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Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 1–14

PRMT2, a member of the protein arginine methyltransferasefamily, is a coactivator of the androgen receptor

Rene Meyer 1, Siegmund S. Wolf ∗, Maik Obendorf ∗

Gynecology & Andrology, MHCII, Schering AG/Jenapharm,Otto-Schott-Str. 15, D-07745 Jena, Germany

bstract

The basal transcriptional activity of nuclear receptors (NRs) is regulated by interactions with additional comodulator proteins (coac-ivator/corepressor). Here, we describe a new androgen receptor (AR)-associated coactivator, PRMT2, which belongs to the arginine

ethyltransferase protein family. To search for AR-interacting proteins a fragment of the AR was used in a library screen exploiting theeast two-hybrid technique and identifying the C-terminal region of PRMT2. We demonstrated that PRMT2 acts as a strong coactivator of theR, had modest or none influence on transcriptional activation mediated by other NRs. Interestingly, PRMT2 interaction with the estrogen

eceptor (ER) was strongly dependent on the cellular background, thus, suggesting the involvement of additional, differentially expressedoregulators. We also demonstrated synergistic interaction of PRMT2 with other known nuclear receptor coactivators, such as GRIP1/TIF-. Potentiation of AR-mediated transactivation by PRMT2 alone and in synergism with GRIP1 was prevented by a competitive inhibitor ofethyltransferase activity. The PRMT2 expression profile overlaps with the distribution of AR, with strongest PRMT2 abundance in androgen

arget tissues. Immunofluorescence experiments showed that the intracellular localization of PRMT2 depends on the presence of the cognateeceptor ligand. Under androgen-free conditions, both AR and PRMT2 are confined to the cytoplasm, whereas in the presence of androgensoth proteins colocalize and translocate into the nucleus. Treatment with the AR antagonist hydroxyflutamide results in nuclear translocation

f the AR, but not the coactivator PRMT2. Thus, it appears that the ligand-dependent AR conformation is essential for the recruitment anduclear translocation of PMRT2 which acts as AR-coactivator, presumably by arginine methylation.

2007 Elsevier Ltd. All rights reserved.

eywords: Coactivator; Androgen receptor; Methyltransferase; Cellular localization

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. Introduction

The androgen receptor (AR) is a member of the nucleareceptor (NR) superfamily that translocates into the nucleusfter ligand binding and interacts with DNA response ele-ents to regulate target gene transcription. Transactivation

nd transrepression activities of the AR are associated with

he recruitment of corepressors and coactivators. Several AR-nteracting proteins which alter the transcriptional activity

∗ Corresponding authors. Tel.: +49 30 468 18360/16288;ax: +49 30 468 98360.

E-mail addresses: Siegmund.Wolf@Schering.de (S.S. Wolf),aik.Obendorf@Schering.de (M. Obendorf).1 Present address: Department of Molecular and Cellular Biology, Baylorollege of Medicine, One Baylor Plaza MS130, Houston, TX 77030, USA.

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f AR have so far been reported in the literature [1–4] andre updated elsewhere (http://www.mcgill.ca/androgendb).early all known aspects of AR behavior within the cell

e.g. stabilization of the unliganded receptor in cytoplasm,ntracellular transport and nuclear translocation, receptor pro-ein turnover, interaction with DNA, histones, or generalranscription factors) involve the participation of regulatoryroteins. Most of these factors are not confined to interac-ion with the AR, but also partner other nuclear receptorsr transcription factors. In fact, the cooperation of sev-ral coactivators of different classes (e.g. CBP/p300, the160 family with histone acetylation activity, methyltrans-

erases, or ATPases such as the SWI/SNF complexes) iseeded for an efficient activation of transcription by all NR’s5,6]. These complexes cause local remodeling of the chro-atin structure in the promoter region and help to recruit

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NA polymerase II to elicit target gene transcription. Inddition, histone modifications may contribute to epigenetichanges and thereby long-term regulation of target genes5,7,8].

Arginine methylation of histones, RNA binding proteins,nd transcriptional regulators is a common posttransla-ional modification involved in chromatin remodeling [6,9].here are two different classes of protein arginine methyl-

ransferases (PRMT) in mammals, type I and II, whichatalyze asymmetric and symmetric dimethylations, respec-ively [10,11]. So far, seven PRMT family members haveeen identified in mammals, with PRMT1, PRMT3, PRMT4,nd PRMT6 belonging to the type I class, and PRMT5nd PRMT7 of the type II class [12]. The existence ofn eighth PRMT was assumed recently [13–15]. Althoughhe methyltransferase action of PRMT2 has been con-rmed, the enzyme has not been assigned to a definedlass yet [16]. However, PRMT2 shows strong similarityith the members of the type I class [17]. Beside homo-

ogous domains, including the adenosyl methionine bindingomain and the methyltransferase catalytic core, individ-al family members display certain unique features. Forxample, PRMT2 is the only member containing a Srcomology 3 (SH3) domain [13]; PRMT1 apparently methy-ates target proteins within an RGG- or RXR-rich consensusequence [18,19], whereas the coactivator-associated argi-ine methyltransferase 1 (CARM1), also called PRMT4, doesot require any consensus sequence in the target proteins20]. In the intricate process of chromatin remodeling andranscriptional regulation synergistic interactions betweenoactivators [21–23], including cooperation of PRMT fam-ly members with GRIP1 and CBP/p300 complexes [24,25]ave been documented.

The results described herein provide evidence that PRMT2s a coactivator of the AR-mediated transactivation. Our dataeveal protein–protein binding and recruitment of PRMT2 byhe AR in the presence of an AR agonist. The cooperationf PRMT2 with GRIP1 in the AR-mediated transactiva-ion results in potentiation of the transcriptional activity ofR, which resembles the cooperation between CARM1 andRIP1 [22,25]. We also demonstrate an agonist-induced

oncomitant translocation of the AR and PRMT2. Neither,RMT2 recruitment by the AR, nor nuclear colocalizationf this coactivator became apparent in the presence of anR antagonist. Inhibition of the presumed methyltransferase

ctivity of PRMT2 clearly decreases its coactivator effi-acy, either alone or synergistically with GRIP1. PRMT2cts as a weak coactivator of the progesterone (PR), butot the mineralocorticoid (MR) and glucocorticoid receptorGR) in the cell lines tested. Potentiation of estrogen recep-or (ER)-mediated transcriptional activation by PRMT2 isnconsistent, and apparently depends on the cellular con-

ext. These data indicate that PRMT2 acts as a coactivatorf the AR in its agonistic conformation and probably partic-pates in synergistic interactions with other nuclear receptoromodulators.

