SUMO-Dependent Compartmentalization in Promyelocytic Leukemia Protein Nuclear Bodies Prevents the Access of LRH-1 to Chromatin

Posttranslational modification by SUMO elicits a repressiveeffect on many transcription factors. In principle, sumoylationmay either influence transcription factor activity on promoters,or it may act indirectly by targeting the modified factors tospecific cellular compartments. To provide direct experimentalevidence for the above, not necessarily mutually exclusive models,we analyzed the role of SUMO modification on the localizationand the activity of the orphan nuclear receptor LRH-1. We demonstrate,by using fluorescence resonance energy transfer (FRET) and fluorescencerecovery after photobleaching (FRAP) assays, that sumoylatedLRH-1 is exclusively localized in promyelocytic leukemia protein(PML) nuclear bodies and that this association is a dynamicprocess. Release of LRH-1 from nuclear bodies correlated withits desumoylation, pointing to the pivotal role of SUMO conjugationin keeping LRH-1 in these locations. SUMO-dependent shuttlingof LRH-1 into PML bodies defines two spatially separated poolsof the protein, of which only the soluble, unmodified one isassociated with actively transcribed target genes. The resultssuggest that SUMO-PML nuclear bodies may primarily functionas dynamic molecular reservoirs, controlling the availabilityof certain transcription factors to active chromatin domains.

INTRODUCTION

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Introduction

The selectivity of gene expression is mainly controlled by thelimited ability of transcription factors to access the genome.Besides alterations of chromatin structure, other processes,such as the positioning of genes within the nucleus and compartmentalizationof the proteins that regulate their expression, have been implicatedin the pathways determining the specificity of gene activation(9, 15, 30). Covalent attachment of the small-ubiquitin-relatedmodifier (SUMO) to proteins has been characterized as a modificationwith diverse effects, including targeting of the substratesto specific nuclear territories (17, 42, 46, 52). For example,sumoylation is required for the targeting of the cytoplasmicnuclear import factor RanGAP1 to the nuclear pore complex orto mitotic spindles and kinetochores in dividing cells (24,32, 33). Furthermore, under conditions that allow SUMO conjugation,several proteins, including transcription factors, have beenfound to localize in nuclear speckles (38, 40, 41), althoughdirect evidence for the identity of the proteins at the nuclearspeckles as SUMO-modified ones is still missing.

These sites of accumulations often coincide with the nuclearbodies associated with promyelocytic leukemia protein nuclearbodies (PML-NBs). PML-NBs are nuclear matrix-associated structures,ranging in size from 0.2 to 1 µm (7, 52). Besides PML,several other proteins accumulating in these structures havebeen identified. These include SUMO-1, Sp100, Daxx, Rb, BLM,CBP, and p53, suggesting roles in DNA replication and repair,cell cycle control, apoptosis, and transcription (1, 6, 23,29, 50, 51). SUMO modification of several of the above components,including the PML protein itself, is important for their recruitmentto the PML-NBs and consequently for the proper formation ofthe nuclear domain (52). Although several lines of evidencesuggest that PML-NBs may play a role in the regulation of geneexpression (3, 5, 47, 52), the relationship between SUMO modification-dependentcompartmentalization of transcription factors into these structuresand their function on target genes is less well understood.

To address this question, we studied the role of SUMO modificationon the localization and the activity of the orphan nuclear receptorLRH-1 (for liver receptor homologue 1; also known as FTF andCPF). LRH-1 plays a crucial role in liver development duringearly embryogenesis in controlling cell proliferation and renewalof intestinal crypt cells and in the regulation of cholesterol-bileacid homeostasis in adult hepatocytes (8, 12, 20, 31, 36). Herewe show that LRH-1 is reversibly modified by SUMO-1 in vitroand in vivo. We present evidence for a SUMO-dependent sequestrationof LRH-1 into PML-NBs, which precludes its access to activechromatin domains.

MATERIALS AND METHODS

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Materials and Methods

Plasmids and antibodies. The open reading frame of the human LRH-1 cDNA was subclonedinto pCDNA-myc (Invitrogen) and pEYFP (Clontech) vectors. Site-directedmutagenesis was performed with the GeneEditor kit (Promega).Glutathione S-transferase (GST)-SUMO-1, GST-Ubc9, GST-PIASx ${alpha}$ ,and pCMV-Flag-PIASx ${alpha}$ plasmids were provided by N. Kotaja andJ. Palvimo, GST-SAE1/2 was provided by R. Hay, GST-RanBP2 wasprovided by F. Melchior, and pSG-PML-III and green fluorescentprotein (GFP)-PML-III were provided by H. Will. pMT-HA-SUMO-1,pECFP-SUMO-1, pEYFP-SUMO-1, and pCMV-Flag-SuPr-1 were generatedby amplifying the open reading frame of SUMO-1 missing the lastfour amino acids and the entire open reading frame of SuPr1by PCR, followed by ligation into pMT-HA, pECFP, pEYFP, andpCMV-Flag vectors, respectively. All constructs were verifiedby DNA sequencing.

Anti-SUMO-1 (FL-101), anti-myc (9E10 and A-14), antihemagglutinin(anti-HA; Y-11), anti-PML, and anti-Cy3-labeled PML (PG-M3)were from Santa Cruz Biotechnology; anti-Pol-II (8WG16) wasfrom Covance; anti-histone 3 (ab8580) was from Abcam; and anti-Flag(M2) and anti-Lamin B were from Sigma. A polyclonal antibodyagainst LRH-1 was raised by immunization of New Zealand Whiterabbits with bacterially expressed recombinant human LRH-1 protein.In Western blots and immunofluorescence assays horseradish peroxidase-conjugatedanti-rabbit and anti-mouse immunoglobulin G (Jackson Laboratories)and anti-rabbit or anti-mouse AlexaFluor568 or AlexaFluor488(Molecular Probes) were used as secondary antibodies, respectively.

siRNA-mediated knockdown of SUMO-1. Double-stranded small interfering RNA (siRNA) for targetinghuman SUMO-1 and control siRNA containing scrambled sequencewere purchased from Santa Cruz Biotechnology (sc-29498 and sc-37007).HepG2 cells were transfected with the siRNAs at 100 nM finalconcentrations by using the jetSI-ENDO kit (Polyplus). Extractpreparations and immunofluorescence analyses were performed72 h after transfection.

