The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG … · These are the PC4...

Published OnlineFirst January 24, 2012.Mol Cancer Res Lai Sum Leoh, Bart van Heertum, Jan De Rijck, et al. Transcriptional ActivityMethyl CpG Binding Protein MeCP2 and Influences Its The Stress Oncoprotein LEDGF/p75 Interacts with the

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DNA Damage and Cellular Stress Responses

TheStressOncoprotein LEDGF/p75 Interactswith theMethylCpG Binding Protein MeCP2 and Influences ItsTranscriptional Activity

Lai Sum Leoh1, Bart van Heertum3, Jan De Rijck3, Maria Filippova1, Leslimar Rios-Colon1, Anamika Basu1,Shannalee R. Martinez1, Sandy S. Tungteakkhun1, Valeri Filippov1, Frauke Christ3, Marino De Leon1,Zeger Debyser3, and Carlos A. Casiano1,2

AbstractThe lens epithelium–derived growth factor p75 (LEDGF/p75) is a transcription coactivator that promotes

resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense finespeckles autoantigen of 70 kDa (DFS70) and has been implicated in cancer, HIV-AIDS, autoimmunity, andinflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, weinitiated an analysis of its interactions with other transcription factors and the influence of these interactions onstress gene activation.We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact withMeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. Theseinteractions were established by several complementary approaches: transcription factor protein arrays, pull-downand AlphaScreen assays, coimmunoprecipitation, and nuclear colocalization by confocal microscopy. MeCP2 wasfound to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75modestly enhancedMeCP2-induced Hsp27pr transactivation in U2OS osteosarcoma cells, whereas this effect wasmore pronounced in PC3 prostate cancer cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells.Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediatedHsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. Theseresults suggest that the LEDGF/p75–MeCP2 interaction differentially influences Hsp27pr activation dependingon the cellular and molecular context. These findings are of significance in understanding the contribution of thisinteraction to the activation of stress survival genes. Mol Cancer Res; 1–14. �2012 AACR.

IntroductionLEDGF/p75 is a stress response protein with increasing

relevance to cancer, HIV-AIDS, autoimmunity, inflamma-tion, and eye disease. LEDGF/p75 and its short splicevariant p52 are derived from the same PSIP1 gene and wereoriginally identified as transcription coactivators that inter-act with the RNA polymerase II transcription complex (1).LEDGF/p75 is also known as the dense fine speckledautoantigen of 70 kDa (DFS70), which is targeted byautoantibodies in various human inflammatory conditions

(2). Although initially proposed to be a growth factor for lensepithelial cells (3), subsequent studies revealed that LEDGF/p75 is a stress survival protein that protects against oxidativestress–induced cellular damage and death (4, 5). As a keycellular cofactor for HIV-1 replication, LEDGF/p75 bindsthe HIV-1 integrase (IN) through its C-terminal integrase-binding domain (IBD) and tethers it to the chromatin,facilitating lentiviral integration to transcriptionally activeregions of the host genome (6–8).LEDGF/p75 is emerging as an oncoprotein in various

human cancers. It is targeted by autoantibodies in patientswith prostate cancer and is overexpressed in prostatetumors and other human malignancies, including chemo-therapy-resistant human acute myelogenic leukemia (9–12). In addition, it can be found as a fusion protein withNUP98 in patients with leukemia (13). Its overexpressionin tumor cells attenuates lysosomal cell death inducedby stressors that trigger oxidative stress (e.g., certainchemotherapeutic drugs, TNF, and serum starvation;refs. 5, 10, 12, 14). LEDGF/p75 also enhanced thetumorigenic potential of HeLa cells in xenograft models(10).

Authors' Affiliations: 1Center for Health Disparities and Molecular Medi-cine and Department of Basic Sciences, 2Department of Medicine,Loma Linda University School of Medicine, Loma Linda, California; and3Laboratory for Molecular Virology and Gene Therapy, Division ofMolecular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium

Corresponding Author: Carlos A. Casiano, Center for Health DisparitiesandMolecular Medicine, Loma Linda University School of Medicine, LomaLinda, CA 92350. Phone: 909-558-1000, ext. 42759; Fax: 909-558-0196;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0314

�2012 American Association for Cancer Research.

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LEDGF/p75 is thought to promote cell survival understress by transcriptionally activating genes encoding protec-tive proteins such as Hsp27, aB-crystallin, peroxiredoxin 6(PRDX6), and VEGF-c (15–17). Presumably, LEDGF/p75transactivates these stress genes by binding to heat shockelements (HSE) and stress elements (STRE) in their pro-moter regions (15–17). In leukemia cells, LEDGF/p75interacts with the menin/MLL-HMT (mixed lineage leu-kemia-histone methyltransferase) transcription complex totransactivate cancer-associated genes and facilitate leukemictransformation (18).The transcriptional and prosurvival activities of LEDGF/

p75 are attenuated by TGF-b1 (19), Bcl-2 (20), SUMOyla-tion (21), and caspase-mediated cleavage (5, 22). Theprosurvival function of LEDGF/p75 is also negatively reg-ulated by alternative splicing as its short splice variant p52antagonizes its transcriptional activity and induces apoptosisin cancer cells (22). Although it is not clear how LEDGF/p75 is upregulated in the presence of stress, recent studiesimplicated transcription factors SP1 and STAT3 in thisprocess (23, 24).LEDGF/p75 and p52 share amino (N)-terminal amino

acids (1–325 aa); however, p52 has an intron-derivedcarboxyl (C)-terminal tail (CTT, 326–333 aa) implicatedin its proapoptotic activity (1, 22). The N-terminal regionshared by both proteins contains a PWWP domain (1–93aa), a structural entity implicated in chromatin binding,HIVintegration, protein–protein interactions in the chromatin,transcriptional repression, and DNA methylation (25–30).The N-terminal region also has a positively charged domain(CR1) immediately after the PWWPdomain that is followedby a nuclear localization signal (NLS) and two AT-hooksequences that cooperate with the PWWP domain forchromatin binding (31, 32). A second charged region (CR2),also designated the supercoiled DNA recognition domain(SRD; 200–336 aa), facilitates LEDGF/p75 binding toactive transcription sites (33). The C-terminus ofLEDGF/p75 (347–429 aa) encompasses both the IBD andthe autoepitope recognized by human anti-LEDGF/p75autoantibodies (8, 34). This region is involved in pro-tein–protein interactions and binding to HSE in promoterregions (35–38). Both the N- and C-terminal regions ofLEDGF/p75 contribute to its transcription and stress sur-vival functions (5, 38).Understanding the mechanisms by which LEDGF/p75

promotes tumor cell resistance to cell death and chemother-apy requires detailed knowledge of its cellular functions,particularly its interactions with other cancer-associatedproteins and its target genes. To date, only a few cellularinteracting partners of LEDGF/p75 have been identified.These are the PC4 transcription factor, menin/MLL, theCdc7 activator of S-phase kinase (ASK), the pogZ transpo-sase, and the myc-interacting protein JPO2 (18, 35–37).Using transcription factor protein arrays, we identifiedseveral candidate interacting partners of LEDGF/p75.Among these, methyl CpG–binding protein 2 (MeCP2)was of particular interest because, like LEDGF/p75, it hasbeen linked to prostate cancer (39). MeCP2 belongs to the

family of methyl CpG–binding proteins and is mutated inRett syndrome, a childhood neurodevelopmental disease(40). MeCP2 is a transcription factor that is generallyconsidered to bind methylated CpG islands to represstranscription. However, recent evidence indicates thatMeCP2 binds DNA regardless of methylation status andacts as a transcriptional modulator that remodels chromatinto activate or repress genes depending on the cellular andmolecular context (40–42). In this study, we characterizedthe interaction between LEDGF/p75 and MeCP2 in vitroand in cancer cells.We provide evidence that theN-terminalPWWP-CR1 region of LEDGF/p75 binds to MeCP2 andinfluences its transcriptional function.

