at 1. Each point represent mean ± SD of at least two independent determinations performed in duplicate.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present paper, we identified ELL as a novel interacting partner of hMR. ELL belongs to a class of elongation factors that included elongin, SII, transcription factor IIF, or P-TEFb and multiple ELL homolog proteins (20). Those factors regulate the activity of the transcription complex through increasing Pol II processivity by preventing Pol II premature arrests or increasing Pol II catalyzing rates mainly by suppressing transient pausing (20). Yeast two-hybrid isolation, GST pull-down experiments and transient transfection assays demonstrated that ELL-MR interaction requires the NTD of MR, which displays important functional properties. We and others reported two distinct ligand-independent transactivation domains, AF1a and AF1b, and a central ID (15, 17). Several lines of evidence suggest that ELL may contact the region spanning from amino acids 163–602 with an interface, which presumably overlaps the junction between the ID and the AF1b (see Fig. 1AGo) but remains to be more precisely defined. The functional characterization of this interaction indicated that ELL constitutes a specific coactivator of the AF1b transactivation function of hMR, as revealed by its binding to and activation of the N454 hMR but not of the {Delta}AB mutant (Fig. 3CGo). Thus, ELL could be considered as an AF1b counterpart of the RNA helicase A (RHA), which interacts with the extreme N-terminal AF1a domain (16). It is tempting to propose that MR, through its NTD, recruits several specific cofactors that serve as bridging factors with Pol II, such as RHA, which directly interacts with CBP/p300 (15, 16, 25), but also elongation factors necessary to achieve the ordered, sequential, and combinatorial formation of a fully active transcriptional complex as recently described for ER{alpha} (26).

We demonstrated that ELL acts as a bona fide transcriptional coactivator of hMR on consensus and natural GRE. This finding constitutes the first report of a direct physical and functional interaction between a nuclear receptor and a transcriptional elongation factor. In addition, the coactivating properties of ELL were selectively restricted to MR, because ELL drastically inhibited GR-mediated transcription without affecting those of AR and PR. Of note these effects were demonstrated in the three different cell types tested (rabbit kidney RC.SV3, simian fibroblast COS1, and murine cortical collecting duct KC3.AC1 cell lines) indicating that ELL action on steroid receptors is independent of the cellular context. This discriminating impact on steroid receptor functions led us to propose for ELL, not only a determinant role in the regulation of Pol II processivity but, as a new concept, a role of transcriptional selector of steroid receptor action.

We have previously demonstrated that part of the MR specificity was conferred by its NTD with the repressive action of protein inhibitor of activated STAT (signal transducer and activator of transcription) 1 (PIAS1) on MR, but not on GR-mediated transactivation, in part through SUMO (small ubiquitin-like modifier) modifications of MR (17). A recent publication reports the modulation of AR transcriptional function upon SUMO-dependent recruitment of coregulators (27). Here, we demonstrated that ELL coactivator properties on MR were totally independent of sumoylation status of MR. More importantly, we showed that ELL plays a discriminating role on the balance between activation/repression of steroid receptor action. The dynamics of the preinitiation complex (PIC) formation could explain these differences. Indeed, it is known that the stage of ELL entry into the sequential and combinatorial assembly of the PIC greatly influences the transcriptional effect of ELL on Pol II-mediated mRNA synthesis (28). When added before the PIC formation, ELL significantly inhibits transcriptional initiation events by aborting the third phosphodiester bond of the nascent transcripts. In contrast, ELL greatly increases Pol II activity when added after formation of the PIC. Considering our results, it is likely that MR incorporates ELL after the formation of the transcriptionally productive complex, whereas GR may recruit ELL within the PIC before its assembly. This selectivity achieved by ELL at the transcriptional level constitutes an additional molecular mechanism implicated in the specificity of the mineralo- vs. glucocorticoid signaling pathways beside that conferred by the 11ßHSD2, the agonist-dependent receptor conformation (10), and the ligand-selective coactivator recruitment (16). Furthermore, we showed that ELL and hMR are specifically coexpressed in vivo in aldosterone target cells of the distal nephron, emphasizing its central role of transcriptional selector of the mineralocorticoid pathway. Incidentally, real-time PCR analysis revealed that in the highly differentiated renal cortical collecting KC3AC cell line (24), ELL expression is rapidly increased after aldosterone treatment. A recent report showed that the gene expression of another member of ELL protein family, ELL2, was stimulated by glucocorticoids in human osteosarcoma cells (29), indicating that ELL homologs are corticosteroid target genes, thus constituting an intriguing hormonal regulatory loop. Taken together, our findings allow us to gain further mechanistic insights on how MR and GR can differentially activate the expression of specific target genes.