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& Molecular Biology 107 (2007) 1–14

. Materials and methods

.1. Plasmid constructions

The full-length cDNAs for AR, GR, MR or PR wereloned into the pSG5 vector to generate pSG5-AR, pSG5-R, pSG5-MR and pSG5-PR, respectively. For ER� the

xpression plasmid pHEGO-ER was kindly provided by P.hambon (IGBMC, Illkirch, France). The hormone respon-

ive MMTV-promoter upstream of the luciferase reporterene (pAH-luc) was a gift from S. Nordeen (Universityf Colorado, Denver, USA). Two estrogen response ele-ents were cloned in front of the luciferase reporter gene

pERE2-luc) [26]. The pSG5 expression plasmid for CARM1as a generous gift from M. Stallcup (University of South-

rn California, Los Angeles, USA). The yeast matchmakerystem and two-hybrid plasmids pVA3-1, pTD1-1, pGBT9nd pACT2 including the fetal brain cDNA library, wereurchased from Clontech (Palo Alto, USA). The match-aker pACT2 plasmid contains the cDNA library, including

he clone-encoding fragment aa 271–433 from PRMT2ARAP74-pACT). For the mammalian two-hybrid systemCMV-AD from Stratagene (La Jolla, USA) containinghe NF�B activation domain (pCMX-NF�B-AD) or theP16 activation domain (pCMV-VP16-AD) were used. The

solated cDNA fragments from the pACT2-library werexcised with SacI + XbaI and ligated into pCMV, pCMV-ADnd pCMX-NF�B-AD. Full length PRMT2 was ampli-ed from human universal or brain cDNAs (Clontech)sing specific primers (forward: 5′ gcg aat tca tgg caaat cag gtg act g 3′ and reverse: 5′ ggg agc tca act gtctc tcc aga tgg gg 3′) and cloned either into the plas-id pCMX [27], pCMV-AD or, to generate eGFP fusion,

cDNA3.1/NT-GFP-TOPO (Invitrogen, Paisley, UK). TheRMT2-GST-fusion plasmids were generated by ligating

he corresponding cDNA from the pCMV-AD plasmid intohe BamHI/XhoI sites of the pSP72-GST plasmid (Promega,

annheim, Germany). The plasmid GST-SHP was createdy fusing the XhoI fragment containing the cDNA of SHProm the pCDM8-SHP plasmid (kindly provided by D.

oore, Baylor College of Medicine, Houston, USA) into theGEX-KG plasmid. The GST-ER� fragment fusion plasmidncluding the AF2 domain of ER� and resulting in the plas-

id pGEX-KG-ER-AF2 (aa 263–595) was kindly providedy S. Oesterreich (Baylor College of Medicine, Houston,SA).

.2. Yeast two-hybrid assay

A fragment of the human AR cDNA coding formino acids 325–919 was ligated into the pGBT9-plasmidpGBT9-AR(325–919)] and used as bait according to the

anufacturer’s protocol (Clontech) to screen a match-aker human fetal brain cDNA library. Colonies were

rown on selection medium (lacking Ade and amino acidseu, Trp, His) containing 10−6 M DHT (dihydrotestos-

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R. Meyer et al. / Journal of Steroid Bioch

erone), and the positive clones were verified using theuxotrophic marker genes and �-galctosidase measure-ents. The inserts of identified positive clones were directly

nalyzed by PCR and sequenced. The interactions wereonfirmed by retransformation of the previously isolatedACT-cDNA plasmids together with the bait plasmidGBT9-AR(325–919) in the presence or absence of hor-one.

.3. Mammalian cell transfection

Cells were cultured in DMEM (for SH-SY5Y) orPMI 1640 (for PC3 and PC3-ARwt) medium con-

aining 15% serum, 200 mM l-glutamine, and 5 mg/lenicillin/streptomycin. Five days prior to all tests, mediumas changed to phenol red free medium with 10% charcoal-

reated (steroid-depleted) fetal calf serum. For transfections,× 105 cells were seeded in 6-well plates and transfectedith the indicated plasmids for 16 h using lipofectin accord-

ng to the instructions of the manufacturer (Invitrogen,aisley, UK). Cells were harvested 24 h after hormonal

reatment, and luciferase activity was determined usinghe manufacturer protocol (Promega). Values were nor-

alized by measurement of total protein (Bradford) inhe same samples. Values represent means (±S.D.) ofriplicates from at least three independent transfections.tudent’s t-test was used to determine significant changes

n receptor activity due to coactivator activity with all-values being smaller than 0.01, if not indicated other-ise.Functional knockdown of PRMT2 with siRNA was

erformed in 293T and T47D cells cultured in 5%tripped serum containing DMEM Zink option (Invitro-en, Paisley, UK). 2 × 105 cells were seeded in 6-welllates and transfected with a final concentration of 100 nMiRNA for 16 h using lipofectamine 2000 according to thenstructions of the manufacturer (Invitrogen). For controls00 nM non-targeting siRNA (D-001210-02-05, Dharma-on, Chicago, USA) and for specific knockdown 50 nMon-targeting and 50 nM PRMT2-specific siRNA (genomemart pool M-004033-00, Dharmacon) were combined.ormones were added as indicated 24 h after siRNA trans-

ection and total RNA was harvested after additional4 h using RNeasy Mini Kit (74104, Qiagen, Valencia,SA).

.4. Immunofluorescent staining and confocalicroscopy

Hep-2 cells were seeded onto gelatin-coated (2%) cover-lips and placed in 6-well plates before transient transfectionith the PRMT2-eGFP fusion plasmid (PRMT2-GFP). After

4 h transfected cells were treated with hormones. Formmunofluorescence, cells were fixed in 3.7% formalde-yde for 10 min, washed with PBS, permealized with 0.25%riton X-100, and incubated after washing with the 441

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& Molecular Biology 107 (2007) 1–14 3

nti-AR antibody (Santa Cruz Biotechnology, Santa Cruz,SA) solution (1:200). Cells were washed three times and

ncubated with a secondary anti-mouse antibody coupled tohodamine (Jackson Immunresearch, West Grove, USA).NA was stained with TO-PRO-3 (1:500) from Invitro-en (Paisley, UK) for 10 min. Finally, slides were mountedith Vectashield (Vector Labs, Burlingame, USA). Confocalicroscopy was performed on a Zeiss ConfoCor2 (LSM 510ETA) observing excitations of Rhodamine at 543 nm and

GFP at 488 nm.