Materials and MethodsCell culture, antibodies, and plasmidsU2OS, PC3, 293T, and HeLa cells were obtained from

the American Type Culture Collection and cultured inMcCoy's 5A medium or RPMI-1640 (Gibco), supplemen-tedwith 2mmol/L L-glutamine and penicillin/streptomycin,and 10% FBS. PC3 cells stably expressing LEDGF/p75(14), were grown in RPMI-1640 with 10% (v/v) FBS, 20mg/mL of gentamicin, and 0.5 mg/mL of Geneticin. Cellswere grown with 5% CO2 at 37�C.The following antibodies were used: mouse monoclonals

anti-LEDGF/p75-p52 (BD Biosciences), anti-b-actin (Sig-ma); rabbit polyclonals anti-LEDGF/p75 (Bethyl Labora-tories), anti-MeCP2 (ProteinTech Group), anti-HA (SantaCruz Biotechnology), anti-eGFP and goat polyclonals anti-eGFP (produced in the laboratory of Z. Debyser), anti-GFP(Santa Cruz Biotechnology), anti-GST (Pharmacia Bio-tech), anti-Flag-HRP (Sigma) and rat monoclonal horserad-ish peroxidase (HRP)-conjugated anti-HA (Roche Diagnos-tics).Human antibodies to LEDGF/p75were a gift fromDr.Eng M. Tan (Scripps Research Institute, La Jolla, CA).Plasmid pET28a-dfs70 encoding His-LEDGF/p75 was a

kind gift from Dr. Edward Chan (University of Florida,Gainesville, FL). Plasmids pDEST-GST-MeCP2 andpcDNA-Flag-MeCP2 were a kind gift fromDr. Adrian Bird(University of Edinburgh, Edinburgh, UK). PlasmidspKB6H-p52, pMal-p2x-BRD4-Ct, and p-eGFP-BRD4-Ctwere generated in the laboratory of Z. Debyser. PlasmidspCruzHA-LEDGF/p75, pCruzHA-p52, and pGL3-Hsp27pr-Luc were generated as described (22). PlasmideGFP-p52 was cloned by replacing the LEDGF/p75 cDNAin p-eGFP-LEDGF/p75 vector with the LEDGF/p52cDNA at XhoI and BamHI restriction sites.

Purification of recombinant LEDGF/p75, p52, andMeCP2Glutathione S-transferase (GST)-tagged MeCP2 was

produced from pDEST-MeCP2 in Escherichia coli BL21grown in the presence of sorbitol and betaine. Expressionwas induced in lysogeny broth (LB) medium at 37�C byaddition of 0.5 mmol/L isopropyl b-D-1-thiogalactopyrano-side (IPTG). Cells harvested 3 hours after induction werelysed by sonication in core buffer (50mmol/L Tris-HCl, 0.5mol/L NaCl, pH 7.5, 10 mmol/L EDTA, 10 mmol/L

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EGTA, 1% Triton X-100, and 20 mg/mL lysozyme). Thefusion protein, captured on glutathione agarose (Sigma) orglutathione sepharose beads (GE Healthcare Life Sciences),was eluted with 20 mmol/L glutathione in core buffer.Histidine (His)-tagged LEDGF/p75 and p52were expressedfrom pET28a-dfs70 and pKB6H52 in E. coli BL21, respec-tively. Expression was induced with 1 and 3 mmol/L IPTG,respectively, at 37�C for 3 hours. Bacteria were lysed bysonication in B-PER bacterial protein extraction reagent(Thermo Scientific). The recombinant proteins were cap-tured on nickel columns (Novagen) or TALON His-TagPurification Resins (Clontech) and washed with 20 mmol/LHEPES, 0.5mol/LNaCl, 2mmol/L KCl, 1%NP-40, and 5mmol/L imidazole. Proteins were eluted with 20 mmol/LHEPES, 137 mmol/L NaCl, 2 mmol/L KCl, 300 mmol/Limidazole, and dialyzed using D-tube dialyzer (Novagen).

Transcription factor arraysTwo transcription factor protein arrays that were com-

mercially available at the time we initiated these studies,Active Protein Array (Active Motif) and TranSignal ProteinArrays I-III (Panomics), were used for identifying interactingpartners of LEDGF/p75 following the manufacturer'sinstructions. These arrays contained 170 transcription fac-tors and coactivators, as well as RNA polymerase II, spottedon membranes in duplicates or triplicates. Briefly, mem-branes were blocked with 5% milk in Tris-buffered salinewith Tween-20 (TBS-T) buffer for 1 hour. RecombinantHis-LEDGF/p75 was incubated overnight with the mem-branes, and after washes with TBS-T, the membranes wereprobed with human anti-LEDGF/p75 autoantibody for 2hours. Following washes with TBS-T, the membranes wereincubated with HRP-conjugated secondary antibodies, andprotein interaction signals were detected by chemilumines-cence (Amersham).

Pull-down assaysGST or GST-MeCP2 proteins bound to glutathione

beads were blocked in HEPES buffer (20 mmol/L HEPES,pH 7.4, 2 mmol/L DTT, 137 mmol/L NaCl, 2 mmol/LKCl, 5% glycerol) with 0.1% bovine serum albumin (BSA)at 4�C for 1 hour. His-LEDGF/p75 or His-p52 were thenincubated with the beads at 4�C for 1 hour inHEPES buffer.The beads were then collected by centrifugation at 5,000rpm for 30 seconds at 4�C, the supernatant was discharged,and the beads were washed 2 times with 1 mL of HEPESbuffer þ 0.1% NP-40 followed by HEPES buffer þ 0.5%NP-40 þ 0.5 mol/L NaCl. Bound proteins were eluted inSDS-PAGE sample buffer, separated by SDS-PAGE (10%Bis–Tris gel), and detected by immunoblotting.