Immunoprecipitations, Western blots and in vitro SUMO conjugation assays. Cells were lysed by resuspension in lysis buffer containing1.72% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl (pH 6.7),10% glycerol, 0.33% NP-40, 0.33% sodium deoxycholate, 10 mMN-ethylmaleimide (NEM), 1 mM phenylmethylsulfonyl fluoride,2 µg of aprotinin/ml, and 10 µg of E64/ml, followedby mild sonication and centrifugation at 12,000 x g. The extractswere either analyzed directly on Western blots or, after a 17-folddilution with a buffer containing 50 mM HEPES (pH 7.9), 5% glycerol,0.1% NP-40, 150 mM NaCl, 0.1 mM EDTA, 10 mM NEM, 1 mM phenylmethylsulfonylfluoride, 2 µg of aprotinin/ml, and 10 µg of E64/ml,subjected to immunoprecipitation as described previously (45).Chromatin immunoprecipitation (ChIP) assays were performed asdescribed previously (21, 44) except for the cross-linking step.For the latter, mouse livers were perfused successively withphosphate-buffered saline (PBS), 1% formaldehyde, and 0.125M glycine for 10 min. Cross-linked nuclei were purified by centrifugationthrough a sucrose gradient as described previously (45). Inre-ChIP assays, after the protein G-Sepharose beads from theprimary immunoprecipitation were washed, bound complexes wereeluted from the beads by incubation with 10 mM dithiothreitolat 37°C for 30 min. After a 50-fold dilution with 1x sonicationbuffer, the eluates were subjected to immunoprecipitation withthe second antibody. Reporter assays were performed as describedpreviously (28).

In vitro sumoylation reactions were performed by using 1 µlof in vitro-translated, ³⁵S-labeled LRH-1 in a 15-µl reactionmixture containing 50 mM Tris-HCl (pH 8.0), 4 mM MgCl₂, 1 mMdithiothreitol, and 2 mM ATP, 50 ng of GST-SUMO-1, 25 ng ofGST-SAE1/2, 25 ng of GST-UBC9, and 1 µg of GST-RanBP2,or GST-PIAS-1, or GST-PIASx ${alpha}$ . After incubation at 30°C for1 h, the reaction products were analyzed by SDS-polyacrylamidegel electrophoresis (PAGE).

Nuclear matrix isolation was carried out essentially as describedin references 22 and 37, except that all buffers were supplementedwith protease inhibitor cocktail (Roche), 10 mM NEM, and 10µg of E64/ml. Briefly, after being washed with PBS, thecells were resuspended in CSK buffer (see below) and incubatedfor 3 min on ice. After centrifugation for 3 min at 5,000 xg, the supernatant (Nucleocytoplasm) was separated, and thepellet was resuspended in CSK buffer containing 1 mg of RNase-freeDNase/ml. After incubation at 37°C for 15 min, the reactionmix was adjusted with ammonium sulfate to 0.25 M final concentrationand centrifuged again. The supernatant (chromatin fraction)was saved, and the pellet was extracted with 2 M NaCl. Completeremoval of histones and DNA was verified by SDS-PAGE and agarosegel electrophoresis. The final insoluble pellet (nuclear matrix)was resuspended in SDS sample buffer. Equal proportions of eachfraction were separated by SDS-PAGE and analyzed in Westernblots.

Quantitative assessment of Western blot signals was performedwith a Fujifilm LAS-1000 luminescent image analyzer.

Immunofluorescence and confocal microscopy. Cells were plated on poly-D-lysine-coated glass coverslips andtransfected with various combinations of expression vectorsby using the PolyPlus transfection reagent. At 24 to 36 h aftertransfection the cells were either used for live cell imagingor fixed with 2% paraformaldehyde. Immunostainings were performedas described previously (28). Fluorescence images were obtainedon Zeiss Axioscope 2 Plus microscope outfitted with a Bio-RadRadiance 2100 laser scanning system and Lasersharp-2000 imagingsoftware.

For fluorescence resonance energy transfer (FRET) analysis thefollowing filter settings were used. Cyanfluorescent protein(CFP) fluorescence was observed by using argon laser 457-nmexcitation and 488/10 band-pass emission filters, whereas yellowfluorescent protein (YFP) fluorescence was observed by usingargon laser 514-nm excitation and 570LP band-pass emission filters.Photobleaching of YFP fluorescence was achieved by irradiationof the selected cells for 1 min with the 514-nm excitation filterat maximum intensity. Sequential images before or after photobleachingwere collected, and pixel intensities of the different regionswere analyzed by using NIH Image-J software. FRET efficiencywas expressed as the percent increase of prebleach CFP fluorescencein the individual areas compared to that observed after YFPphotobleaching.

For fluorescence recovery after photobleaching (FRAP) analysis,the coverslips were placed in PBS and observed by using argonlaser 514-nm excitation and 545/40 band-pass emission filtersfor YFP and argon laser 488-nm excitation and 515/30 band-passemission filters for GFP. An approximately 1-µm² spotwas bleached for 2 s at 50 to 60% intensity setting of the appropriateexcitation laser, and serial images were collected over a 60-speriod. Fluorescence intensities of the bleached areas fromfive independent experiments were normalized to out-of-focuscontribution and are expressed as a percentage of the measuredprebleach intensities.