.5. In vitro SAM binding assay

GST or the GST-fusions of PRMT2 or AR-LBD werexpressed in BL21-DE3 (Invitrogen) and incubated with0 �Ci of S-adenosyl-l-[methyl-3H]methionine (3H-SAM)n methylation buffer (20 mM Tris–HCl, 150 mM NaCl,mM MgCl2, pH 7.5). After incubation for 120 min at7 ◦C, GST-fusion proteins bound to glutathione–sepharoseB beads (Amersham, Little Chalfont, UK) were washedhree times in PBS buffer and remaining radioactivity deter-

ined.

.6. Glutathione-S-transferase (GST) pulldownxperiments

In vitro transcription/translation of AR or PRMT2 waserformed with 35S-methionine using the TNT-T7 reticu-ocyte lysate system according to the instructive protocolPromega). GST-fusion plasmids were transformed intoL21-DE3 cells (Invitrogen). Expression of GST-fusion pro-

eins was induced with 0.2 mM IPTG for 2–3 h in LB-mediumt OD600 = 0.6. After cell lysis equal amounts of GST-fusionroteins were bound to glutathione–sepharose 4B beadsAmersham) and incubated overnight with the in vitro 35S-ethionine-labeled proteins. Beads were carefully washed

hree times with buffer (800 mM NaCl, 80 mM Hepes, 8 mMDTA, 28 mM �-mercaptoethanol, 40% glycerol) and ana-

yzed by 10% SDS-PAGE followed by autoradiography.

.7. Northern blot analysis

Multi-tissue blots were obtained from a commercialupplier (Clontech). For the analysis of PRMT2 expres-ion in cell lines RNA from 2 × 107 cells was isolated byRNeasy Midi Kit’ extraction (Qiagen, Hilden, Germany).he isolated RNA was separated on a 1% agarose-

ormaldehyde gel and transferred onto Hybond N nylon

random primer labeling system according to the man-facturer’s protocol (Amersham). After hybridization andashing the membranes were exposed to film for 3–days.

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R. Meyer et al. / Journal of Steroid Bioch

.8. Quantitative real time polymerase chain reactionQ-RT-PCR)

Isolated RNA was reverse transcribed using SuperScriptI reverse transcriptase (Invitrogen) and random hexamerrimers according to manufacture instructions. Q-RT-PCRas performed on an ABI Prism 7700 Sequence detector andrimers were designed to span over an exon/intron/exon bor-er and validated with SYBR Green assay (melting curvenalysis and amplification efficiency at least 95%). Specificrobes for TaqMan analysis were FAM labeled standardrobes with BHQ or TAMRA as quencher and ROX Dyes internal reference. Relative expression levels were calcu-ated using the ��CT method using �-actin expression aseference gene. The following sequences were used: �-actin:or: CCC TGG CAC CCA GCA C, rev: GCC GAT CCA CACGA GTA C, probe (FAM-TAMRA): ATC AAG ATC ATTCT CCT CCT GAG CGC; AR: for: GCT TCT ACC AGCCA CCA AGT T, rev: GGT CAA AAG TGA ACT GATCA GC, no probe, SYBR Green only; PRMT2: for: GGACA TCA GTC TCT TCT GTG CA, rev: GTG CTG TGCAT CTC ACT GG, no probe, SYBR Green only. Oligonu-leotides for NKX3.1 or TMPRSS2 were purchased from aommercial source (HS00171834 m1 and HS00237175 m1,espectively, Applied Biosystems, Foster City, USA).

. Results

.1. Isolation of PRMT2 as an AR-interacting protein

Screening of a human fetal brain library with GAL4 DBDusions including a fragment of the AR yielded more than00 positive clones representing nearly 80 distinct c-DNAs,s identified by growth on the appropriate selection mediumnd confirmed by �-gal assay. Several known AR interactingroteins, such as �-actin [28], Hsp90 [29,30], and steroideceptor coactivator-1a (SRC-1a) [31–33], were found, thusorroborating the validity of the yeast two-hybrid assay used.ith this system, however, we also isolated several novel AR-

nteracting proteins. In the presence of 10−6 M DHT, a clearpecific interaction between a large fragment containing theNA binding domain (DBD) and the ligand binding domain

LBD) of the human AR (aa 325–919) and a protein initiallyamed androgen receptor associated protein 74 (ARAP74)as documented.The sequence for ARAP74 found in the isolated clone

rom the fetal brain cDNA library is identical to a fragmentf the NCBI entry HSRNAAM (X99209) coding for themino acid sequence 271–433 of the protein CAA67599. Thisequence is almost identical to a protein from the arginineethyltransferase (PRMT) family member, namely PRMT2

cDNA: U80213; protein: AAB48437); the difference com-rises 5 amino acids, due to 2 frame shifts in the cDNA.o clarify which sequence is expressed in human tissues, theull-length cDNA was amplified by PCR from human univer-

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& Molecular Biology 107 (2007) 1–14

al, as well as from human brain cDNA, and sequenced. Inll cases a sequence identical to HSRNAAM was identifieddata not shown). The discrepancy between the sequencingesults and the PRMT2 gene bank entry U80213 might beue to a naturally occurring mutation or a sequencing fluke.lthough we use the name PRMT2 throughout the paper,

hus indicating its similarity to the PRMT family, the corre-ponding mRNA and protein sequences are described by theenbank entries X99209 and CAA67599, respectively.

.2. PRMT2 interacts directly with AR in vitro and inivo

In order to verify the interaction between PRMT2 andR, the yeast two-hybrid plasmids (pGBT9-AR (325–919),ACT-PRMT2, pACT-SRC1a) were retransformed into theeast strain PJ69-4A and compared with control transfor-ations (pVA3-1, pTD1-1) in the presence and absence of

he hormone DHT. As expected, all plasmid transformationsesulted in yeast clones which could grow on full mediumFig. 1A). Unlike the control, however, only the yeast cellshich had been transformed with the plasmids pGBT9-AR

325–919) containing the AR fragment (aa 325–919), thelasmid pACT-PRMT2 containing the PRMT2 fragment (aa71–433), or the plasmid pACT-SRC1a containing the SRC-a fragment (aa 618–1441) could grow and form coloniesn selection medium containing the hormone (Fig. 1A). Noolony growth on selection medium was seen when the plas-id pACT-PRMT2 and the empty bait vector pGBT9 were

ransformed into yeast, thereby excluding the possibility of airect interaction of PRMT2 with the GAL4 plasmid or theeast promoter. Furthermore, colony growth did not occurn the absence of the AR-agonist DHT, indicating a ligandependency of the AR/PRMT2 interaction. Yeast containinghe positive control plasmids pVA3-1 and pTD1-1 grow, asxpected, on all media, regardless of DHT presence (Fig. 1A).hese results confirmed the initial screen and the interactionetween AR and PRMT2 or SRC-1a in yeast cells.