Analysis of protein–protein interactions by AlphaScreenassayThe AlphaScreen assay was conducted according to the

manufacturer's protocol (Perkin Elmer). Briefly, reactionswere conducted in 25 mL final volume in 384-well Optiwellmicrotiter plates. The reaction buffer contained 25 mmol/LTris-HCl pH 7.4, 150 mmol/L NaCl, 1 mmol/L MgCl2,

0.1% Tween-20, 0.1% BSA. Varying concentrations ofGST-MeCP2 in bacterial lysate and purified Flag-LEDGF/p75 were incubated in 15 mL reaction volume at4�C for 1 hour. The concentration of GST-MeCP2 in thelysates was estimated using BSA standards on SDS-PAGEgels stained with Coomassie Blue. Equal volume of bacteriallysate not expressing GST-MeCP2 was used as negativecontrol. Subsequently, 5 mL of the diluted donor (glutathi-one) and acceptor (Flag) beads were added. After incubationfor 1 hour in the dark, light emission was measured in theEnVision reader (Perkin Elmer) and analyzed using theEnVision manager software.

Transient and stable transfection293T cells were transfected in 10-cm3 plates by poly-

ethyleneimine (PEI) transfection. Ten micrograms of plas-midDNAwas used to transiently transfect cells at more than50% confluency. U2OS and PC3 cells were transfectedusing TransIt 2020 (Mirus) transfection reagent. Trans-fected cells were grown for 24 to 48 hours before analysis.Stable PC3 clones overexpressing LEDGF/p75 were gener-ated by transfecting cells with pcDNA-LEDGF/p75, orempty pcDNA vector for controls, and growing them inselection media containing Geneticin (Calbiochem) asdescribed (14).

CoimmunoprecipitationCells were collected 48 hours posttransfection and lysed in

radioimmunoprecipitation assay (RIPA) buffer (Santa CruzBiotechnology). Antibodies were incubated with cell lysatesfor 1 hour before protein A/Gþ agarose beads (Santa CruzBiotechnology) were added. The beads were collected bycentrifugation and washed 3 times with core buffer [50mmol/L Tris, pH 7.5, 300 mmol/L NaCl, 1 mmol/LMgCl2, 5% glycerol, complete protease inhibitor EDTAfree (Roche), and 0.1%NP-40]. 293T cells were transientlytransfected with p-eGFP-LEDGF/p75 and pcDNA-Flag-MeCP2. Whole-cell lysates were collected 24 hours post-transfection and lysed in core buffer with 1% Triton X-100.Antibody against GFP was incubated with cell lysate over-night before protein G sepharose beads (GE Healthcare)were added. Precipitation of Flag-MeCP2 was done usingFlag-agarose beads. Immunoprecipitated proteins weredetected by immunoblotting using appropriate antibodies.

Confocal microscopyCells were cotransfected with different combinations of

plasmids encoding eGFP-tagged LEDGF/p75, HcRed-tagged p75 or p52, or Flag-tagged MeCP2. Cells were thenfixed with 4% formaldehyde and permeabilized with 0.5%Triton X-100. Ectopically expressed Flag-MeCP2 was visu-alized by incubation with anti-Flag antibodies, followed byincubation with corresponding secondary antibodies labeledwith either fluorescein isothiocyanate (FITC) or rhodamine.For visualization of endogenous LEDGF/p75 and MeCP2proteins, fixed/permeabilized cells were coincubated withhuman anti-LEDGF/p75 and rabbit anti-MeCP2 antibo-dies, both used at 1:200 dilution. After incubation with

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LEDGF/p75 and MeCP2 Interact in Cancer Cells





respective secondary antibodies labeled with FITC or rho-damine, cells were mounted with medium containing 40,6-diamidino-2-phenylindole (DAPI; Vectashield). Confocalmicroscopy was conducted in a Zeiss LSM 710 NLOmicroscope with 63� oil immersion objective and appro-priate filters. Images were analyzed using ImageJ software.

Luciferase-based transcription reporter assaysHsp27pr luciferase transcription reporter assays were

conducted as described previously (22). Briefly, cells werecotransfected with plasmids encoding the proteins of interestor empty vector, and pGL3-Hsp27pr. At 48 hours post-transfection, cells were lysed and luciferase assays wereconducted using the Luciferase Assay System (Promega).Relative light units were obtained in a MicroLumatPlus Lb96V luminometer (Berthold Tech), and luciferase valueswere normalized to protein concentration of lysates fromcells cotransfected with the empty vectors and pGL3-Hsp27pr. Student t-test analysis was conducted usingMicrosoft Excel. Experiments were repeated at least 3 times.

LEDGF/p75 knockdown by RNA interferenceTransient knockdown of LEDGF/p75 was carried out

using synthetic siRNA oligos as described previously (43).The siLEDGF/p75 sequence corresponded to nucleotides1,340 to 1,360 (50-AGACAGCAUGAGGAAGC-GAdTdT-30) with respect to the first nucleotide of the startcodon of the LEDGF/p75 open reading frame. AmbionSilencer Negative Control siRNA #1 was used as scrambledcontrol. siRNAs were transfected into cells using siQuest(Mirus). LEDGF/p75 knockdown was verified byimmunoblotting.

Chromatin immunoprecipitation assaysU2OS cells were fixed in 1% formaldehyde for 10minutes

and subjected to chromatin immunoprecipitation (ChIP)assay using ChIP-IT Express Enzymatic kit (Active Motif).Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), anti-MeCP2 antibodies (07–013, Millipore), and rabbit IgG(Santa Cruz Biotechnology) were used to immunoprecipi-tate protein–chromatin complexes. PCR was carried outusing primers to amplify Hsp27 promoter: set A, forward50-CGC TTA AGC ACC AGG GCC GG-30 and reverse50-CCGGCCCTGGTGCTTAAGCG-30; set B, forward50-CTGGGCTCAAGCACCAGACTC-30 and reverse50-CAAATGAATTCGAGAGCGCGACGC-30; set C, for-ward 50-CAGGGTTTTGCTCTGTAG CC-30 and reverse50-CCACACGCGTGTGAGATAGAATGTG-30; and setD, forward 50-CTCTGCCTTCTGGGTTCAAG-30 andreverse 50-TTGAACCCCGGTGAGTAGAG-30.

ResultsIdentification of MeCP2 as a candidate interactingpartner of LEDGF/p75We hypothesized that LEDGF/p75 cooperates with tran-

scription factors to facilitate the activation of stress genes. Toidentify novel cellular interacting partners of LEDGF/p75,we screened 170 different transcription factors for LEDGF/

p75 binding using transcription factor protein arrays thatwere commercially available at the time we initiated thesestudies (Panomics and Active Motif). Purified recombinantHis-tagged LEDGF/p75 (Fig. 1A) was incubated withrecombinant transcription factors spotted on membranes,and protein–protein interactions were identified using aspecific human autoimmune serum against LEDGF/p75(Fig. 1B), followed by signal detection with chemilumines-cence (Fig. 1C). We detected moderate to strong protein–protein interaction signals with 17 different transcriptionfactors, with the strongest reactivity corresponding to theLEDGF/p75–MeCP2 interaction (Fig. 1C). One array(Active Motif) yielded strong signals with transcriptionfactor PC4 and RNA polymerase II subunits (data notshown), consistent with the early report that LEDGF/p75copurifies with these proteins (1).