Because ELL has been identified as a fusion partner gene of MLL in the (11, 19)(q23;p13.1) translocation (30) and also directly implicated in the development of acute myeloid leukemia (31, 32), it was of great interest to further investigate whether MR and/or GR functions might be modulated by MLL-ELL fusion protein. It has been shown that ELL contains two separable binding domains for EAF1, which by itself supports leukemic activity of the MLL-ELL protein (31). The C-terminal EAF1 interaction domain of ELL is necessary and sufficient for the initiation of leukemogenesis mediated by MLL-ELL. A second EAF1 interacting domain is also present in the N terminus of ELL. The EAF2 protein, a functional homolog of EAF1, also binds to this N-terminal interaction domain within ELL (33). Of interest, the MLL-ELL fusion protein does not retain the potential to bind to EAF2. In addition, ELL has been reported to contain a domain in its carboxy terminus that is required for the interaction with, and the functional inhibition of, p53 transcriptional activity (34) and for apoptotic effects (35). Functional assays with various fusion or truncated ELL proteins clearly demonstrated that both N- and C-terminal domains of ELL are required for MR potentiation effects, suggesting that elongation activity and protein interactions are both important for MR-mediated functions. Future studies specifically exploring the elongation properties of ELL will be crucial for a better understanding of its role played in gene regulation. Of particular interest, point mutations of ELL NTD demonstrated the major importance of the leucine in position 214, which is embedded in a VLHL214LALR motif spanning from amino acids 211 and 218, in potentiating MR transactivation. This motif, located in the elongation domain of ELL, is highly conserved between human, mouse, Xenopus, and Drosophila ELL family proteins (36, 37, 38) (see Fig. 4EGo), suggesting a major functional role of this region presumably involved in the elongation activity of ELL protein and/or constituting a site contact surface for protein-protein interaction. Interestingly, this critical region is present in hELL1 and hELL2 but absent in hELL3, which lacks the 235 first amino acids, and the expression of which is restricted to the testis (39), raising the possibility that ELL family proteins differentially affect gene transcription through physical interaction and functional interference with distinct proteins in a tissue-specific manner.

With respect to the pathophysiological significance of ELL-MR interaction, a recent report demonstrated that the NTD of MR prevents dexamethasone-induced GR-mediated apoptosis of prelymphocyte B cells (40). The authors of this interesting study made the assumption that MR and GR could, in concert, trigger the balance between proliferation and cell death through the action of shared cofactors. Considering our results, we propose that ELL might be such a candidate plus the fact that preliminary immunohistochemical studies indicate that ELL and MR indeed colocalize in lymphoid cells (data not shown). Along this line, in target cells where MR and GR coexist, ELL may actually exert a crucial role in determining MR- and GR-mediated effects. This might be also particularly relevant in the central nervous system in which pro- and antiapoptotic actions of these corticosteroid receptors, most notably through their NTDs, determine neuronal fate (18, 41).

In sum, ELL represents a unique example of remarkable selectivity based upon opposite modulation of steroid receptor functions, whereas generally most of the other known coregulators do not have a discriminating action on nuclear receptors. Our data open a new field of investigations concerning the role of MR-mediated gene expression, through the regulation of Pol II activity by specific elongation factors acting as coactivator, and its implication in pathophysiological processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
hMR N-terminal fragments were generated by PCR using pRS-hMR as template (42) and high-fidelity polymerase Taq platinium (Invitrogen, San Diego, CA) as described elsewhere (17). hMR was also subcloned from pcDNA3-hMR using HindIII and XhoI sites into pCMV2-FLAG at HindIII and SalI sites. Other vectors included N454-hMR in pcDNA3 (21), hMR{Delta}5,6 in pcDNA3 (22), {Delta}ABhMR (kindly provided by Dr. Rafestin-Oblin), hGR or hPR or hAR in eukaryote expression vectors, pSV-ß-galactosidase, and pGL3-GRE2-TATA-Luc. ELL digested from pCMV2-FLAG vector was subcloned into pcDNA3 and in frame into pGEX-KG, using NotI and BamHI/XbaI restriction sites, respectively. All truncated ELL constructs were described previously (31, 43). Leucine to valine mutations were obtained by PCR-based targeted single-point mutation using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) with pCMV2-FLAG-ELL as a matrix. Mutated plasmids were sequenced to verify the correct mutation.