The interaction of AR with PRMT2 was confirmed byST pulldown assay in vitro. Two different PRMT2-GST-

usion constructs were used, GST-PRMT2 and GST-PRMT2-, containing either the full length PRMT2 (aa 1–433) or the-terminal sequence (aa 271–433). In vitro 35S-methionine

abeled AR was incubated with GST or GST-fusion proteinsGST-PRMT2, GST-PRMT2-C, GST-AR, GST-SHP) and theinding analyzed by SDS-PAGE. In the GST pulldown assay,nteractions between 35S-AR, and GST-AR including the ARragment aa 538–919 (Fig. 1B, lane 6), or GST-SHP con-aining full-length SHP (Fig. 1B, lane 2) were detected, ingreement with other publications [34,35]. Concordantly, theinding of AR to GST-SHP was ligand-independent, whereashe binding of AR to the GST-AR (aa 538–919) was only pos-

ible in the presence of the AR agonist DHT (Fig. 1B, lanesand 6). The binding of the full length PRMT2 to in vitro

ranscribed and translated 35S-labeled AR was independentf the ligand (Fig. 1B, lane 4). The interaction between AR

R. Meyer et al. / Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 1–14 5

Fig. 1. Ligand dependent interaction of AR with PRMT2. AR shows liganddependent interaction with PRMT2 in yeast and in GST pulldown assay. (A)PJ69-4A yeast cells were transformed with the indicated plasmids and grownon either full medium, or selection medium lacking Ade, His, Leu, Trp in thepresence or absence of the AR-agonist DHT. The positive control (pVA3-1and pTD1-1) could grow on all media. The isolated SRC-1a and PRMT2fragments were only in the presence of 10−6 M DHT able to interact withAR to promote yeast growth on selection media. (B) In vitro 35S-methioninelabeled AR was incubated with the indicated GST-fusion proteins either inthe presence or absence of 10−7 M DHT. The AR specifically binds to SHP,Pat

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Fig. 2. PRMT2 expression in human tissues and cell lines. (A) Multi-tissueblots including 23 human organs (indicated from 1 to 23) were hybridizedwith 32P-labeled fragments of PRMT2 (825–1299 bp) or �-actin (loadingcontrol) and exposed for 5 days to film. The arrows indicate the 2.4 kbPRMT2 RNA (top) and the �-actin RNA (bottom). (B) Verification ofPRMT2-mRNA expression in cell lines by Northern blot analysis using 32P-lSp

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RMT2 and the C-terminal half of PRMT2 (aa 271–433), but not to GSTlone (lane 3). A clear ligand dependence interaction could be observed forhe homodimerisation of AR (lane 6).

nd PRMT2-C was much weaker and also ligand indepen-ent (Fig. 1B, lane 5). As AR did not bind to GST (control)lone (Fig. 1B, lane 3), a specific direct interaction with SHPFig. 1B, lane 2), AR (Fig. 1B, lane 6), and PRMT2 (Fig. 1B,anes 4 and 5) could be confirmed.

.3. PRMT2 expression in major androgen target tissuesnd cell lines

Northern blot analysis of PRMT2 expression revealedhat a 2.4 kb transcript is present in several human tissuesFig. 2A). High abundance was found in several androgenarget organs (e.g., heart, prostate, and skeletal muscle); thetrongest expression was documented in the ovary and spinalord. PRMT2 expression could not be found in lung, livernd thymus tissues.

Analysis of RNA samples isolated from different cell linesFig. 2B) revealed that PRMT2 transcripts are not consti-utively highly expressed in any of the cells tested in theresent study; which does not rule out a very low expression

(dCt

abeled PRMT2-DNA (825–1299 bp). Human cell lines (PC3, PC3-ARwt,H-SY5Y) were transfected with either 5 �g pCMV control (-), 1 or 5 �gCMV-PRMT2, (1 or 5) and blots exposed to film for 48 h.

hat is not detectable by Northern blot analysis. However,ose-dependent recovery of PRMT2 mRNA is detectablepon transfection with different amount of pCMX-PRMT21 and 5 �g). It should be mentioned, that neither of the ‘par-nt’ cell lines PC3 and SH-SY5Y expressed endogenous ARetectable in Northern blot analysis (Fig. 2B and data nothown).

.4. PRMT2 and AR interaction in mammalian cell lines

The transactivation of the MMTV promoter-driveneporter gene was dose-dependently induced by the andro-en (DHT) up to 6-fold in prostate cancer cells (PC3-ARwt),egardless of the co-expression of the control plasmidsCMV-VP16-AD or pCMX-NF�B-AD (data not shown).ransfection of PC3-ARwt cells with the plasmid constructCMV-AD-SRC1a, which contains the activator domain ofP16 (VP16-AD) fused in frame to the SRC-1 fragment

aa 618–1441) identified by the yeast two-hybrid screen,esulted in a further 5–6-fold increase of androgen-dependentransactivation (data not shown). These findings confirm theinding of the SRC-1a fragment to the AR and its previouslyescribed coactivator efficacy [31–33].