LEDGF/p75 interacts with MeCP2 in vitroTo confirm the interaction between LEDGF/p75 and

MeCP2 in vitro, pull-down experiments were carried outusing recombinant proteins. Beads with bound GST-MeCP2 or GST were incubated with His-LEDGF/p75,and interactions were detected by immunoblottingusing anti-GST antibody. His-LEDGF/p75 was pulleddown with GST-MeCP2 but not with GST (Fig. 2A). A

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Figure 1. Identification of candidate interacting partners of LEDGF/p75using transcription factor protein arrays. A, Coomassie blue–stainedSDS-PAGE gel showing purified His-LEDGF/p75. E. coli BL21 strain wastransformed with pET28a-dfs70 encoding His-LEDGF/p75 and inducedwith IPTG. Lysate was passed through a nickel column to purify His-LEDGF/p75. B, immunoblot showing the specificity of the humanautoantibody against LEDGF/p75 used as detection reagent in thetranscription factor protein arrays. The autoantibody reacts specificallywith LEDGF/p75 in a PC3 prostate cancer cell lysate. WB, Westernblotting. C, transcription factor arrays were used to identify candidateinteracting transcription factors of LEDGF/p75. Purified His-LEDGF/p75was incubated with transcription factors spotted on membranes. Proteininteractions were detected with human anti-LEDGF/p75 autoantibodyand chemiluminescence. A section of the transcription factor arraymembrane containing MeCP2 is shown.


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protein–protein interaction assay using the AlphaScreenTechnology (Perkin-Elmer) was used for additional confir-mation of the LEDGF/p75–MeCP2 interaction in vitro. Toprevent false-positive signals due to rapid degradation ofpurified recombinant GST-MeCP2, we used GST-MeCP2induced in the E. coli BL21 bacterial lysate. Enhancedbacterial expression of GST-MeCP2 was attained in thepresence of sorbitol and betaine. Cross-titration of increasingconcentrations of recombinant Flag-LEDGF/p75 andGST-MeCP2 showed binding between both proteins over a widerange of concentrations (Fig. 2B). Optimum binding wasobserved between 11 nmol/L Flag-LEDGF/p75 and esti-mated 1 nmol/L GST-MeCP2 (Fig. 2C).

LEDGF/p75 interacts with MeCP2 in cellular modelsThe LEDGF/p75–MeCP2 interaction was confirmed in a

cellular system by coimmunoprecipitation (co-IP) assays.

Plasmids encoding Flag-MeCP2 and different eGFP-taggedproteins were cotransfected in 293T cells (for high trans-fection efficiency). Expressed proteins were then immuno-precipitated with anti-GFP antibody and pulled down byproteinG-agarose beads. Immunoprecipitated proteins wereanalyzed by immunoblotting with antibodies against eGFPand Flag tags. Immunoblotting analysis showed MeCP2interaction with eGFP-LEDGF/p75 but not with eGFP-HIV-IN or the irrelevant protein eGFP-BRD4-Ct (carboxylterminal fragment of the bromodomain containing protein4; Fig. 3A). To verify that LEDGF/p75 andMeCP2 interactendogenously in cancer cells, co-IP experiments were carriedout using U2OS osteosarcoma cells, which express highendogenous levels of LEDGF/p75 (22). U2OS cell lysateswere incubated with antibody against LEDGF/p75, and theimmunoprecipitated proteins were detected by immuno-blotting. Endogenous LEDGF/p75 and MeCP2 were

Figure 2. LEDGF/p75 interacts with MeCP2in vitro. A, pull-down assays with His-LEDGF/p75 and GST-MeCP2. RecombinantHis-LEDGF/p75 was incubated with GST orGST-MeCP2 bound to glutathione beads andpulled down proteins were analyzed byimmunoblotting using antibodies specificfor GST or LEDGF/p75. His-FADD(Fas-associated protein with death domain) wasused as negative control. Protein input wasdetermined by immunoblotting of whole-cellextracts. �, degraded GST-MeCP2. WB,Western blotting. B, cross-titration for Flag-LEDGF/p75 and GST-MeCP2 interaction asmeasured by AlphaScreen assay. Interactionwas measured at different concentrations ofFlag-LEDGF/p75 as indicated on the verticalaxis andGST-MeCP2as indicated on the x-axis.C, an estimated 1 nmol/L MeCP2 was sufficientto interact with 11 nmol/L LEDGF/p75. E. coliBL21 lysate not expressing GST-MeCP2 wasused as a negative control. Results in (B) and (C)are representative of 3 independentmeasurements.

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detected in immunoprecipitates of endogenous LEDGF/p75 but not in immunoprecipitates of an IgG controlantibody (Fig. 3B).The LEDGF/p75–MeCP2 interactionwas also confirmed

by reciprocal co-IP and immunoblotting in PC3 prostatecancer cells (Fig. 3C). Whole-cell lysates from PC3 cellsstably overexpressing LEDGF/p75 (PC3p75) or fromuntransfected PC3 cells (PC3) were incubated with antibodyagainst MeCP2, and the immunoprecipitated endogenousproteins were detected by immunoblotting with antibodiesto MeCP2 or LEDGF/p75 (Fig. 3C, middle). In the recip-rocal experiment, the PC3p75 and PC3 cells were incubatedwith antibody against LEDGF/p75, and the immunopreci-pitated endogenous proteins were detected by immunoblot-ting with antibodies to MeCP2 or LEDGF/p75 (Fig. 3C,right). Endogenous LEDGF/p75 andMeCP2 were detectedin immunoprecipitates of endogenous MeCP2 but not in

immunoprecipitates of an IgGcontrol antibody (Fig. 3D).Asan additional control, we sought to confirm that the anti-LEDGF/p75 and MeCP2 antibodies were recognizing dif-ferent endogenous proteins in PC3 cells. For these experi-ments, LEDGF/p75 was knocked down in PC3 cells bytransient transfection of siRNA oligos (sip75). Immunoblotsrevealed that endogenous LEDGF/p75 was dramaticallyreduced by sip75 but not by the control scrambled duplexoligos (siSD;Fig. 3E).The endogenous levels ofMeCP2werenot affected, indicating that the anti-LEDGF/p75 and anti-MeCP2 antibodies were recognizing different proteins.Confocal microscopy analysis was conducted to examine

the intracellular colocalization of LEDGF/p75 andMeCP2.U2OS and HeLa cells were transiently transfected withdifferent combinations of tagged LEDGF/p75 and MeCP2constructs. Figure 4A shows that coexpression of eGFP-p75and Flag-MeCP2 in U2OS cells gave an identical nuclear

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Figure 3. co-IP of LEDGF/p75 andMeCP2. A, 293T cells cotransfectedwith Flag-MeCP2 and eGFP-LEDGF/p75, eGFP-HIV-IN, oreGFP-BRD4-Ct expressionconstructs were lysed 24 hoursposttransfection. Proteins in celllysates were immunoprecipitated(IP) with antibody against GFP,resolved by SDS-PAGE, anddetected by immunoblotting usinganti-GFP and anti-Flag antibodies.B, endogenous proteins inU2OS cell lysates wereimmunoprecipitated with mousemonoclonal anti-LEDGF/p75antibody, aswell as an irrelevant IgGas negative control.Immunoprecipitated proteins weredetected by immunoblotting withrabbit anti-LEDGF/p75 and anti-MeCP2 antibodies. �, degradedMeCP2. Protein input wasdetermined by immunoblotting ofwhole-cell extracts. C, proteins inlysates from PC3 cells or cellsstably overexpressingLEDGF/p75 (PC3p75) wereimmunoprecipitatedwith rabbit anti-MeCP2 and mouse monoclonalanti-LEDGF/p75 antibodies.Immunoprecipitated proteins weredetected by immunoblotting withrabbit anti-LEDGF/p75 and anti-MeCP2 antibodies. D, endogenousproteins in PC3 cell lysates wereimmunoprecipitatedwith rabbit anti-MeCP2 antibody, as well as anirrelevant IgG as negative control.Immunoprecipitated proteins weredetected by immunoblotting withanti-LEDGF/p75 and anti-MeCP2antibodies. E, immunoblot of totalproteins from PC3 cells with (sip75)and without (siSD) siRNA-mediatedknockdown, using rabbit antibodiesto LEDGF/p75 and MeCP2. WB,Western blotting.


