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J. Biol. Chem., Vol. 278, Issue 40, 38780-38785, October 3, 2003
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From the
Institut für Humangenetik and Institut für Zellbiologie, Universitätsklinikum, Hufelandstrasse 55, D-45122 Essen, Germany and ¶Department of Repoduction & Development, Erasmus Medical Center, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands
Received for publication, June 13, 2003 , and in revised form, July 22, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The human and mouse TRPS1/Trps1 proteins are highly similar and have an unusual combination of different zinc-finger motifs (Fig. 1A) including a GATA-type and IKAROS-like zinc fingers (1). Malik et al. (2) have shown that TRPS1 is a repressor of GATA-factor mediated gene transcription. The IKAROS-like C2H2 zinc fingers of TRPS1 constitute a dimerization domain (6). The latter zinc fingers (as well as the zinc fingers 4-6) are possibly also involved in the binding of the dynein light chain 8 (LC8a), which we have recently identified as a TRPS1-binding partner in a yeast-two-hybrid screen (7). LC8a decreases the repressional function of TRPS1 (7). It is involved in various intracellular processes of which Bcl-2-regulated apoptosis may be relevant for the pathogenesis of the tricho-rhino-phalangeal syndrome. In addition to LC8a, other putative binding partners of TRPS1 were identified. Here we report that TRPS1 interacts with the RING finger protein RNF4, which is a co-regulator of several different gene transcription mechanisms (8-12). For instance, RNF4 interacts with the androgen receptor and functions as a co-activator of androgen-mediated transcription (8). Our findings suggest that RNF4 may modulate TRPS1 transcription and the function of the gene product.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-Galactosidase Assay—DNA sequences encoding amino acids (aa)1 6-65 of Rnf4, which are responsible for the interaction with Trps1, were inserted in-frame into a galactose-inducible VP16 vector (p415GALNVP0) and co-transfected with truncated Trps1 fragments fused in-frame to the lexA DNA-binding domain. The truncated Trps1 fragments (F6, F9, and F10) were generated by PCR using the plasmid lexA-Trps1-F5 as template and inserted into the bait vector. Transfected L40 yeast cells were grown in selective medium to an A600 of 0.4-0.5, precipitated, and resuspended in 1.2 ml of buffer Z (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM 2-mercaptoethanol, pH 7.0). From this suspension, 100-150 µl (VE) were diluted to 1 ml with buffer Z and cells were lysed by the addition of 15 µl of chloroform and 10 µl of 0.1% (w/v) SDS. The mixture was equilibrated at 30 °C for a few minutes, and the reaction was started by adding 200 µl of ONPG (4 mg/ml o-nitrophenol galactose in 0.1 M K2PO4, pH 7.0). After a defined reaction period (e.g. tR = 1-3 h), the reaction was stopped by the addition of 500 µl of 1 M Na2CO3. The reaction was cleared of insoluble material by centrifugation, and the optical density was measured at 420 and 550 nm. The -galactosidase activity was calculated in units (U) by the equation U = 1000 x A420 - 1750 x A550/A600 x VE x tR (13).
Co-immunoprecipitation—For immunochemical analysis, the entire coding region of the human TRPS1 was inserted in-frame into the green fluorescent protein (GFP) expression vector pEGFP-N3 (Clontech). The full-length human RNF4 cDNA amplified from a fetal brain Marathon-ready cDNA library (catalog number 7402-1, Clontech) (5'-CCCAAGCTTACAATGAGTACAAGAAAGCG-3'; 5'-CGGAATCCTATATAAATGGGGTGGTAC-3') was cloned in-frame into a FLAG expression plasmid (pFLAG-N3). COS-1 cells were co-transfected with both plasmids by electroporation. For immunoprecipitation analysis using the lysate of transfected COS-1 cells, a volume equivalent to 100 µg of protein was dissolved in 1 ml of Dignam D buffer (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, 75 mM NaCl, 100 mM KCl, 0.2 mM EDTA, 3% (w/v) bovine serum albumin). For the studies on endogenous level, a volume equivalent to 1 mg of total protein from primary human fibroblast cell extracts was dissolved in 1 ml of incubation buffer (20 mM HEPES, pH 7.9, 75 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 mM Na3VO4). The solutions were precleared with 30 µl of preequilibrated protein A-Sepharose slurry for 30-60 min.