Transfection of PC3-ARwt cells with the fusion con-truct pCMX-NF�B-AD-PRMT2-C, including the activationomain of NF�B and the C-terminal fragment of PRMT2

aa 271–433), resulted in 5–6-fold coactivation of the AR-ependent reporter transcription, thereby indicating that the-terminal sequence of PRMT2 is sufficient for the interac-

ion with the AR (Fig. 3A). Similar results were obtained

6 R. Meyer et al. / Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 1–14

Fig. 3. PRMT2-C (aa 271–433) interacts with AR, and PRMT2 acts as coactivator for a nuclear receptor subgroup. (A) Prostate cells (PC3-ARwt) weretransfected with the reporter plasmid pAH-luc (1.0 �g), and pCMX (0.5 �g) or equal molar amount of the indicated plasmids. Relative luciferase activitywas determined 24 h after treatment with increasing amount of DHT (0; 0.01; 0.1; 1; 10; 100 nM) and normalized to total protein. Increase in AR-mediatedtransactivation by full length PRMT2 (pCMX-PRMT2) and the fusion construct of PRMT2-C (aa 271–433) with the NF�B-AD (pCMX-NF�B-AD-PRMT2-C)is shown. The C-terminal fragment of PRMT2 (pCMX-PRMT2-C) did not potentiate AR-mediated transactivation in comparison to control plasmid (pCMX).(B) Human neuroblastoma cells (SH-SY5Y) were transfected with either expression plasmids for AR (0.75 �g), PR (0.5 �g), GR (0.5 �g), or MR (0.5 �g), thereporter plasmid (pAH-luc), and equal molar amount of the indicated control plasmid (pCMV-VP16-AD), or the expression plasmids for SRC-1 (pCMV-AD-SRCI), the fusion of NF�B with PRMT2-C (pCMX-NF�B-AD-PRTM2-C) or full length PRMT2 (pCMX-PRMT2). After transfection cells were treated withdifferent amounts (0; 1; 10 nM) of reference agonists (DHT for AR; promegestone for PR; dexamethasone for GR; aldosterone for MR) for 24 h and luciferaseexpression was measured and normalized to total protein.

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sing a different activation domain (VP16-AD) fusion (dataot shown). However, lack of differences between the pCMXontrol and pCMX-PRMT2-C suggests that the C-terminalragment of PRMT2 is not sufficient to potentiate AR-ediated transcription on its own (Fig. 3A). Since the-terminal PRMT2 fragment (aa 271–433) does not con-

ain the SH3 and the S-adenosyl-methionine (SAM) bindingomains of the mature protein [14,36], but binds in vivond in vitro to AR, it seems that the latter domains are notritical prerequisites for PRMT2/AR interaction. However,he fact that the C-terminal fragment of PRMT2 without

fused transactivation domain did not influence the AR-ediated transcription suggests an important role for theRMT2 N-terminal region (aa 1–270) for its coactivationunction.

Transfection of full-length PRMT2 (aa 1–433) in PC3-Rwt cells increased the AR-dependent transactivation 7–8

imes and corroborates the role of PRMT2 as a coactivatorFig. 3A). Similar observations were made in cells devoidf constitutive AR (PC3 (data not shown) and SH-SY5YFig. 3B), following transient transfection with PRMT2 andSG5-AR expression plasmids.

Data obtained in mammalian cells indicate that PRMT2cts as a ligand-dependent coactivator of the AR, and the-terminus of PRMT2 is of critical importance for this coac-

ivation. Potentiation of AR-driven transcription by eitherull-length PRMT2 or the fusion construct of PRMT2-C-erminus and NF�B-AD also indicates that ligand-dependenteceptor activation is a prerequisite for the recruitment of thisrotein and manifestation of coactivator effects.

.5. Functional PRMT2 knockdown is associated witheduced expression of known AR regulated genes in twoifferent and unrelated AR expressing cell lines

To validate the physiological significance of the here inransient transfection experiments observed coactivation ofhe AR by PRMT2, we chose two independent cell linesxpressing both the AR and PRMT2 together on its own,or functional siRNA mediated knockdown experiments. Wenalyzed 293T cells by quantitative real time polymerasehain reaction (Q-RT-PCR) for expression of the androgeneceptor (not shown) and accordingly TMPRSS2 mRNA ispregulated in the presence of 1 nM DHT in this cell lineFig. 4A). Functional knockdown of PRMT2 expression byiRNA as confirmed by Q-RT-PCR (data not shown) attenu-tes the TMPRSS2 expression. In an independent knockdownxperiment T47D cells, known to express the AR [37], weresed. Here, the functional siRNA mediated knockdown ofRMT2 (data not shown) resulted in a reduced expres-ion of the AR target gene NKX3.1 (Fig. 4B). Treatment

ith non-targeting siRNA or PRMT2 siRNA had no sig-ificant effect on AR or ß-actin expression as determinedy Q-RT-PCR (data not shown) in neither T47D nor 293Tells.

tbbt

& Molecular Biology 107 (2007) 1–14 7

.6. PRMT2 cross reactivity with other nucleareceptors

As most of the previously described putative AR coac-ivators also interact with other nuclear receptors [1–4,38],e examined this possibility for PRMT2 in SH-SY5Y neu-

oblastoma (Fig. 3B) or PC3 prostate cells (data not shown)sing the coactivator SRC-1a (aa 618–1441) as non-selectiveositive reference [39–41]. In settings employing transientransfection of different nuclear receptors such as AR, PR,R, or MR, and their cognate agonists, it could be shown

hat the presence of the non-selective coactivator SRC-1aroduces 2–4-fold enhancement of ligand-induced reporterranscription (Fig. 3B). Transfection of SH-SY5Y cells withhe corresponding NRs and either the plasmid pCMX-RMT2 or the fusion construct pCMX-NF�B-PRMT2-Cmplified AR-, but not GR- and MR-mediated transcrip-ional activation (Fig. 3B). For PR a weak potentiation2-fold) of PRMT2 was found (Fig. 3B). Similar results werebtained using the human prostate cell line PC3 (data nothown).

.7. Binding of PRMT2 to ERα and its cell-specificoactivation

Estrogen-induced reporter transcription (pERE-luc) wasignificantly potentiated by SRC-1 introduction in PC3nd SH-SY5Y cells transfected with an ER� expres-ion vector (pHEGO-ER). Transfection of cells with thexpression plasmids for ER� and pCMX-PRMT2 or pCMX-F�B-AD-PRMT2-C dramatically increased ER-dependent

ransactivation only in the neuroblastoma cell line SH-Y5Y and had no effect in the prostate cell line PC3Fig. 5A). These data demonstrate that amplification of ERignaling by PRMT2 strongly depends on the cellular back-round.

The possibility for direct interactions between PRMT2nd ER� was examined in GST pulldown experiments usinghe C-terminal region of ER� (Fig. 5B). In vitro transcribednd translated 35S-methionine-labeled PRMT2 was incu-ated with purified GST alone or GST-ER� fusion protein.RMT2 was able to bind to the AF2 domain (aa 263–596) ofR� only in the presence of estrogen (Fig. 5B, lane 1) andas not able to bind to GST alone (Fig. 5B lane 2).