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localization pattern. White arrows pointing to nuclei expres-sing only eGFP-p75 or only Flag-MeCP2 indicated that theobserved colocalization was not due to bleeding of fluores-cence from one channel into the other (Fig. 4A). The nuclearcolocalization of LEDGF/p75 and MeCP2 could be betterappreciated in nuclei of U2OS coexpressingHcRed-p75 andFlag-MeCP2, or in mitotic HeLa cells coexpressing eGFP-p75 and Flag-MeCP2 (Fig. 4B and C, respectively). Thenuclear colocalization of endogenous LEDGF/p75 andMeCP2 was also established in U2OS and PC3 cells bycoincubating each cell line with both human antibody toLEDGF/p75 (green) and rabbit antibody to MeCP2(red; Fig. 4D and E). Both proteins displayed a distinctivenuclear speckled pattern that colocalized with DAPI-stainedchromatin.

LEDGF/p52 also interacts with MeCP2Asmentioned above, LEDGF/p75 and p52 share their N-

terminal region, which contains the PWWP domain, CR1and CR2, NLS, and AT-hooks that collectively facilitateDNA binding (Fig. 5A). To determine whetherMeCP2 alsobinds to p52, we conducted pull-down assays in whichrecombinant His-p52 was incubated with GST or GST-MeCP2 beads. Immunoblotting showed that His-p52 waspulled down by GST-MeCP2 but not by GST (Fig. 5B).Confocal microscopy showed partial colocalization ofHcRed-p52 with Flag-MeCP2 inU2OSnuclei coexpressingthese tagged proteins (Fig. 5C). White arrows pointing tonuclei expressing only Flag-MeCP2 indicated that theobserved colocalization was not due to the bleeding offluorescence from one channel into the other (Fig. 5C).

Figure 4. Nuclear colocalization ofLEDGF/p75 and MeCP2 by confocalmicroscopy. U2OS cells weretransiently cotransfected withplasmids encoding Flag-MeCP2 andeGFP-LEDGF/p75 (A), or thecombination of HcRed-LEDGF/p75andFlag-MeCP2 (B). HeLa cellsweretransiently cotransfected withplasmids encoding Flag-MeCP2 andeGFP-LEDGF/p75 (C). White arrowsin (A) point to cells that were stainedwith eGFP-LEDGF/p75 but not withFlag-MeCP2, or vice versa, ruling outthe possibility of bleeding from onechannel into the other. Ectopicallyexpressed MeCP2 was detected 48hours posttransfection using anti-Flag antibodies and visualized withFITC-labeled secondary antibody.Endogenous colocalization ofLEDGF/p75 and MeCP2 wasobserved in U2OS (D) and PC3 (E)cells after coincubation with humananti-LEDGF/p75 and rabbit anti-MeCP2 antibodies, followed bydetection with correspondingsecondary antibodies. Nuclei werestained with DAPI, and fluorescentsignals were analyzed by confocalmicroscopy.

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Taken together, these results indicated that p52 also interactswith MeCP2 and pointed to interaction of this protein withthe N-terminal region of LEDGF/p75.

The N-terminal region of LEDGF/p75 mediates theinteraction with MeCP2In light of the fact that LEDGF/p75 and p52 share the

same N-terminal region (1–325 aa), we sought to map theminimal interacting region of these proteins with MeCP2.To accomplish this, deletion constructs comprising differentregions of LEDGF/p75 were used. co-IP was conducted incells transiently cotransfected with plasmids encoding Flag-MeCP2 and one of the following eGFP-tagged constructs:LEDGF/p75 (1–530 aa), LEDGF/p52 (1–333 aa), PWWPdomain (1–93 aa), DPWWP (94–530 aa), D1–325 (325–530 aa), or IBD (347–429 aa; Fig. 6A). co-IP with anti-Flagantibody followed by detection with anti-GFP antibodyshowed that Flag-MeCP2 coprecipitated with both full-length LEDGF/p75 and p52 but not with any of thetruncated LEDGF/p75 constructs (Fig. 6B and C).Because both LEDGF/p75 and p52 interacted with

MeCP2, and the PWWP domain alone or DPWWP didnot interact with MeCP2, we concluded that additionalregions in the N-terminal portion of these proteins may beneeded for MeCP2 binding. To identify these regions,deletion constructs consisting of the PWWP domain alone(1–93 aa), or in combination with its downstream CR1region (1–141 aa), were used to examine MeCP2 binding.U2OS cell lysates containing endogenous MeCP2 were

incubated with the following Flag-tagged recombinant pro-teins: LEDGF/p75, PWWP-CR1 (1–141 aa), or PWWP(1–108 aa; Fig. 6A). Pull-down was done using anti-Flagagarose beads. Immunoblotting analysis with anti-Flag anti-body or anti-MeCP2 antibody showed that endogenousMeCP2 was pulled down with Flag-LEDGF/p75 andFlag-PWWP-CR1 but not with Flag-PWWP (Fig. 6D).This suggested that the extreme N-terminal region (1–141aa) of LEDGF/p75 mediates its interaction with MeCP2.

MeCP2 transactivates the Hsp27 promoterThe interaction of LEDGF/p75 and p52 with MeCP2

suggested that these proteins are part of a transcriptioncomplex that activates and regulates stress genes such asHsp27. To examine this, we first determined whetherMeCP2 transactivates the Hsp27 promoter (Hsp27pr) inluciferase reporter assays. U2OS cells were transientlycotransfected with pGL3-Hsp27pr-Luc and pcDNA emptyvector, pcDNA-Flag-MeCP2, pCruz empty vector, pCruz-HA-LEDGF/p75, or pCruz-HA-p52. Transient expressionof Flag-MeCP2 transactivated Hsp27pr at higher levels(14-fold; Fig. 7A) than those induced by LEDGF/p75 andp52 (2- to 3-fold; Fig. 7B).To establish whether transactivation of Hsp27pr by

LEDGF/p75 and MeCP2 was mediated by their bindingto this promoter, ChIP assay was conducted. On the basisof previous reports, LEDGF/p75 was predicted tobind HSEs and STREs located in the proximal region(�185 to �111) of Hsp27pr (15), whereas MeCP2 was

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Figure 5. LEDGF/p52 interacts withMeCP2. A, schematic domainstructure of LEDGF/p75 and p52.ATH, AT-hook. CR, chargedregion. CTT, carboxy terminal tail.IBD, integrase-binding domain.NLS, nuclear localization signal. B,pull-down assaywas conducted asdescribed in Fig. 2A usingrecombinant His-LEDGF/p52.�, degraded MeCP2. Proteininput was determined byimmunoblotting of whole-cellextracts. WB, Western blotting.C, LEDGF/p52 partially colocalizeswith MeCP2 in the cell nucleus.White arrows point to cells thatwere stained with Flag-MeCP2 butnot with HcRed-p52, ruling out thepossibility of bleeding from onechannel into the other.Colocalization assay wasconducted as described in Fig. 4.