The supernatant of the transfected COS-1 cell extracts was then incubated with 0.2 mg of an anti-FLAG antibody (Santa Cruz Biotechnology) for 30 min and 30 µl of preequilibrated 50% (v/v) protein A-Sepharose slurry for another 90 min. To the fibroblast cell extracts, 20 µl of a monoclonal anti-TRPS1 antibody LK2B7F6 were added and incubated for 12-16 h. Subsequently, 70 µl of the preequilibrated protein A-Sepharose slurry were added and the mixture was incubated for another 90 min.
The Sepharose beads were sedimented and washed three times with incubation buffer and resuspended in 4x SDS gel-loading buffer (62 mM Tris, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, 0.005% (w/v) bromphenol blue).
All of the incubations mentioned above were carried out at 4 °C and with constant movement using a head-over-tail rotor. The precipitates were analyzed by SDS-PAGE and Western blotting using an anti-FLAG antibody, the rabbit anti-TRPS1 antiserum SN652 (14), and a rabbit anti-RNF4 antiserum (kindly provided by J. Palvimo) (8).
Intracellular Localization—Primary human fibroblasts were directly seeded on glass coverslips. The cells were harvested after 24-48 h in cold phosphate-buffered saline, fixed with methanol, and washed with phosphate-buffered saline and with IF buffer (10 mM Tris, pH 7.5, 300 mM NaCl, 0.05% (v/v) Tween 20). In the next step, the coverslips were blocked for 30 min in 0.5% (w/v) bovine serum albumin in IF buffer, incubated with primary antibody for 1 h, washed twice with IF buffer, and again incubated with secondary antibody, and after washing with IF buffer, they were mounted on slides with AQUAMOUNT (BDH Chemicals, Product number 36086). Cell nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride Blue. Endogenous TRPS1 and RNF4 were detected by using rabbit antisera and stained with a purified Alexa-labeled anti-rabbit antibody. Cells were analyzed by confocal laser-scanning microscopy.
Reporter Gene Assays—Transient transfection assays including a constitutively lacZ expressing plasmid were performed in COS-7 cells essentially as described previously (2, 15). The 
D3 promoter construct (provided by R. Shivdasani, Boston, MA) contains multiple repeats of the sequence AGATAA upstream of the firefly luciferase cDNA. Transfections were performed using FuGENE 6 (Roche Applied Science), 0.1-0.5 µg of the D3 reporter plasmid, 0.25 µg of the TRPS1 expression plasmid, 0.5 µg of the XGATA4 expression plasmid, and 0.25-0.5 µg of the RNF4 expression plasmid. 2 µg of total DNA was used for each transfection. The individual DNA mixtures were adjusted with respective amounts of empty plasmid. After 48 h, cells were lysed at 4 °Cin25 mM Tris, pH 7.8, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 2 mM dithiothreitol, 0.3 mM phenylmethylsulfonyl fluoride, and 2 µg/ml aprotinin. The luciferase activity was measured by luminometry (LUMAT LB9507) after dilution of the lysate 1:3 or 1:5 in 25 mM glycyl glycine, 15 mM MgSO4, 15 mM K2HPO4, 4 mM EGTA, 40 µM ATP, 40 µM dithiothreitol, and 0.3 µg/ml luciferin. Relative luciferase activity was determined by comparing averaged luciferase:-galactosidase ratios and was expressed relative to results from control transfections with the empty vector.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To confirm our yeast two-hybrid data and to narrow down the Rnf4 interaction region in the Trps1 protein, we performed another yeast in vivo assay. In addition to the original Trps1 bait construct (lexA-Trps1-F5), we used three other truncated fragments of Trps1 (Fig. 1C) as baits. Fragment F6 (Trps1 aa 635-1184) lacks the C-terminal IKAROS-like double zinc finger. Fragment F9 (aa 635-984) bears a larger C-terminal truncation but still includes the GATA DNA-binding zinc finger. The smallest fragment, F10 (aa 635-819), contains only the predicted zinc fingers 4-6 (Fig. 1C).