In situ permeabilization experiments were performed by treatingthe cells with 0.5% Triton X-100 containing CSK buffer (10 mMPIPES [pH 6.8], 300 mM sucrose, 100 mM NaCl, 3 mM MgCl₂, 1 mMEGTA) for 1 min at room temperature. Permeabilization was stoppedby fixing the cells with 2% paraformaldehyde or by extractionwith lysis buffer.

RESULTS

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Results

LRH-1 is modified by SUMO-1 in vitro and in vivo. The LRH-1 protein has three conserved consensus sumoylationsites ( ${Psi}$ KXE) at amino acid positions 146, 224, and 264. In orderto demonstrate that LRH-1 can be modified by SUMO, we performedWestern blot analysis with lysates from HEK293 cells, whichwere transfected with HA-tagged SUMO-1 and myc-tagged wild type,or SUMO consensus-site mutant forms of LRH-1. A slower-migratingLRH-1 species ( $~$ 90 kDa) was observed only when LRH-1 was coexpressedwith SUMO-1 and the known SUMO E3 ligase PIASx ${alpha}$ (25) (Fig. 1A,lane 3). The fact that this band corresponds to SUMO-modifiedLRH-1 is demonstrated by its dependence on the coexpressionof SUMO-1 and PIASx ${alpha}$ (Fig. 1A, lanes 1 to 3) and by its disappearanceupon coexpression of SuPr-1 (Fig. 1A, lane 4), which is a knownSUMO protease (3). Furthermore, we could detect a band withthe same mobility in myc-LRH-1 immunoprecipitates with an HAantibody recognizing HA-tagged SUMO-1 and in HA-SUMO-1 immunoprecipitatesby using a myc antibody recognizing myc-tagged LRH-1 (Fig. 1B).When the same analyses were performed with the different sumoylationconsensus site LRH-1 mutants, the slower-migrating band disappearedonly in the case of K224R, suggesting that lysine 224 of LRH-1is the major SUMO conjugation site (Fig. 1A, lanes 6 to 8).

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FIG. 1. LRH-1 is modified by SUMO-1 in vivo. (A) Extracts of HEK-293 cells transfected with the indicated expression vectors were analyzed in Western blots with a myc tag-specific antibody to detect modified and unmodified LRH-1. (B) myc-LRH-1 and HA-SUMO-1 proteins were immunoprecipitated with ${alpha}$ myc or ${alpha}$ HA antibody, and the presence of LRH-1 and SUMO-1 in the immunoprecipitates was detected in Western blots by using ${alpha}$ LRH-1, ${alpha}$ myc and ${alpha}$ HA antibodies as indicated. (C) Endogenous LRH-1 from C3A-HepG2 cells was detected in Western blot by an ${alpha}$ LRH-1 antibody (left panel) or immunoprecipitated with ${alpha}$ LRH-1 antibody and analyzed in Western blots with ${alpha}$ SUMO-1 antibody. As a control, nonimmune (Non-Imm.) antiserum was used for immunoprecipitation. (D) C3A-HepG2 cells were transfected with SUMO siRNA and control siRNA as indicated. Cell lysates prepared 72 h after transfection were analyzed in Western blots with antibodies recognizing SUMO-1, HNF4, TFIIB, and LRH-1 (left and middle panels). Part of the extracts was immunoprecipitated with ${alpha}$ LRH-1 antibody and analyzed in Western blots with ${alpha}$ SUMO-1 antibody (panel at right).

In order to examine the modification of endogenous LRH-1 proteins,we performed Western blot analysis with nuclear extracts fromC3A-HepG2 cells. A slower-migrating ${alpha}$ LRH-1-reactive band ( $~$ 90kDa) was observed in addition to the major 55-kDa species, whichcorresponds to unmodified LRH-1 protein. The identity of the90-kDa band as SUMO-conjugated LRH-1 was confirmed by Westernblot analysis of anti-LRH-1 immunoprecipitates with an ${alpha}$ SUMO-1antibody (Fig. 1C). Additional evidence for the sumoylationof endogenous LRH-1 was provided by the analysis of cells transfectedwith SUMO-1 siRNA. In SUMO-1-specific siRNA-transfected cellsendogenous SUMO-1 was efficiently knocked down and the slower-migratingband in simple Western blots stained with anti-LRH-1, or inLRH-1 immunoprecipitates probed with anti-SUMO-1, disappeared(Fig. 1D). The specificity of the assay was confirmed by theunaltered expression of LRH-1, or HNF-4 and TFIIB, which wereused as additional controls (Fig. 1D).

Next, we tested whether LRH-1 could also be a substrate forSUMO modification in vitro. We used an in vitro sumoylationassay using purified recombinant GST-SUMO-1 protein, E1 (SAE1/2),E2 (Ubc9) enzymes, and various E3 ligases (RANBP2, PIAS1, andPIASx ${alpha}$ .). As substrates, we used in vitro-translated ³⁵S-labeledwild-type LRH-1 or its different SUMO consensus site mutantderivatives. We could detect SUMO attachment to radiolabeledLRH-1 only when SUMO-1, SAE1/2, Ubc9 and PIAS1, or PIASx ${alpha}$ ligaseswere present in the reaction (Fig. 2, lanes 4 and 5). As expected,unlike the K146R or the K264R mutants, the K224R mutant formof LRH-1 was not modified in vitro (Fig. 2, lanes 8 to 13).Collectively, these results suggest that LRH-1 can be modifiedby SUMO-1 on lysine 224 by the PIAS family of E3 ligases andthat sumoylated LRH-1 is a substrate for SuPr-1, which can hydrolyzethe SUMO moiety from the protein.