Yeast Two-Hybrid Screening and Analysis of Interaction Specificity
Yeast two-hybrid screening was performed as described (17). Briefly, pLexA-163–437 plasmid was used as bait to screen a human kidney cDNA library (Matchmaker GAL4 two-hybrid) cloned into a pACT2 vector (CLONTECH, Palo Alto, CA). Clones were selected on triple selective medium (DO-WLH) containing 5 mM 3-amino-1,2,4 triazole to lower background. Positive clones were tested for ß-galactosidase activities. Plasmids were rescued from His+/LacZ+ colonies through classical procedures. Specific protein-protein interactions were analyzed by yeast mating tests, and selected plasmids were then sequenced.

GST Pull-Down Assays
GST pull-down experiments were performed as described elsewhere (17). BL21-gold (DE3) Escherichia coli bacteria (Stratagene) were transformed with GST-fused proteins cloned into pGEX vector. In vitro translated proteins by TnT-T7 Quick Coupled Transcription/Translation kit (Promega Corp., Madison, WI), labeled with [35S]methionine (Amersham Pharmacia Biotech, Orsay, France), were added to immobilized GST-fused proteins and were incubated for 1 h at 4 C. After washes with HKEN buffer (25 mM HEPES, pH 7.9; 60 mM KCl; 1 mM EDTA; 0.5% Nonidet P-40; 1 mM dithiothreitol; 1 mM phenylmethylsulfonylfluoride; 5 µg/ml Leupeptine), interacting proteins were eluted in 2x Laemmli loading buffer, boiled, separated onto 10% acrylamide SDS-PAGE, and autoradiographed.

Immunoprecipitations and Western Blots
Human embryonic kidney (HEK) 293 cells were transiently cotransfected with hemagglutinin (HA)-ELL and either FLAG-MR, FLAG-PCAF, or FLAG-EAF1. Cells were lysed in NETN (100 mM NaCl; 20 mM Tris, pH 8.0; 1 mM EDTA; and 0.2% NP-40) with a protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). To precipitate the complexes, supernatants were precleared with protein A/G agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then incubated overnight with the {alpha}-FLAG-M2 antibody (Sigma) at 1:500 and protein A/G agarose beads. The beads were washed five times with NETN, boiled in sample buffer, and fractionated by SDS-PAGE. Western blot experiments were performed using mouse anti-FLAG (1:1000), rabbit N499 anti-hGR (1:1000) kindly provided by M.J. Garabedian, and mouse anti-{alpha} tubulin (1:1000). Goat secondary antibodies were coupled to peroxidase and signals were detected by chemiluminescent Immun-Star reagents (Bio-Rad Laboratories, Inc., Marnes la Coquette, France).

Cell Culture and Transient Transfection
RC.SV3 cells (44), M cells (21), and COS1 cells were cultured and transfected as previously described (17). All products for cell culture were purchased from Invitrogen. For transient transfections, cells were seeded at 4 x 105 cells per well in steroid free medium for 6 h and transfected by the calcium phosphate precipitation method. After a 24-h hormone incubation period, cells were harvested, and ß-galactosidase and luciferase assays were performed as described elsewhere (17). Luciferase activities were normalized to ß-galactosidase activities and expressed as percent of relative transcriptional activities compared with controls.