Yeast L40 cells, which carry a lacZ gene under the control of a stretch of eight lexA-operators, were co-transfected with different lexA-Trps1 fusion constructs and a galactose-inducible reporter plasmid encoding aa 6-65 of the Rnf4 fused in-frame to the VP16 activation domain. The proper expression of each lexA-Trps1 construct as well as the expression of the VP16-Rnf4 construct was examined by Western blot analysis using anti-lexA or anti-VP16 antibodies (Fig. 1B).
As shown in Fig. 3A, Trps1 fragments F5 and F6 interact with the Rnf4 N terminus with a similar strength, whereas fragments F9 and F10 do not interact at all. Thus, the Rnf4-binding region of the Trps1 protein could be narrowed down to 200 amino acids (aa 985-1184, Fig. 3B). This region does not contain any of the predicted zinc finger motifs of the TRPS1 protein.
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TRPS1 and RNF4 Form Stable Complexes in Mammalian Cells—All of the yeast assays described above were performed using the mouse Trps1 and Rnf4. For all of the further investigations, we used the human orthologues TRPS1 and RNF4. The human RNF4 is only 190 aa in length and 90% identical to the mouse Rnf4 (12). The entire coding regions of TRPS1 and RNF4 were inserted in-frame into a GFP or a FLAG expression plasmid to obtain the TRPS1-GFP and the RNF4-FLAG fusion proteins, respectively. To test whether TRPS1 and RNF4 are able to form stable complexes in mammalian cells, we co-transfected COS-1 cells with both plasmids. As controls, we used each construct co-transfected with the empty GFP or FLAG vector, respectively.
The expression of the TRPS1-GFP fusion construct was examined by Western blot analysis using an anti-TRPS1 antiserum (SN652) (14). Fig. 4A, lane 2, illustrates the proper expression of the TRPS1-GFP fusion construct in cells co-transfected with RNF4-FLAG. No signal was found in extracts of cells co-transfected with empty GFP and RNF4-FLAG (Fig. 4A, lane 1). Similar results were obtained by using a commercial anti-GFP antibody (data not shown). To detect the RNF4-FLAG fusion protein, a commercial anti-FLAG antibody (Santa Cruz Biotechnology) was used. We found a strong RNF4-FLAG fusion protein signal in RNF4-FLAG/TRPS1-GFP-co-transfected cells (Fig. 4B, lane 1), whereas no signal was detectable using extracts from COS-1 cells co-transfected with empty FLAG plasmid and the TRPS1-GFP construct (Fig. 4B, lane 2).
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To determine whether TRPS1 and RNF4 interact, we incubated cell extracts with Sepharose-coupled anti-FLAG antibodies. After several washing steps, we analyzed the eluates on Western blots with the anti-TRPS1 antiserum and were able to detect a TRPS1 signal in extracts of COS-1 cells co-transfected with TRPS1-GFP and RNF4-FLAG (Fig. 4C, lane 1) but not in extracts from cells where either RNF4-FLAG or TRPS1-GFP was absent (Fig. 4C, lanes 2 and 3). These findings indicate that TRPS1 and RNF4 do not only interact in yeast but are also able to form complexes in mammalian cells.
We next used primary human fibroblasts to test whether an interaction between the endogenous TRPS1 and RNF4 proteins can take place. Expression of TRPS1 and RNF4 could be readily detected in cell extracts using the anti-TRPS1 SN652 antiserum (Fig. 4D) and an anti-RNF4 antiserum (kindly provided by J. Palvimo) (Fig. 4E, lane 1) (8). To analyze the TRPS1-RNF4 interaction, a monoclonal anti-TRPS1 antibody (LK2B7F6) was successfully used to prepare immunoprecipitates from cell extracts. We were able to detect RNF4 in these precipitates with the anti-RNF4 antiserum (Fig. 4E, lane 2), demonstrating that the endogenous TRPS1 and RNF4 proteins physically interact and can be co-precipitated from human fibroblasts.