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FIG. 2. Modification of LRH-1 by SUMO-1 in vitro. ³⁵S-labeled wild-type LRH-1 and its mutant forms were synthesized in vitro and incubated in a reaction reconstituted with the indicated recombinant proteins. The reaction products were separated by electrophoresis in SDS-10% polyacrylamide gels and visualized by autoradiography.

Sumoylated LRH-1 is localized in discrete nuclear dots. To study the subnuclear localization of LRH-1, we expresseda YFP-LRH-1 fusion protein in HeLa cells. A diffuse nuclearstaining was observed (Fig. 3A). However, when the wild-typeYFP-LRH-1 was coexpressed with SUMO-1 and PIASx ${alpha}$ , which, as judgedby Western blot analysis leads to the sumoylation of ca. 50%of the protein (data not shown), a substantial part of the fluorescencesignal was accumulated in dot-like structures in the majorityof the cells. No such accumulation was observed with the K224Rmutant protein or in SuPr-1-expressing cells, suggesting thatthe observed relocalization of LRH-1 was dependent on sumoylation(Fig. 3A). Although such SUMO-dependent subnuclear redistributionhas been observed with other transcription factors (9, 38, 40),the question of whether the proteins accumulating at the nucleardots correspond to SUMO-modified ones has not been directlyaddressed before. To this end, we performed FRET after photobleachingassays (25), with CFP-tagged SUMO-1 and YFP-tagged LRH-1 proteins.FRET is based on the ability of a higher-energy donor fluorophore(CFP) to transfer energy to a lower-energy acceptor molecule(YFP), causing sensitized fluorescence of the acceptor and simultaneousquenching of the donor fluorescence, when the two moleculesare in close proximity. If FRET occurs, photobleaching of YFPshould lead to an increase in the emission of the CFP protein.An average of 8% increase in CFP emission was observed onlyat the nuclear dots but not in the areas outside them (Fig.3B, A1 and A2 datum set). The significance of this increaseis substantiated by the absence of FRET signal in control experimentswhere either YFP-LRH-1 K224R was coexpressed with CFP-SUMO-1and PIASx ${alpha}$ , or YFP-LRH-1wt was coexpressed with CFP-SUMO-1 withoutPIASx ${alpha}$ (Fig. 3B, B and C datum set). Because FRET occurs onlywhen the donor and acceptor molecules are within 50 to 100 Åof each other (25), these results can be interpreted eitheras a true molecular interaction between wtLRH-1 and SUMO-1 oras an interaction of LRH-1 with other sumoylated proteins. Thelatter scenario can be excluded by the absence of FRET signalin any of the randomly selected nuclear locations under conditionswhere LRH-1 sumoylation does not occur (e.g., in YFP-LRH-1 K224R-transfectedcells or in cells not transfected with PIASx ${alpha}$ ). Therefore, weconclude that SUMO-modified LRH-1 is localized solely at thenuclear dot areas, pointing to the existence of two populationsof LRH-1 molecules (sumoylated and nonsumoylated), which aredistributed into distinct nuclear territories.

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FIG. 3. SUMO-modified LRH-1 is localized in discrete nuclear dots. (A) HeLa cells were transfected with the indicated expression vectors and observed by confocal microscopy. Representative images of cells from several experiments are shown. (B) HeLa cells were transfected with pECFP-SUMO-1, pEYFP-LRH-1, and pCMV-Flag-PIASx ${alpha}$ . Sequential CFP and YFP fluorescence images were recorded before and after 1 min of photobleaching of YFP fluorescence by 514-nm laser line. FRET efficiency was expressed as the percent increase of prebleach CFP fluorescence after YFP photobleaching in the nuclear dot areas (A1) and similar-sized areas outside the nuclear dots (A2). In the control experiment B, the cells were cotransfected with pECFP-SUMO-1, pEYFP-LRH-1 K224R, and pCMV-Flag-PIASx ${alpha}$ , whereas in the control experiment C, the cells were cotransfected with pECFP-SUMO-1 and pEYFP-LRH-1 without the PIASx ${alpha}$ expression vector. The differences in fluorescence intensity in similar-sized nuclear areas were calculated as described above. The graph shows mean values and standard errors from the indicated number (N) of measurements, which were obtained from six to eight different cells.

The sites where sumoylated LRH-1 accumulates correspond to PML-containing nuclear bodies. In order to further characterize the identity of the nucleardomains where sumoylated LRH-1 accumulates, we performed colocalizationassays with PML, which is known to be concentrated in similarspeckles in a sumoylation-dependent manner (3, 13). In C3A-HepG2cells endogenous LRH-1 and SUMO-1 were detected throughout thenucleoplasm and at small speckles, which largely overlappedwith endogenous PML-containing nuclear dots (Fig. 4A1 and 2).When endogenous SUMO-1 expression was knocked down, LRH-1 disappearedfrom the nuclear speckles, whereas PML was concentrated in twoto four large nuclear aggregates (Fig. 4A3 and 4).

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FIG. 4. SUMO-modified LRH-1 is localized in PML-containing nuclear bodies. (A) Untransfected and SUMO-siRNA-transfected C3A-HepG2 cells were stained with ${alpha}$ LRH-1 (rows 1 and 3) and ${alpha}$ SUMO-1 (rows 2 and 4). (B and C) HeLa cells, which were transfected with the expression vectors indicated at the right, were observed by direct fluorescence of YFP or immunofluorescence staining with anti-Flag or anti-RNA Pol II antibodies (left panels). In all cases PML-NBs were detected in the same field by immunofluorescence staining with a TRITC (tetramethyl rhodamine isothiocyanate)-conjugated PML antibody (middle panels). Merged images are depicted in the right panels.