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predicted to bind AT-rich repeats in the distal region (Fig.6C; ref. 44). ChIP assays using a specific MeCP2 antibodyrevealed binding of this protein toHsp27pr regions C andD(bp �1,071 to �382), located upstream of the HSE andSTRE consensus sequences (Fig. 7D, region A). On theother hand, LEDGF/p75 bound to the entire Hsp27prtested (bp �1,071 to þ18). ChIP with control rabbitanti-IgGdid not produce any bands, whereasb-actin primersshowed optimal enzymatic digestion of the chromatin.These results indicated that both LEDGF/p75 and MeCp2bind to the Hsp27pr, with overlapping binding sites at thedistal regions of the promoter.

LEDGF/p75 and p52 modulate the transcriptionalactivity of MeCP2We sought to determine whether LEDGF/p75 and p52

modulate the ability of MeCP2 to transactivate Hsp27pr.

Luciferase reporter assays were conducted with coexpressionof Flag-MeCP2 and HA-LEDGF/p75 or Flag-MeCP2 andHA-p52. Coexpression of HA-LEDGF/p75 and Flag-MeCP2 in U2OS cells enhanced Hsp27pr transactivationlevels above those induced by Flag-MeCP2 alone, althoughthis effect wasmarginal (8- vs. 7-fold increase, respectively; P� 0.05) and only at the low LEDGFp75/MeCP2 plasmidratio (1:6; Fig. 7A).On the other hand, coexpression of Flag-MeCP2 with HA-p52 significantly reduced Hsp27pr activ-ity (�40%; Fig. 8B), suggesting that p52 represses MeCP2transcriptional activity.LEDGF/p75 is a transcription coactivator of RNA poly-

merase II transcription (1), whereas MeCP2 participates inchromatin remodeling events leading to recruitment of RNApolymerase II to activate gene transcription (reviewed inrefs. 41, 42). In light of this, we had expected that ectopiccoexpression of HA-LEDGF/p75 and Flag-MeCP2 would

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Figure 6. The N-terminus of LEDGF/p75 interacts with MeCP2. A, diagram of LEDGF/p75 deletion constructs used tomap interaction regions. ATH, AT-hook.CR, charged region. IBD, integrase-binding domain. NLS, nuclear localization signal. B, Flag-MeCP2 binds to eGFP-LEDGF/p75 but not to eGFP-taggedtruncatedLEDGF/p75constructs.293Tcellsectopicallyoverexpressing the taggedproteins labeled in theblotwere immunoprecipitated (IP)withGFPantibodyand visualized by immunoblotting with antibodies to GFP and Flag. C, Flag-MeCP2 binds to eGFP-tagged LEDGF/p75 and LEDGF/p52 but not to truncatedconstructs. Proteins ectopically overexpressed in U2OS cells were immunoprecipitated with Flag antibody and visualized with both anti-GFP and anti-Flagantibodies. �, degradationproduct of LEDGF/p75.D, recombinant Flag-LEDGF/p75, Flag-PWWP-CR1 (1–141aa), andFlag-PWWP (1–101aa)were incubatedwith U2OS cell lysate. Proteins pulled down with anti-Flag affinity matrix were detected by immunoblotting. Endogenous MeCP2 in the cell lysate was pulleddown (PD) by Flag-LEDGF/p75 and Flag-PWWP-CR1. Absence of recombinant proteins (U2OS only) served as negative control. WB, Western blotting.







result in a robust enhancement ofHsp27pr activity inU2OScells. However, because neither synergistic nor additiveeffects were observed, we decided to transiently silenceendogenous LEDGF/p75 using siRNAwhile overexpressingFlag-MeCP2. Surprisingly, U2OS cells overexpressing Flag-MeCP2 but depleted of endogenous LEDGF/p75 displayedrobust and significant increase (3-fold) in Hsp27pr activa-tion compared with cells transfected with control siRNAs,suggesting that LEDGF/p75 may repress MeCP2-drivenHsp27pr activity in these cells (Fig. 8C).In view of the fact that both LEDGF/p75 and MeCP2

have been implicated in prostate cancer cell growth andsurvival (9, 11, 14, 39), we also examined the effects of theircoexpression on Hsp27pr activation in PC3 cells. First, weevaluated Hsp27pr activation in PC3 cells stably overex-pressing LEDGF/p75 and observed activation levels thatwere 3- to 5-fold above cells stably transfected with emptypcDNA vector (Fig. 8D and E). In contrast to what weobserved in U2OS cells, transient Flag-MeCP2 overexpres-sion in PC3 cells stably transfected with empty pcDNA

vector did not significantly enhance Hsp27pr activationcompared with cells without Flag-MeCP2 transfection (Fig.8E, pcDNA). However, PC3 cells overexpressing bothLEDGF/p75 (stably) and Flag-MeCP2 (transiently) signif-icantly enhanced Hsp27pr activation compared with cellswithout Flag-MeCP2 transfection (Fig. 8E, pcDNA-p75).This enhancement was more robust (10-fold) than thatobserved in U2OS (see Fig. 7B). Interestingly, PC3 cellswith transient Flag-MeCP2 overexpression but depleted ofLEDGF/p75 also showed a significant increase in Hsp27practivation, compared with cells transfected with controlsiRNAs (2-fold) or cells with without Flag-MeCP2 over-expression (1.75-fold; Fig. 8F). However, this LEDGF/p75-mediated transcriptional repression effect was modest com-pared with that observed in U2OS cells.