.8. PRMT2 binds S-adenosyl-l-methionine (SAM) ands sensitive to S-adenosyl-l-homocystein (SAH)nhibition

Functional projections of the homology of PRMT2 withlass I type arginine methyltransferases, especially in the cat-lytic core and the SAM binding site [17] were examined by

he capacity of GST-fused recombinant PRMT2 protein toind the methyldonor SAM in vitro. PRMT2 was able toind the arginine transferase substrate whereas no associa-ion was detected with GST and AR-LBD alone and addition

8 R. Meyer et al. / Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 1–14

Fig. 4. Knockdown of PRMT2 in the AR expressing cell lines 293T and T47D resulted in attenuation of the expression of the AR regulated genes TMPRSS2or NKX3.1. (A) 293T cells were transfected with siRNA and relative expression (normalized to �-actin expression using ��CT method) of TMPRSS2 wereanalyzed by Q-RT-PCR in DHT treated and untreated cells. Shown are the median fold changes in TMPRSS2 expression with SEM as error bars. DHT in aconcentration of 1 nM resulted in 3.7-fold induction of TMPRSS2 in case of the non-targeting control siRNA. Using the specific PRMT2 siRNA the expressionwas attenuated to 70% of this induction. (B) T47D cells were transfected with siRNA and relative expression (normalized to �-actin expression using ��CTm eated cea in theP endent

oAop

wioioAPtt(

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3

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ethod) of NKX3.1 were analyzed by Q-RT-PCR in DHT treated and untrs error bars. DHT at 1 nM induced NKX3.1 mRNA expression by 2.5-foldRMT2 siRNA attenuated the NKX3.1 expression to 65% of this DHT dep

f AR ligand did not alter the SAM binding of PRMT2 plusR-LBD (data not shown). However, no active methylationf any human protein could be found for PRMT2, confirmingrevious reports [20,24,42, and data not shown].

Since PRMT2 binds to the methyl group donor SAM,e explored whether inhibition of methyltransferase activ-

ty would influence the function of this protein as coactivatorf AR-mediated transcription. As reported above PRMT2s able to coactivate the AR function. There was no effectf the competitive methyltransferase inhibitor SAH on theR-mediated basal transactivation without the coactivator

RMT2 (Fig. 6A, white columns). Interestingly, exposure to

he inhibitor SAH attenuated the PRMT2 mediated poten-iation of AR transactivation in a dose-dependent mannerFig. 6A, grey columns). The chemical similar methyldonor

rbtP

lls. Shown are the median fold changes in NKX3.1 expression with SEMnon-targeting control siRNA transfected cells. Transfection of the specificinduction.

AM (100 �M) had no effect on AR transactivation in thisystem, indicating sufficient endogens supply of the methyl-onor in these cells. These results emphasize the importancef the methyltransferase activity of PRMT2 for its coactivatorunction.

.9. Synergism of PRMT2 and GRIP1

At least for the ER, the cellular context modulates PRMT2oactivation activity suggesting that interaction with fur-her transcription modulating proteins might play a crucial

ole for PRMT2 mediated NR coactivation. The interplayetween GRIP1, CARM1 or PRMT2 was tested in transac-ivation experiments using the AR-expressing prostate cellsC3-ARwt. In this system PRMT2, CARM1, and GRIP1

R. Meyer et al. / Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 1–14 9

Fig. 5. PRMT2 coactivation of ER� is cell type specific and ligand dependent. (A) SH-SY5Y and PC3-cells were transfected with the reporter plasmidpERE-luc (0.2 �g) and the expression plasmids pHEGO-ER (0.1 �g), as well as pCMX-PRMT2 (0.5 �g), or equal molar amounts of pCMV-VP16-AD, pCMV-AD-SRC1a, or pCMX-NF�B-AD-PRMT2-C(271–433). Relative luciferase activity was determined 24 h after treatment with vehicle, or estradiol (0.1 or 10 nM)a roblastoP his inte1

ecPsvGfpmtbtpa

3m

ffpt

ptditdPtcctil

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nd normalized to total protein. PRMT2 acts as a coactivator of ER in neuRMT2 binds in GST pulldown to the C-terminal AF2 domain of ER� and t). There is no binding of PRMT2 to the GST control (lane 2).

nhanced the AR-dependent transactivation in a hormoneoncentration-dependent manner (Fig. 6B). Coexpression ofRMT2 and GRIP1 in the PC3-ARwt cells resulted in strongynergistic potentiation of AR-mediated transactivation. Pre-iously it was described that coexpression of CARM1 andRIP1 has also a synergistic effect [22,24]. The two PRMT

amily members CARM1 and PRMT2, however, failed toroduce any synergistic or even additive effects on AR-ediated transcriptional regulation (Fig. 6B). At least in these

ransient transfection experiments, PRMT2 and CARM1ehave similar in its capability to interact with and fur-her activate the GRIP1 mediated coactivation, suggesting aossible functional role within similar or overlapping mech-nisms.

.10. Colocalization of PRMT2 and AR insideammalian cells

To analyze the subcellular localization of PRMT2 and to

ollow possible translocalization in a living cell an eGFPusion protein was used. Under hormone-free conditions inhenol red-free medium including charcoal treated serum,he PRMT2-eGFP fusion construct (green) was localized

PAnt

ma (SH-SY5Y), but not prostate (PC3) cells. (B) 35S-Methionine labeledraction is estrogen-dependent (10−6 M estradiol) for ER-AF2 domain (lane

redominantly in the cytoplasm of PC3 (not shown) andhe AR-expressing Hep-2 cells (Fig. 7A). Under these con-itions more than 90% of the AR (red) was also foundn the cytoplasm. However, under hormone free condi-ion, the fluorescence signals related to AR and PRMT2id not overlap, indicating different localization of AR andRMT2 in the cytoplasm (Fig. 7A, Merge). Time-dependent

ranslocation of both, AR and PRMT2 proteins from theytoplasm into the nucleus occurred after exposure of theells to DHT (Fig. 7B). Upon hormone exposure PRMT2ranslocated simultaneously with AR from the cytoplasmnto the nucleus as indicated in the merged image (yel-ow).