The correlation between sumoylation of LRH-1 and its colocalizationwith PML was further corroborated by the analysis of HeLa cellstransfected with various combinations of expression vectorsthat mimic the conditions under which SUMO modification of theectopically expressed YFP-LRH-1 can occur (transfection withwtLRH-1, PIASx ${alpha}$ , and SUMO-1 [Fig. 4B1]) or cannot occur (transfectionwith wtLRH-1 and SUMO-1 without PIASx ${alpha}$ [Fig. 4B3] or transfectionwith LRH-1 K224R with PIASx ${alpha}$ and SUMO-1 [Fig. 4B4]). As expected,colocalization of YFP-LRH-1 with PML in the NBs could be detectedin a SUMO-1- and PIASx ${alpha}$ -dependent manner (Fig. 4B1). Under thesame conditions YFP-SUMO-1 also colocalized with PML (Fig. 4B2).Overexpression of SuPr-1 resulted in a diffuse nuclear distributionof YFP-LRH-1 and YFP-SUMO-1, with some accumulation of the latterat the nuclear periphery (Fig. 4C1 and 2). Although this couldbe consistent with the idea that desumoylation of LRH-1 leadsto its release from PML-NBs, we note that SuPr-1 action mightnot be selective because sumoylation of the PML protein itselfis important for the integrity of nuclear bodies (3, 13, 34).In agreement with a previous study (3), we observed that SuPr-1overexpression resulted in the disruption of PML-NBs and thatPML was relocated in two to four large, abnormally shaped aggregates(Fig. 4C1 to C3). Interestingly, in addition to the diffusenuclear fraction, a clear concentration of endogenous RNA polymeraseII was detected in these SuPr-1-containing and SUMO-deficientstructures, suggesting that they may represent novel PML depositsites functionally different from those of PML-NBs, which containsumoylated proteins but do not accumulate RNA polymerase-II(Fig. 4C4 and 5). Because of the high density of RNA polymeraseII in these SuPr-1-generated territories, it is tempting tospeculate that they may represent hot spots of active transcriptionfor several genes. However, the diffuse nucleoplasmic distributionof LRH-1 in SuPr-1-overexpressing cells suggests that the SuPr-1-mediatedinduction of LRH-1 activity (see below) is more likely a consequenceof the release of LRH-1 from PML-NBs and the concomitant increaseof the free LRH-1 pool than of its relocation to the above RNApolymerase II-dense nuclear regions.

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FIG. 5. Release of LRH-1 from PML-NBs correlates with the loss of SUMO moiety. (A) HeLa cells were transfected with YFP-LRH-1, HA-SUMO, PIASx ${alpha}$ , and PML-III expression vectors. (C) HeLa cells were transfected with YFP-SUMO-1, myc-LRH-1, PIASx ${alpha}$ , and PML-III expression vectors. The cells were incubated in a 0.5% Triton X-100-containing buffer for 1 min before fixation or extraction. Triton X-100 treatments were performed in the presence or absence of 10 mM NEM and 10 µg of E64 isopeptidase inhibitors/ml. Confocal microscopic images (A and C) and Western blot analysis of extracts with ${alpha}$ LRH-1 antibody (B) are shown.

Dynamics of SUMO-dependent association of LRH-1 with PML-NBs. Evidence for the role of desumoylation on the release of LRH-1from PML-NBs was provided by in situ permeabilization experiments.Our rationale was that if the accumulation of SUMO-LRH-1 atPML-NBs has a "storage" function, then decreasing the intranuclearconcentration of LRH-1 might mobilize the molecules locatedin these compartments. To this end, we treated the cells with0.5% Triton X-100 for 1 min and, after fixation, examined thedistribution of YFP-LRH-1, YFP-SUMO-1, and PML proteins. Underthese mild permeabilization conditions, ca. 50% of the LRH-1protein leaked out of the nuclei, whereas the overall morphologyof the cells was preserved. As shown in Fig. 5A, YFP-LRH-1 wascleared from the nuclear dots in Triton X-100-treated cellsand was localized mainly at the surrounding areas. On the otherhand, under the same conditions, PML and YFP-SUMO-1 remainedconcentrated in discrete nuclear speckles (Fig. 5A and C). Importantly,when the detergent treatment was performed in the presence ofthe isopeptidase inhibitors NEM and E64, which prevent the cleavageof SUMO from modified proteins (14), a substantial proportionof YFP-LRH-1 fluorescence remained associated with the PML-containingnuclear dots (Fig. 5A). Parallel Western blot analysis revealedthe loss of SUMO moiety from LRH-1 in Triton X-100-permeabilizedcells and its retention when the treatment was performed inthe presence of isopeptidase inhibitors (Fig. 5B). These resultssuggest that lowering the nuclear concentration of proteinsleads to desumoylation and the release of LRH-1 from the nuclearspeckles, while a large portion of SUMO molecules, which maycorrespond to certain proteins that retained SUMO-1, cleavedSUMO-1, or free SUMO-1, are retained in PML-NBs.

If the observed isopeptidase inhibitor-sensitive desumoylationand release of LRH-1 is mediated by the activation of the SuPr-1family of enzymes, the results of the above experiment are inan apparent contradiction with those obtained in SuPr-1-overexpressingcells, where PML-NBs were disrupted. We have to assume thatthe observed desumoylation of LRH-1 is either not selectiveand is accompanied by at least a partial disruption of the PML-NBsor that it is mediated by other, specific isopeptidases thatdo not cleave the SUMO moiety from PML. We favor the first scenario,which is also supported by the less-compact appearance of PMLdots and the partial loss of SUMO-1 from PML-NBs in Triton X-100-permeabilizedcells (Fig. 5A and C). We also note that high levels of SuPr-1expression over a long period of time may lead to more dramaticeffects compared to those exerted by the activation of the endogenousprotein. In either case, however, the results clearly demonstratethat desumoylation of LRH-1 leads to its dissociation from thePML-NBs.