DiscussionThis study establishedMeCP2 as an interacting partner of

LEDGF/p75 and p52 in vitro and in cellular systems usingseveral complementary approaches. There was consistent

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Figure 7. LEDGF/p75, LEDGF/p52, and MeCP2 transactivate Hsp27 promoter. U2OS cells were cotransfected with pGL3-Hsp27pr-luc and the indicatedamount of (A) empty pcDNA vector or pcDNA-Flag-MeCP2, or (B) pCruzHA, pCruzHA-LEDGF/p75, or pCruzHA-p52 (1 ¼ 1.66 mg DNA). Promoter activitydetermined as luciferase light units/protein is expressed as fold activation compared with control activity (cotransfection of pGL3-Hsp27pr-luc with emptyexpression vectors). Data from each graph are representative of at least 3 independent experiments. Representative immunoblots corresponding tothe reporter assays show protein expression. C, schematic diagram of Hsp27pr showing AT-rich regions, and HSE and STRE sites. PCR primers targetedHsp27pr regions A (bp �271 to þ18), B (bp �480 to �220), C (bp �803 to �382), and D (bp �1,071 to �781). D, ChIP analysis of MeCP2 andLEDGF/p75 binding to Hsp27pr. Formaldehyde-fixed chromatin from U2OS cells was precipitated with nonspecific IgG or antibodies specific for MeCP2 orLEDGF/p75. PCR amplifications of immunoprecipitated DNA derived from U2OS cells were carried out with primer sets specific for Hsp27pr regions A to D.Hsp27pr primers amplified DNA fragments precipitated by LEDGF/p75 antibody or MeCP2 antibody but not by IgG. Primers that target human b-actincontrolled for optimal enzymatic digestion of chromatin. Data represent the average of at least 3 independent experiments.


Leoh et al.





agreement in the results obtained from all the differentapproaches, suggesting specific interaction between theseproteins. The 11 to 1 nmol/L ratio needed for Flag-LEDGF/p75 to bind GST-MeCP2 in the AlphaScreen assay sug-gested weak binding between the 2 proteins under the invitro conditions of this assay. It should be noted that theconcentration of GST-MeCP2 was approximate as thisprotein degraded rapidly when purified, which obliged usto use it in the bacterial lysates for the assays. Alternatively,LEDGF/p75 and MeCP2 may need other nuclear proteinsor native chromatin for stronger interaction.We examined whether the LEDGF/p75–MeCP2 inter-

action influences Hsp27pr activation in luciferase reporter

assays. Hsp27 is a target gene of LEDGF/p75 implicated inprostate cancer resistance to cell death and chemotherapy(15, 22, 45, 46). Overexpression of LEDGF/p75 upregu-lates Hsp27 transcript in cancer cells, correlating with itspromoter transactivation (22). Our data indicated thatbinding of LEDGF/p75 to Hsp27pr was not limited to theregion where HSE and STRE are located (bp�271 toþ18),as binding was observed throughout the entire Hsp27prregion examined (bp �1,071 to �220). This observationsheds some light into the question of whether LEDGF/p75binding to promoter regions is mainly restricted to STREand HSE, as observed by some investigators but not byothers (15, 32). Recently, studies using the DamID

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Figure 8. LEDGF/p75 and p52 influenceMeCP2-induced transactivation of Hsp27pr. A, U2OS cells were cotransfected with pGL3-Hsp27pr-luc, pCruzHA, orpCruzHA-LEDGF/p75, and increasing amounts of pcDNA-Flag-MeCP2 (1 ¼ 1.66 mg DNA). B, U2OS cells were cotransfected with pGL3-Hsp27pr-luc,pCruzHA, or pCruzHA-LEDGF/p52, and increasing amounts of pcDNA-Flag-MeCP2. C, transient knockdown of LEDGF/p75 in U2OS cells was achievedusing specific siRNA oligos. Cells were then cotransfectedwith pcDNA-Flag-MeCP2 and pGL3-Hsp27pr-luc. Promoter activity determined as luciferase lightunits/protein is expressed as fold activation compared with control activity. D, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75were cotransfected with pGL3-Hsp27pr-luc and pMAX-GFP (transfection control). E, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were cotransfected with pGL3-Hsp27pr-luc and pcDNA-Flag-MeCP2. F, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were transfected with siRNA oligos to knockdown this protein. Cells were then cotransfected with pGL3-Hsp27pr-luc and pcDNA-Flag-MeCP2.Promoter activity determined as luciferase light units/GFP is expressed as fold activation comparedwith control activity. Data represent the average of at least3 independent experiments. Representative immunoblots corresponding to the reporter assays showprotein expression. All protein bands in (F)were from thesame blot. �, P < 0.05; ��, P < 0.01.







technology, focusing on the highly annotated ENCODE(encyclopedia of DNA Elements) region, revealed thatLEDGF/p75 binding to DNA occurs primarily downstreamof active transcription unit start sites, is not restricted toSTREs, and correlates with active chromatin markers andRNA polymerase II binding (47). Nevertheless, STREsappear to be important for LEDGF/p75-mediatedVEGF-c promoter transactivation (17). It is plausible thatLEDGF/p75 binding to particular motifs in promoterregions could be influenced by interactions with specifictranscription factors as well as the cellular type andmicroenvironment.Our results revealed that ectopic MeCP2 overexpression

robustly transactivated Hsp27pr in U2OS cells, surpassingthe transactivation levels induced by LEDGF/p75 or p52.This suggested that Hsp27 is a transcriptional target gene ofMeCP2, with LEDGF/p75 and p52 possibly playing aregulatory role. Alternatively, LEDGF/p75 and MeCP2may play redundant roles in activating Hsp27pr, with thecellular and molecular context determining which of theseproteins play a major role in the promoter activation. Whileour ChIP data suggested binding of MeCP2 to upstreamregions of the Hsp27pr, further studies are needed todetermine the exact binding sites, whether or not theyrequire DNA methylation, and whether MeCP2 could beactivating or repressing different transcription factors toactivate this promoter.To our knowledge, this is the first report documenting

MeCP2-induced activation of Hsp27, a key prosurvivalgene in prostate cancer (44, 45). MeCP2 was shown tobe critical for prostate cancer cell growth and tumorige-nicity, presumably by repressing tumor-suppressing genes(39). However, it cannot be ruled out that MeCP2 alsocontributes to prostate tumorigenesis by transactivatingstress survival genes such as Hsp27. In agreement with ourresults, MeCP2 has been shown to directly bind promotersites of proteins and enhance their activation, as shown byits association with transcription activator Creb1 on pro-moters of transcribed genes (42). It is possible that MeCP2interacts with other transcription factors on Hsp27pr in amanner similar to its association with Creb1 at promotersites, contributing to upregulation of Hsp27pr activity inhuman cancer cells.The coexpression of LEDGF/p52 with MeCP2 in U2OS

cells resulted in markedly reduced transactivation ofHsp27pr, suggesting that p52 represses the transcriptionalactivity of MeCP2. Bueno and colleagues (21) recentlyreported that mutations impairing SUMOylation ofLEDGF/p75 increase its transcriptional activity but notthat of p52, suggesting that these splice variants activateHsp27pr via different molecular mechanisms. Consideringthe observed antagonistic functions of LEDGF/p75 (pro-survival) and p52 (proapoptotic; ref. 22), it is possible thatthe latter may directly antagonize LEDGF/p75 andMeCP2by disrupting their binding to Hsp27pr or their interactionwith other transcription components. Binding of p52 toMeCP2 could also form a growth suppressor unit, down-regulating the activation of Hsp27pr, similar to that of the

growth suppressor unit formed by interaction of JunD withmenin (48). Alternatively, reduced Hsp27pr activity may bedue to p52-induced apoptosis, which generates a dominant-negative fragment p38 that interferes with the transcriptionfunction of LEDGF/p75 (22).Contrary to our expectations, we did not detect a robust

cooperation between ectopically expressed MeCP2 andLEDGF/p75 on Hsp27pr activation in U2OS cells.Instead, we observed that transient silencing ofLEDGF/p75 in this cell line induced a dramatic increasein MeCP2-driven Hsp27pr activity. However, in PC3cells, the cooperation between LEDGF/p75 and MeCP2on Hsp27pr activation appeared to be stronger than inU2OS cells, whereas the enhancement of promoter activ-ity by the LEDGF/p75 knockdown was weaker. Theseapparently contradictory results suggest the interestingpossibility that LEDGF/p75 could be a modulator ofMeCP2 transcriptional activity, enhancing or repressingthis activity depending on the cellular and molecularcontext, or the microenvironment. LEDGF/p75 mightbe part of a feedback loop by competing with MeCP2 orblocking other transcription cofactors from promoterregulatory sites, as in the feedback loop observed betweenHSF1 and Hsp27 (49). This would be important toreduce spurious or excessive transcriptional activation ofHsp27 and other stress genes.While the mechanism by which LEDGF/p75 modulates