Treatment with the anti-androgen hydroxyflutamideesulted in a time-dependent translocation of AR intohe nucleus, albeit slightly slower in comparison tohe agonist DHT. However, unlike the AR, nuclearranslocation of PRMT2 was not triggered by anti-ndrogen treatment (Fig. 7B). Therefore, AR and

RMT2 did not colocalize after treatment with theR antagonist hydroxyflutamide and the binding part-ers became separated into different cellular compar-ments.

10 R. Meyer et al. / Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 1–14

Fig. 6. Alteration of PRMT2 coactivator activity by enzyme inhibition and cooperative interaction. (A) Influence of the methyl group donor SAM and thecompetitive inhibitor SAH on basal and PRMT2-enhanced AR-mediated transactivation. PC3-ARwt cells were transfected with equal molar amounts of pCMX(0.5 �g) or pCMX-PRMT2 and pAH-luc (1.0 �g) for 16 h and treated with the indicated concentrations of methyl donor or arginine methyltransferase inhibitorin the presence of vehicle, 0.1 or 10 nM DHT for 10 h; fresh drug supply and medium change were done every 2 h. Bars represent the mean of triplicatesfrom two repeats and show significant decrease with 1 �M as well as 100 �M SAH (*p < 0.05 and **p < 0.01). (B) Cooperative interaction of AR-coactivatorsC -luc (1a h aftert

4

badSppnwsprti

mcaa

ettlmda

ARM1, PRMT2, and GRIP1. PC3-ARwt-cells were transfected with pAHdded in equal molar amounts. AR-mediated transcription was assayed 24riplicates for each of the three independent experiments.

. Discussion

The data presented here demonstrate that PRMT2, a mem-er of the protein arginine methyltransferase family, acts ascoactivator of AR-mediated transcription. As previously

escribed for other nuclear receptor-interacting proteins (e.g.RC-1a), we used the yeast two-hybrid assay as a startingoint for the identification of PRMT2 as an AR-associatedrotein with functional importance in androgen receptor sig-aling. Binding of the C-terminal part of the PRMT2 proteinith the AR was confirmed in different experimental settings,

uch as yeast two-hybrid, mammalian one-hybrid and GST

ulldown assays. Subsequently, evidence for a functionalole and physiological role as a coactivator of AR-mediatedranscription was accumulated in transient transfection exper-ments overexpressing PRMT2 and nuclear receptors, siRNA

plia

.0 �g) and of pSG5 (1.0 �g) plasmid (control). Coactivator plasmids wereapplication of vehicle, 0.1 or 10 nM DHT. Transfection was performed in

ediated knockdown of PRMT2 or androgen induced intra-ellular translocation of a PRMT2-GFP fusion. Severalspects of PRMT2 binding to and modulation of AR behaviorre of particular interest.

The C-terminal domain of PRMT2 (aa 271–433) is appar-ntly sufficient for the association with the AR, whereashe N-terminal domain seems to be critical for coactiva-ion of transcription. The binding of PRMT2 to AR isigand-dependent, as shown in the yeast two-hybrid or the

ammalian one-hybrid assays. These results are in concor-ance with the observed induction of nuclear colocalizationfter AR agonist application. However, results of in vitro GST

ulldown experiments failed to confirm the significance ofigand-induced AR activation for PRMT2 recruitment. Sim-lar contradictions between GST pulldown and cell-basedssays have been previously reported for the AR corepres-

R. Meyer et al. / Journal of Steroid Biochemistry & Molecular Biology 107 (2007) 1–14 11

Fig. 7. Ligand-dependent trafficking and colocalization of AR and PRMT2 in Hep-2 cells. Localization of endogens AR (in red) was determined by Rhodamine-conjugated antibody at 543 nm excitation. PRMT2 localization is visualized in green by means of GFP-fusion with excitation at 488 nm. Yellow color in them ction ofs r AR-af in this fi

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ttportpmbom

erged image depicts colocalization of AR and PRMT2 in the confocal sehowing cells before AR ligand application. (B) Cells were treated with eitheor the indicated time periods. (For interpretation of the references to color

or SHP [34] and other nuclear receptor/cofactor interactions43,44]. These discrepancies might be caused by the exis-ence of more than one site of protein–protein interaction,hose accessibility is not exclusively dependent on an ago-istic conformation of the AR-LBD. In the living cell, someinding motifs might be masked or occupied by other factorshich are not present in the GST pulldown system. Inter-

stingly, there was extensive overlap of PRMT2 behaviorn GST pulldown, mammalian one-hybrid and transcriptionctivation experiments when interactions with the ER wereested. PRMT2 has been previously described as a coacti-ator of the ER [16], but neither GST pulldown nor yeastwo-hybrid assays provided evidence for ligand dependencef this action. Indeed, in this study we show that PRMT2 bind-ng to a LBD-containing AF2-fragment of the ER is stronglynduced by estradiol.

Coactivator effects of PRMT2 display two interesting fea-ures: (i) the protein shows strong preference for the AR butnteracts only marginally or not at all with its close kins PR,

R and GR; (ii) PRMT2 interaction with ER-mediated tran-cription is not ubiquitous and may depend on the host cellackground. We speculate that the PRMT2 amplification ofR, MR and, probably, PR signaling, has minor (if any) phys-

ological importance, with this function possibly exerted byther specific coregulator proteins. As demonstrated in the

resent study, PRMT2 and CARM1 complement, but alsoutually substitute their effects on AR-mediated transcrip-

ional regulation at least in transient transfection experiments.till, we cannot rule out that, as exemplified by the ER, sig-

iPbm

the image. DNA was stained with TO-PRO-3 (blue). (A) Time point zerogonist (DHT) or antagonist (hydroxyflutamide) at a concentration of 10 nMgure legend, the reader is referred to the web version of the article.)

ificant PRMT2 interactions with PR, GR or MR may occurn a different cellular environment. The latter may encom-ass additional cell-specific factors with either ‘permissive’r ‘prohibitive’ influence on the interactions between recep-or and coregulator. This assumption may be true for severaluclear receptors, as coactivator-like PRMT2 interaction haseen reported for PR, PPAR� and RAR� [16].