The kinetics of LRH-1 association with PML-NBs was evaluatedby FRAP experiments. In cells expressing YFP-LRH-1, YFP-SUMO-1,or GFP-PML-III, individual nuclear dot areas and areas outsideof the dots were photobleached by using a 2-s pulse, and thekinetics of the recovery of fluorescence intensity at the bleachedspots were measured. Recovery of YFP-LRH-1 fluorescence in nucleardot areas reached equilibrium at ca. 75% of the prebleach intensitywithin 15 s, pointing to the existence of a highly mobile anda relatively immobile population of the protein (Fig. 6A). Inareas outside the nuclear speckles almost full recovery of YFP-LRH-1fluorescence was observed within 2.5 s (Fig. 6B). The existenceof an immobile fraction in the nuclear speckles and the differencein residence time between the two territories (dots and outsidedot areas) provide further support for the idea that PML-NBsmay function as molecular reservoirs of nuclear LRH-1. In agreementwith a previous report, PML protein residing at NBs was foundto be immobile (Fig. 6D), which is consistent with the natureof structural proteins that contribute to the integrity of NBs(6). Interestingly, however, SUMO-1 was also found to be a relativelyimmobile protein (Fig. 6C). This is most likely due to the sumoylationof immobile PML and other structural components (e.g., SP100)of the NBs, without precluding the possibility that NB-associatedSUMO-1 stably resides in these structures as part of the sumoylationmachinery in reserve for modification of incoming protein substrates.

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FIG. 6. Dynamic association of LRH-1 with PML-NBs. HeLa cells were transfected with the expression vectors indicated at the right, and expression was detected by direct fluorescence of YFP or GFP in live cells. After targeted photobleaching of the boxed areas corresponding to nuclear dots (A, C, and D) or areas outside the nuclear dots (B), serial images were collected every 2.5 s. Images from one experiment at selected time points are shown. The graphs at the bottom represent fluorescence recovery curves for the bleached areas. The results are presented as mean values and standard errors of the percentages of the postbleach fluorescence intensities at the irradiated areas relative to the prebleach intensity from five independent experiments.

Taken together, these results suggest that association of LRH-1with PML-NBs is a dynamic process, which involves rapid sumoylationand desumoylation-driven exchange of the majority of LRH-1 proteinresiding in these structures.

Sumoylation negatively regulates LRH-1 transcriptional activity by excluding it from active chromatin domains. The regulated SUMO-dependent shuttling of LRH-1 into PML-NBsdefines two spatially sequestered pools of the protein withapparently different roles in transcription. Therefore, we analyzedthe functional consequence of sumoylation on the transcriptionalactivity of LRH-1 and on its recruitment to target genes inthe context of chromatin. In transient-transfection assays thesumoylation-deficient mutant of LRH-1 exhibited an approximatelytwofold increased transactivation capacity (Fig. 7A). A similarlevel of increase in wtLRH-1-mediated transcription was observedin SuPr1-transfected cells, suggesting that sumoylation elicitsa repressive effect on the transcriptional activity of LRH-1.Although in our Western blot assays (Fig. 1) we could not observesumoylated LRH-1 in cells transfected with wtLRH-1 alone, thedifferent transactivation potential of wild-type and mutantLRH-1 may be explained by assuming that some of the overexpressedwild-type protein became sumoylated by endogenous SUMO-1 andPIASx ${alpha}$ which, due to the high turnover of the modification, limiteddetection. To clarify this, we performed additional assays bycoexpressing LRH-1 with PIASx ${alpha}$ and SUMO-1. Interestingly, coexpressionof PIASxa alone with either wild-type and the K224R mutant ofLRH-1 increased their transactivation potential (Fig. 7A), suggestingthat PIASx ${alpha}$ can function as a coactivator for LRH-1 independentof sumoylation. However, when SUMO-1 was coexpressed (conditionswhen sumoylated LRH-1 can be detected by Western blot analysis),an eightfold decrease in wtLRH-1-induced promoter activity wasobserved. Importantly, no such repression was evident in LRH-K224R-inducedtranscription (Fig. 7A). In principle, a repressive effect inreporter assays could be the result of a direct action of SUMO-1on LRH-1 properties or an indirect effect brought about themodification of coactivators (e.g., p300) (19) or corepressors(e.g., HDAC) (11, 26, 49), which may modulate LRH-1 activity.Although the results obtained with the sumoylation-deficientLRH mutant argue against the latter possibility, in either ofthe above cases SUMO is presumed to act on promoter-bound LRH-1-containingtranscription complexes. An alternative possibility is thatsumoylated LRH-1 cannot access its target genes because it isspatially sequestered to nuclear locations from which activechromatin domains are excluded. Because, as shown above, sumoylatedLRH-1 is localized in PML-NBs, one way to distinguish betweenthese possibilities is to determine whether sumoylated LRH-1and/or PML can associate with chromatin and LRH-1-regulatedtarget genes.