Immunohistochemistry
Sections (0.5 µm thick) of formol-fixed paraffin-embedded normal renal tissue were deparaffinized by successive baths in xylene and alcohol. Antigen retrieval was performed in a microwave oven at full power in pH 6 citrate buffer for 15 min. Endogenous biotin activity was quenched with the Biotin blocking system (DAKO Corp., Carpinteria, CA). After overnight incubation with the primary antibodies (rabbit polyclonal antibodies anti-MR A4 (45) or anti-ELL (43) at 4 C, endogenous peroxidases were quenched with 3% H2O2 in PBS for 5 min and bound immunoglobulins were revealed with a secondary biotinylated antibody and peroxidase-labeled streptavidin (LSAB2 immunostaining Kit; DAKO). Aminoethylcarbazol (Sigma-Aldrich Chemical Co., Saint Quentin Fallavier, France) was used as a chromogen. Each experiment was performed at least twice in duplicate.

Real-Time PCR
Total RNA from KC3AC1 cells (24) grown on filters was isolated by Trizol extraction technique (Invitrogen). Total RNA (1 µg) was treated with DNase I Amplification Grade procedure (Invitrogen). Random primed cDNA was prepared from total RNA with 200 U of reverse transcriptase using the Superscript II kit (Invitrogen). Real-time PCR carried out on an ABI 7700 Sequence Detector (Applied Biosystems, Foster City, CA) using the qPCR Mastermix Plus for Sybr Green I (Eurogentec, Seraing, Belgium). PCR were performed with 50 ng cDNA in the presence of 2.5 mM MgCl2, 200 µM deoxynucleoside triphosphates, 1.25 U Hot goldstar DNA polymerase, and 300 nM specific primers. Reaction parameters were 95 C for 10 min followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min. For preparation of standards, amplicons were purified from agarose gel and subcloned into pGEMT-easy plasmid (Promega), and then sequenced to verify the identity of each fragment. Standard curves were generated using serial dilutions of linearized standard plasmids, spanning 5 orders of magnitude and yielding correlation coefficients greater than 0.98 and efficiencies of at least 0.98 in all experiments. Amplification of 18S ribosomal was used as internal control for data normalization expressed as the ratio (attomoles of ELL/attomoles of 18S). Primers were as follows: mouse ELL forward (5'-AAGCTGTGTCAGCCACAGAATG-3'); mouse ELL reverse (5'-TGTGCTCGCTGTGTGAAATGT-3'); mouse ribosomal 18S forward (5'-CCCTGCCCTTTGTACACACC-3'); mouse ribosomal 18S reverse (5-CGATCCGAGGGCCTCACTA-3').

Statistical Analysis
Statistical analysis was performed using Student’s t test for unpaired comparisons (InStat version 2.01; GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered to be significant.


    ACKNOWLEDGMENTS
 
We thank Marie-Christine Lecomte for her help in the yeast two-hybrid meanders; Franck Letourneur, Nicolas Lebrun, and Raphael Rambur for their help in sequencing and in preparing the figures, respectively; M. J. Garabedian for the critical reading of the manuscript; and Hughes Loosfelt, Marie-Liesse Asselin, Marc Pallardy, Maria-Christina Zennaro, Marie-Edith Rafestin-Oblin, and Yves de Keyzer for providing us with plasmids.


    FOOTNOTES
 
Present address for L.P.-L.T.: Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, New York 10016. E-mail: Laurent.Pascual-LeTallec@med.nyu.edu.

This work was supported by the Institut National de la Santé et de la Recherche Médicale. L.P.-L.T. was the recipient of a fellowship from the Ministère de l’Education Nationale de la Recherche et de la Technologie and from La Ligue Nationale contre le Cancer. This work was also supported by Grant CA78431 from the National Cancer Institute (to M.T.).

First Published Online January 14, 2005

Abbreviations: AF, Activating function; AR, androgen receptor; CBP, cAMP response element-binding protein (CREB)-binding protein; EAF1, ELL-associated factor 1; ELL, eleven-nineteen lysine-rich leukemia; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; 11ßHSD2, 11ß-hydroxysteroid dehydrogenase type 2; ID, inhibitory domain; LBD, ligand-binding domain; MLL, mixed-lineage leukemia; MR, mineralocorticoid receptor; NTD, N-terminal domain; PCAF, p300 CREB-binding protein-associated factor; PIC, preinitiation complex; Pol II, RNA polymerase II; PR, progesterone receptor; RHA, RNA helicase A.

Received for publication August 24, 2004. Accepted for publication January 4, 2005.


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