The Northern blot results elucidate expression of PRMT2n major AR target organs as testis and prostate, indicatingpotential role in AR-dependent gene activation. Consider-

ng other possible nuclear receptors as binding partners forRMT2 the strong expression in the ovary overlaps besidesR with PR and ER.The binding of PRMT2 to the AR is mediated by its C-

erminus, whereas coactivation function requires additionallyhe participation of the N-terminal part. We tested severalutative substrates for arginine methylation such as histonesr AR (unpublished observations) with negative results, aseported also by others [20,24,42]. For some members ofhe PRMT family, it was observed that their substrates areartly overlapping, e.g. both proteins PRMT1 and PRMT5ethylate histone H4. In general, most substrates for mem-

ers of the PRMT family are DNA or RNA binding proteinsr are in complex with these proteins [13,15]. Thus, arginineethylation often regulates transcription and RNA process-

ng. It was indicated that the methyltransferase activity ofRMT2 may not be obligatory for its coactivator function,ecause PRMT2 is a strong coactivator per se without knownethylation activity [20,24,42]. However, for other family

1 emistry

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embers, such as PRMT1 and CARM1, methylation activitys an important prerequisite of coactivator function and syn-rgistic interaction with other coactivators like CBP and p16022,45,46]. We observed binding of S-adenosy-l-methionineo PRMT2 confirming previous results [20,47]. In addition weemonstrated that binding of the competitive inhibitor SAHecreases PRMT2 coactivator capacity and synergism withRIP1 on AR coactivation. These observations strongly sug-est that arginine methyltransferase activity of PMRT2 maye necessary for the full-scale manifestation of coactivatorffects on AR-mediated transcription, albeit the substrate stillemains elusive. Similar results were reported for PRMT2oactivation of ER-regulated transcription [16], binding ofAM was demonstrated and mutation of the binding sitebrogated most of the coactivator effects on the ER.

Transcriptional comodulators often display mutual influ-nce and interactions with other cellular proteins. In thesetudies such behavior was demonstrated also for PRMT2.ynergistic interactions of PRMT2 with GRIP1 on AR-ediated transactivation resemble those previously described

or CARM1 [24], and the magnitude of individual effectss a hint for a possibility for mutual substitution or redun-ancy. The latter assumption is supported by the lack ofynergistic/additive effects of CARM1 and PRMT2 on theranscriptional outcome, while similarity between the mecha-isms of action of CARM1 and PRMT2 is underscored by thebservation that individual coactivator effects of CARM1 andRMT2 are inhibited by SAH. Our data contradict previouseports on coactivator interactions [16,48] which argue thathe mechanisms of PRMT2-mediated transcriptional con-rol differ from those employed by CARM1 and PRMT1.

hile the latter studies used ER-regulated transcription as aubstrate for examination of coactivator effects, and did notddress the synergism of PRMT2 and GRIP1, hints in supportf our evidence for cooperation of CARM1 and PRMT2 withRIP1 on nuclear receptor coactivation has been reported

24]. The demonstration of the capability of PRMT2 for syn-rgistic interplay with other comodulators strengthens thessumption that, as described here for the ER, manifestationf coactivator effects may depend on intricate comodulatornteractions which are ultimately defined by a specific cellularackground. In similar vein, a role of secondary coactiva-ors has been proposed for PRMT1 and CARM1 [21–23].lthough PRMT2 per se is able to associate with nuclear

eceptors (as demonstrated in GST pulldown experiments),ts contribution to transcriptional activation may be obscuredy co-present primary coactivators.

Under steroid free conditions transfected PRMT2-eGFPusion constructs are nearly exclusively localized in the cyto-lasm of Hep-2 cells, which express the AR on its own.RMT2 has a SH3 domain, often found in proteins involved

n cytoplasmic signaling pathways [49,50]. Nonetheless, the

ocalization in the cytoplasm is a surprising observation,s most comodulators of hormone receptors are primar-ly located in the nucleus. According to our observations,RMT2 colocalizes in the cytoplasm with ligand-activated

PaaC

& Molecular Biology 107 (2007) 1–14

R, and both proteins are subsequently transported into theucleus. In this paper ligand dependent cotranslocation ofR together with its comodulator PRMT2 was described,hereas translocation of AR alone from the cytoplasma to

he nucleus was reported previously [51–53]. As yet, onlyhanges of coactivator localization within the nucleus areescribed, as for example demonstrated for AR/GRIP1 inter-ction [48]. However, the simultaneousness of PRMT2 andR translocation does not lend support for a role of PRMT2

n the transport of the AR into the nucleus. Indeed, as PRMT2acks an own nuclear localization signal consensus sequence,t is likely piggyback-transported into the nucleus by thegonist-activated AR. The latter assumption is supported byhe observation that nuclear translocation of the antagonist-ound AR is not accompanied by PRMT2 trafficking. Thenly so far confirmed function of PRMT2 is coactivation ofuclear receptors as reported here for AR. PRMT2 exertsts effect as AR-coregulator upon being transferred fromhe cytoplasm to the nucleus as a result of agonist binding.hus, unlike with most known nuclear receptor coregula-

ors, PRMT2 lays down an intriguing example of coregulatorecruitment and trafficking out of a cytoplasmic pool bygonist-activated receptor.

PRMT2 is the third member of arginine methyltransferaseamily for which a comodulation of nuclear receptors haseen reported. Although its methyltransferase substrate(s)emain to be identified, these data are indicative of moreeneral importance of arginine methylation in transcrip-ional regulation. Arginine methyltransferase activity haseen demonstrated in yeast, thus suggesting a physiologi-al role in eukaryotes. The elusive methylation substrate ofRMT2 alludes that this protein is either unconventional orxperimental conditions in vivo are of critical importance forhe maintenance of the enzyme activity. It is also possible thatrior modification of the target protein is necessary. Identifi-ation of the substrates of PRMT2 appears crucial to the fullnderstanding of its function. However, even with the currentimited knowledge, we can summarize that PRMT2 is uniqueithin the arginine methyltransferase family. It is the only

amily member which depends on the agonist-activated ARor nuclear receptor) for nuclear translocation and manifes-ation of coactivator activity. The ligand-dependent nuclearedistribution of PRMT2, the still unknown function of itsH3 domain, and abundant presence in androgen target

issues warrant further interest in its physiological role inuclear receptor signaling.

cknowledgements

We are grateful to Sabine Herrmann and Monika Kindleror excellent technical assistance. Special thanks to Vladimir

atchev for his constructive discussion, Peter Hemmerichnd Gerhard Wieland (IMB, Jena, Germany) for providingccess to their confocal microscope. We also thank Pierrehambon, David Moore, Steven Nordeen, Steffi Oesterreich,

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nd Michael Stallcup for their gifts of plasmids and Berndiederanders (Friedrich-Schiller University, Jena Germany)

or his support.

eferences

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