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FIG. 7. SUMO-modified LRH-1 is not associated with transcriptionally active LRH-1 target genes in vivo. (A) Sumoylation has a repressive effect on the transactivation potential of LRH-1. HeLa cells were transfected with the SHP-luc reporter containing the 500-bp proximal promoter region of the SHP gene in front of firefly luciferase cDNA, together with the indicated expression vectors. The bars represent mean values and standard errors of normalized luciferase activities from three independent experiments and are expressed as the fold increase over the values obtained from cells transfected with the reporter alone. (B) C3A-HepG2 cells were sequentially extracted with Triton X-100 (Nucleocytoplasm), DNase I, and (NH₄)₂SO₄ (Chromatin), and 2 M NaCl. Equal aliquots of each fraction and the solubilized pellet (Nucl. Matrix) were subjected to SDS-PAGE and, after being blotted to nitrocellulose membranes, were stained with the indicated antibodies. (C and D) ChIP assays were performed with the indicated antibodies by using cross-linked, soluble chromatin from mouse livers. LRH-1 target promoters (CYP7A1, CYP8B, and SHP genes) in the immunoprecipitates were detected by radioactive PCR. (E) ${alpha}$ SUMO-1 (top panel) or ${alpha}$ LRH-1-immunoprecipitated (bottom panel) complexes (1st IP) were eluted from the protein G-Sepharose beads and, after dilution, were subjected to a second immunoprecipitation step with the indicated antibodies (2nd IP) and analyzed by radioactive PCR.

To this end, we first performed a nuclear protein fractionationprocedure as described in references 22 and 37. In the firstfractionation step, soluble nucleocytoplasmic proteins are removedby extraction with Triton X-100. Chromatin and associated proteinsare then released by DNase I digestion and extraction by 0.25M ammonium sulfate. After being washed with 2 M NaCl, the remaininginsoluble material in the pellet is composed of nuclear matrixproteins (22, 37). Equal amounts of the different fractionswere analyzed by Western blot analysis, with antibodies to LRH-1,PML, RNA polymerase (Pol) II, and lamin B (Fig. 7B). As expected,RNA Pol II was distributed mainly in the "chromatin" fractionand to a lesser extent in the "nucleocytoplasmic" fraction.Lamin B and sumoylated PML were detected only at the "nuclearmatrix" fraction. These results verify that the different fractionsare essentially pure or that only minimal cross-contaminationoccurred during the procedure. The majority of unmodified LRH-1was detected in the detergent-soluble and chromatin fractions.Importantly, SUMO-modified LRH-1 was solely observed in thefinal insoluble fraction (Fig. 7B), indicating that sumoylatedLRH-1 is associated with the nuclear matrix but not with DNA.Although the above data argue against the possibility that sumoylatedLRH-1 is targeted to gene regulatory regions, they do not entirelyexclude it. It is still possible that, due to its tight associationwith components of the nuclear matrix, chromatin-bound SUMO-LRH-1could not be liberated by DNase treatment or that the DNase-I-releasedprotein was somehow desumoylated.

Therefore, we addressed the issue by using an independent approach.We performed ChIP assays with soluble chromatin prepared frommouse liver cells that were cross-linked in their native tissueenvironment. We analyzed LRH-1, SUMO-1, and PML occupancy onthree well-characterized LRH-1-regulated genes (Cyp7A1, Cyp8B,and SHP). As expected, LRH-1 occupancy was observed at the promoterregions of all three genes (Fig. 7C and D). Interestingly, however,positive ChIP signals could be detected in anti-SUMO-1-immunoprecipitatedchromatin, but not in that obtained with anti-PML. To determinewhether the observed SUMO-1 signal corresponds to sumoylatedLRH-1 and to find a correlation between SUMO-1 occupancy andthe activation state of the genes, we performed sequential ChIPassays. In these experiments SUMO-1-containing, or LRH-1-containingchromatin purified by the first immunoprecipitation was elutedfrom the protein G-Sepharose beads and subjected to a secondimmunoprecipitation with antibodies recognizing SUMO-1, LRH-1,histone 3, or RNA Pol II. As shown in Fig. 7E, histone H3, butnot LRH-1 or RNA Pol II, could be detected on the SUMO-1-associatedSHP promoter. Furthermore, SUMO-1 was absent in LRH-1-occupiedchromatin, which contained histone H3 and RNA Pol II. This findingclearly demonstrates that sumoylated LRH-1 is not associatedwith its target genes. Because RNA Pol II recruitment is a molecularindicator of the activation state of genes, the results pointto the existence of two populations of hepatocytes or allelesat any given time: one with active promoters, occupied by unmodifiedLRH-1 and RNA Pol II, and another with inactive promoters, occupiedby an unidentified sumoylated protein(s) but lacking LRH-1 andRNA Pol II.

DISCUSSION

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Discussion

LRH-1 is an orphan member of the nuclear receptor superfamily,which plays a pivotal role in early endodermal development,whereas in adults it controls the expression of enzymes involvedin lipid and bile acid homeostasis (12, 16, 20, 31, 36, 39).In addition, LRH-1 has also been implicated in cell proliferationvia cross talk with the ß-catenin signaling pathway(8). The functioning of LRH-1 in such diverse biological processeshighlights the importance of identification and explorationof potential signaling pathways that may modulate its activity.

We demonstrate here that LRH-1 is a direct substrate for theSUMO conjugation machinery and that sumoylation can modulateits activity. LRH-1 is reversibly modified by SUMO-1 at a singlelysine residue, located at the hinge domain of the protein.Several lines of evidence described here suggest that SUMO modification-mediatedinhibition of LRH-1 activity is due to its spatial sequestrationfrom active chromatin domains. First, we demonstrate that sumoylatedLRH-1 is localized in PML-containing nuclear bodies but notin nuclear territories outside them. Second, sumoylated LRH-1is not associated with chromatin and its actively transcribedtarget genes. Third, desumoylation, which leads to the dissociationof LRH-1 from PML-NBs, increases its transcriptional activity.