TRPS1 and RNF4 Are Located in Distinct Structures of the Nucleus—As shown previously, TRPS1 is a nuclear protein, which is located in large nuclear bodies (Fig. 5A) (see Ref. 7). The endogenous RNF4 protein has been found in the nucleus and the cytoplasm (8, 12). Because we were able to co-immunoprecipitate TRPS1 and RNF4 from human fibroblast cell extracts, we decided to evaluate the intracellular localization of both proteins within these cells. We used the rabbit anti-TRPS1 antiserum and an anti-rabbit Alexa-labeled secondary antibody to show the intracellular localization of TRPS1 (Fig. 5A). The number of TRPS1 containing nuclear bodies varied between 1 and 4 per cell. The intracellular distribution of RNF4 was investigated by using the rabbit anti-RNF4 antiserum and the Alexa-labeled anti-rabbit antibody. As shown in Fig. 5B, RNF4 is homogeneously distributed in the cytoplasm but also located in numerous small bodies in the nucleus. The RNF4-containing nuclear bodies are clearly distinct from those in which TRPS1 is found. Co-localization experiments could not be performed because both available antisera were generated in rabbit, which does not allow a specific staining of both proteins with a secondary anti-rabbit antibody.
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RNF4 Inhibits the Repressional Function of TRPS1 on GATA-mediated Transcription—Using a luciferase reporter assay, Malik et al. (2) have demonstrated that TRPS1 represses GATA-dependent gene activation (2). We used this reporter assay to analyze whether RNF4 has any influence on the function of TRPS1. For this purpose, we transfected COS-7 cells with the reporter plasmid D3, which contains the luciferase cDNA under the control of multiple AGATAA elements (15). The luciferase activity of D3-transfected cells was arbitrarily set as 1.0 (Fig. 6, column 1). Co-transfection with the transcriptional activator XGATA4 resulted in a 5.7-fold increased activity of the reporter (Fig. 6, column 2). This activation was reduced by adding a TRPS1 expression plasmid, demonstrating the function of TRPS1 as a transcriptional repressor (Fig. 6, column 3). The TRPS1-mediated repression could be completely relieved by adding an equal amount of the RNF4 expression plasmid (Fig. 6, column 5). The addition of a higher amount of RNF4 expression plasmid resulted in an even higher luciferase activity (Fig. 6, column 7), suggesting that RNF4 acts as a co-activator on XGATA4 in this system. This was proved by the increased luciferase activity (7.2- and 7.5-fold) upon co-expression of XGATA4 with increasing amounts of RNF4 but without TRPS1 (Fig. 6, columns 4 and 6). RNF4 does not activate expression of the reporter in the absence of XGATA4 (Fig. 6, column 8). In summary, these results suggest that the interaction with RNF4 modulates the activity of TRPS1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Transiently expressed TRPS1 and RNF4 are found exclusively in the nuclei of NIH-3T3 and COS-1 cells where they are homogeneously distributed (2, 8, 9, 11, and our data not shown). In contrast, endogenous RNF4 occurs in the cytoplasm as well as in numerous small structures within the nuclei of human fibroblasts (Fig. 5B), which was also observed by Moilanen et al. (8) in CV-1 cells. The endogenous TRPS1 protein also shows a non-homogeneous distribution within the nuclei of human fibroblasts, but the TRPS1-containing structures are less numerous and much bigger than the RNF4-containing structures (Fig. 5, A and B). This finding makes it probable that the bulk of the two proteins are involved in separate intracellular processes. Because of the lack of appropriate antibodies, we could not investigate whether the different structures overlap partially. However, our co-immunoprecipitation studies demonstrate that the two proteins do interact in human fibroblasts.
RNF4 is the second protein known to interact with TRPS1 and binds within a 200 amino acid region (aa 985-1184) of the transcription factor. It does overlap with only two base pairs with the recently identified region B of TRPS1-LC8a interaction (region A: aa 635-723; region B: aa 1182-1281) (7) and has no predicted protein-protein interaction motif.
During the last years a variety of proteins, which associate with RNF4 have been identified. RNF4 contains two distinct regions, which are known to be protein-protein-interacting domains (Fig. 2). The RING finger at the C terminus is necessary for the interaction to the TATA-binding protein (8), SPBP (11), and Sp1 (9). The second protein-interacting domain to which TRPS1 (Fig. 2) binds lies in the N-terminal part of RNF4. This region binds transcriptional repressors such as Gscl (12) and POZ-AT hook-zinc finger protein (PATZ) (10) as well as steroid receptors (8). In the above mentioned interactions, RNF4 undertakes several diverse functions. It can either act as a transcriptional co-repressor (10) or as a co-activator (8, 9, 11).