MeCP2 transcriptional activity is unknown, it should beemphasized that the PWWP-CR1 region of LEDGF/p75appears to be responsible for binding to MeCP2. ThePWWP domain of LEDGF/p75 has been shown to coop-erate with the NLS and AT-hooks downstream the CR1region to facilitate interaction with DNA/chromatin (31–33). Recently, this domain was shown to lock into thechromatin cargo proteins such as HIV-IN that are boundto the IBD domain of LEDGF/p75 (30). PWWP domainsbelong to the Royal family of protein domains that regulatechromatin remodeling and function (29). In DNA methyl-transferases, the PWWP domain is essential for heterochro-matin targeting, possibly by facilitating interaction withmethylated histones (25, 26). The PWWP domain ofhepatoma-derived growth factor, a cancer-related proteinthat shares sequence homology with LEDGF/p75, isrequired for binding to chromatin and the transcriptionalcorepressor C-terminal–binding protein (CtBP), whichresults in transcriptional repression of specific genes (27,28). Thus, PWWP domains are emerging as structuralentities that bind toDNA, histones, and transcription factorsto regulate gene expression.It is possible that LEDGF/p75 could tether MeCP2 to

transcriptionally active sites in the chromatin via thePWWP-CR1 region. However, given the documented roleof PWWPdomains in transcriptional repression, it would betempting to speculate that this region may negatively reg-ulate the activity of MeCP2 and other interacting transcrip-tion factors. Singh and colleagues (38) provided evidencethat the N-terminal region of LEDGF/p75 has negativetranscriptional regulatory elements and proposed that


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LEDGF/p75, like other transcription proteins, bear repres-sive as well as activation domains, with the repressive domainbeing the PWWP domain (38). Interestingly, chromosomaltranslocations in acute leukemia produce a NUP98-LEDGF/p75 fusion protein lacking the N-terminal regionof LEDGF/p75 (13, 18), suggesting the possibility that thisPWWP-CR1–deficient protein induces deregulated onco-gene transcription.To our knowledge, this is the first report implicating the

N-terminal region of LEDGF/p75 in protein–protein inter-actions. To date, all the interacting partners of LEDGF/p75,namely, HIV-IN, JPO2,menin/MLL, ASK, and pogZ, havebeen shown to interact with the C-terminal region of theprotein, particularly, with the IBD domain. Through thisinteraction, some of these proteins are recruited to tran-scriptionally active sites in the chromatin (18, 30). In the caseof menin/MLL, the interaction with LEDGF/p75 actuallyleads to the activation of cancer-related genes involved inleukemogenesis (18).In conclusion, we validated MeCP2 as a cellular interact-

ing partner of both LEDGF/p75 and p52 and a transacti-vator of Hsp27pr. Follow-up studies should determine ifrational mutation of specific sites in the PWWP-CR1domain of LEDGF/p75 (e.g., Pro-Trp-Trp-Pro motif,SUMOylation sites, etc.) results in loss of MeCP2 bindingto LEDGF/p75 or Hsp27pr or in deregulated stress pro-moter activity that is mediated byMeCP2 or other LEDGF/p75-interacting transcription factors. It should also be deter-mined if the LEDGF/p75 PWWP domain is required forMeCP2 binding to chromatin and if the complex betweenthese proteins bind tomethylated or demethylated promoterregions. Additional studies will be needed to define ifLEDGF/p75, p52, and MeCP2 are part of a larger dynamictranscriptional complex that regulates the expression of stresssurvival genes depending on the cellular context and themicroenvironment. Our results have implications for under-

standing the contribution of LEDGF/MeCP2 containingtranscription complexes in the regulation of gene expressionin cancer, HIV-AIDS, Rett syndrome, and other diseases inwhich these proteins have been implicated.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsDevelopment of methodology: L.S. Leoh, B. vanHeertum, J. de Rijck, M. Fillipova,S.S. Tungteakkhun, V. FillipovAcquisition of data: L.S. Leoh, B. van Heertum, J. de Rijck, M. Fillipova, L.Rios-Colon, A. Basu, S.R. Martinez, S.S. Tungteakkhun, V. FillipovAnalysis and interpretation of data: L.S. Leoh, B. van Heertum, J. de Rijck, M.Fillipova, S.S. Tungteakkhun, V. Fillipov, F. Christ, M. De Leon, Z. Debyser, C.A.CasianoExperimental design: F. Christ, M. De Leon, Z. Debyser, C.A. CasianoProvided facilities: F. Christ, M. De Leon, Z. Debyser, C.A. CasianoWriting of the manuscript: L.S. Leoh, C.A. Casiano

AcknowledgmentsThe authors thank Dr. Sean Wilson and Monica Rubalcava (Loma Linda

University, Loma Linda, CA) for assistance with confocal microscopy and Dr. MelissaMcNeely, Dr. Antonio Gallo, Christine de Kogel, and Jonas Demeulemeester(Leuven) for the generation of plasmids and recombinant proteins, and technicalassistance. They also thank Melanie Mediavilla-Varela, Terry Brown-Bryan, FabioPacheco, Vidya Ganapathy, Papri Chatterjee, Evyn Adkins, Lauren Melendez, andBobby Jimenez (Loma Linda University) for technical assistance in the initial stages ofthis work and Drs. Rik Gijsbers (Leuven), Penelope Duerksen-Hughes, ThomasLinkhart, and Nathan Wall (Loma Linda University) for excellent suggestions.

Grant SupportThis work was supported by grants NIH-NCMHD 5P20MD001632 (M. De

Leon and C.A. Casiano), KU Leuven Research Council grant OT/09/047(Z. Debyser), and Flanders Research Foundation (FWO) grant G.0530.08(Z. Debyser). Confocal microscopy imaging was conducted in the LLUSM AdvancedImaging and Microscopy core facility, supported by NSF grant MRI-DBI-0923559(S.M. Wilson) and the Loma Linda University School of Medicine.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received June 30, 2011; revisedDecember 27, 2011; acceptedDecember 28, 2011;published OnlineFirst January 24, 2012.

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The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG … · These are the PC4...

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