Various functions have been attributed to PML-NBs, includinga role in transcriptional regulation (52). Although the precisemechanism by which PML-NBs regulate gene expression is unknown,accumulating evidence on various transcription factors has ledto the proposal of speculative models, which assume that PML-NBsmay function either as "storage sites," which can titrate theconcentration of soluble factors in the nucleus, or as sitesof specific posttranslational modifications (e.g., sumoylation),or as scaffolds into which transcription complexes assemble(52). Our results provide experimental evidence for links betweenthe proposed functions by demonstrating a dynamic, sumoylation-dependentassociation of LRH-1 with the PML-NBs. The sumoylation machineryat these sites may play an operational role in the supply ofactive LRH-1 for transcription, as demonstrated by the findingthat the release of LRH-1 from PML-NBs requires enzymatic removalof the SUMO moiety. Although the mode of activation of SUMOproteases in response to nuclear protein concentration is notknown, it is clear that their function is central to the establishmentof a dynamic equilibrium between the two pools of the LRH-1protein: the one localized at PML-NBs and the other an unmodifiedsoluble pool situated outside the PML-NBs. In this way, SUMO-mediatedcompartmentalization may play a role in regulating the actualnuclear concentration of the protein available for transcriptionin addition to or more likely in combination with the pathwaysthat control the production and degradation of LRH-1.

The PML protein itself is also subject to SUMO modification,which is required for the proper formation of the nuclear body(3, 13, 35, 53). Interestingly, global desumoylation of proteinsbrought about by overexpressing a SUMO protease or by knockingdown endogenous SUMO-1 expression results in relocalizationof PML into a small number of nuclear structures, which unlikethe regular PML-NBs accumulate RNA Pol II. These novel PML depositsites may therefore represent hot spots of active transcriptionof genes although, because LRH-1 was not concentrated in theseterritories, it is unlikely that they contribute to the desumoylation-mediatedactivation of LRH-1 target genes. Nevertheless, the observationilluminates the plasticity of specific territories within thenuclear environment, where proteins can accumulate in responseto different stimuli and possibly exercise distinct biologicalfunctions. Consistent with this notion is the recent demonstrationof association of PML with nucleoli in response to specificstress signals (2).

SUMO modification of transcription factors is becoming increasinglyrecognized as an important pathway regulating gene expression(10, 27, 34, 38, 40, 41, 48). Several models have been put forwardas possible mechanism(s) for SUMO modification-dependent transcriptionaleffects, including competition with other modifications, modulationof interactions with DNA and other proteins on the promoters,or the above-mentioned "sequestration model," whereby sumoylationleads to the localization of the transcription factor to subnucleardomains where it cannot access its target genes (4, 17, 18,42, 46, 52). Although the models described above are not necessarilymutually exclusive, probably no generally applicable mechanismexists and, depending on the modified factor, sumoylation mayelicit its transcriptional effects by several means. This scenariois corroborated by our sequential ChIP assays, which detectedSUMO-1 or LRH-1 on the promoters but not the two proteins together.The results are consistent with the idea that two populationsof hepatocytes or promoter alleles exist at any given time:one with active promoters, occupied by unmodified LRH-1 andRNA Pol II, and another, with inactive promoters occupied byunidentified sumoylated protein(s), which lack LRH-1 and RNAPol II. This points to the parallel involvement of a direct,LRH-1 modification-independent function of SUMO-1 at the promoters.The recent finding that histone H4 and, to a lesser extent,histones H2A, H2B, and H3 can be modified by SUMO (43), whichat least in transfected reporters mediates gene silencing, isin line with our detection of histones but not of an activeRNA Pol II-containing preinitiation complex on the SUMO-associatedpromoter (Fig. 7 and data not shown). Although our results donot provide definite evidence for the presence of sumoylatednucleosomes on the repressed promoters, these results clearlyshow that sumoylated proteins (histones or others) can be associatedwith chromatin, which, as indicated by the absence of PML ChIPsignals on these regulatory regions, most likely reside outsidethe PML-NBs. In this respect we note that a significant amountof SUMO-1 fluorescence signal can be detected throughout thenucleoplasm, outside of the PML-NBs (Fig. 3, 5, and 6). SUMOmodification of some of these proteins may affect preinitiationcomplex formation at the promoters by recruiting corepressoractivities. Examples for such direct repression have been providedby studies on Elk-1 and histones, whose sumoylation at promoterscan facilitate the recruitment of HDAC or HP-1 repressor proteins(43, 49).

Our results on LRH-1 point to a fundamentally different controlmechanism for the set of transcription factors that are modifiedby SUMO at PML-NBs. Sumoylation of these factors leads to their"storage" at PML-NBs, and thus they become spatially sequesteredfrom active chromatin. SUMO-dependent compartmentalization ofLRH-1 is a reversible and highly dynamic process, which mayplay a fine-tuning role in controlling the availability of thisfactor to chromatin. PML-NBs, where sumoylated LRH-1 accumulates,are not regarded as sites of active transcription since theyare devoid of DNA, but nascent RNAs and transcriptionally activegenomic loci are concentrated in their immediate periphery (31,47). In light of this and the evidence provided here, we proposethat sumoylation may act as a "modus operandi" for the formationof dynamic reservoirs of transcription factors in the vicinityof active chromatin domains. Shuttling of transcription factorsbetween these nuclear domains may represent an important controlmechanism of gene expression.

ACKNOWLEDGMENTS

We are indebted to R. Hay, F. Melchior, N. Kotaja, J. Palvimo,and H. Will for providing reagents; N. Katrakili for technicalassistance; N. Tavernarakis for help with confocal microscopy;and P. Hatzis for helpful comments on the manuscript.

This study was supported by grants from EU (QLRT-2000-01513and LSHG-2004-502950) and from GSRT (01ED509).

FOOTNOTES

* Corresponding author. Mailing address: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, P.O. Box 1527, Vassilika Vouton, 711 10 Herakleion, Crete, Greece. Phone: 30-2810-391163. Fax: 30-2810-391101. E-mail: talianid{at}imbb.forth.gr.

REFERENCES

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