In the presence of androgen, for example, RNF4 strongly activates androgen receptor (AR)-dependent transcription (8). Interestingly, TRPS1 expression is repressed in androgen-dependent human prostate cancer cells (LNCaP-FGC) by physiological androgen concentrations and repression is mediated by the AR (4, 5). Furthermore, after androgen withdrawal, the Trps1 expression in the rat ventral prostate increases significantly and is associated with the regression of the prostate and the occurrence of apoptosis. RNF4 is expressed in adult mouse, rat, and human prostate (8, 12), but it is not known whether it is expressed in the LNCaP-FGC cells. If RNF4 is involved in TRPS1 repression, we assume that this repression does not affect the TRPS1 gene directly but is mediated via other AR-RNF4-regulated proteins because RNF4 normally activates AR-dependent transcription.
Analyses by Fedele et al. (10) revealed two novel functions of RNF4. They found that RNF4 enhances basal transcription from a minimal c-myc promoter in a concentration-dependent manner. The activation was reversed to a strong co-repression when the transcriptional repressor PATZ was added (10). Because TRPS1 is a repressor of transcription, we had expected a co-repressional effect of the RNF4 binding. However, in the reporter gene assay we found a complete suppression of the repressional activity of TRPS1 upon the addition of RNF4 (Fig. 6, columns 5 and 7). Our studies revealed a so far unknown function for RNF4. Obviously, it acts as a co-activator on GATA factor-mediated gene transcription. RNF4 enhanced transcription by 26-32% (Fig. 6, columns 4 and 6), but this effect is too small to account for the reversal of TRPS1-induced transcriptional repression. Therefore, we assume that the latter is caused by an interaction between RNF4 and TRPS1.
In contrast to the findings of Fedele et al. (10), Moilanen et al. (8) showed that full-length RNF4-SNURF fused to the Gal4-DNA-binding domain does not activate a minimal promoter controlled by Gal4-binding sites in yeast or mammalian cells. They consequently suggested that SNURF does not have intrinsic transcription activation function (8). In agreement with this suggestion, we found that RNF4 is not capable of activating gene transcription from an artificial GATA promoter in the absence of XGATA4 (Fig. 6, column 8).
Based on current knowledge, it seems clear that RNF4 functions as a more general co-regulator, possibly as a bridging factor participating in the coordination of the activities of multiple transcription signals of upstream factors. One of these factors may be TRPS1. However, it remains elusive whether RNF4 is involved in the pathogenesis of the tricho-rhino-phalangeal syndromes because it is not known whether RNF4 is expressed, for example, in chondrocytes or hair follicles. The TRPSs are caused by haploinsufficiency (TRPS I and TRPS II) or by a dominant negative effect of a mutant TRPS1 protein (TRPS III) (see Ref. 17.). RNF4 and TRPS1 are also expressed in many tissues, which are not obviously affected in the TRPSs. Possibly, these tissues are not as susceptible to concentration imbalances as those affected in the TRPSs.
RNF4 is similar to the previously identified multifunctional LC8a (7), a suppressor of TRPS1-mediated gene repression. Analyses of the other potential TRPS1-interacting proteins will show whether there are even more suppressors of TRPS1, which interact in a developmental stage or tissue-specific manner with TRPS1 to modulate its function.
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FOOTNOTES |
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|| To whom correspondence should be addressed. Tel.: 49-201-7234555; Fax: 49-201-7235900; E-mail: hj.luedecke{at}uni-essen.de.
1 The abbreviations used are: aa, amino acid; GFP, green fluorescent protein; PATZ, POZ-AT hook-zinc finger protein; SNURF, small nuclear RING finger protein.
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ACKNOWLEDGMENTS |
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D3) constructs, Jorma J. Palvimo for providing the RNF4 antiserum, Diane von Holtum and Stephanie Gro for expert technical assistance, and Albert O. Brinkmann for continuous support. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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