Impaired Retinoic Acid (RA) Signal Leads to RAR�2 Epigenetic Silencing and RA Resistance

MOLECULAR AND CELLULAR BIOLOGY, Dec. 2005, p. 10591–10603 Vol. 25, No. 230270-7306/05/$08.00�0 doi:10.1128/MCB.25.23.10591–10603.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Impaired Retinoic Acid (RA) Signal Leads to RAR�2 EpigeneticSilencing and RA Resistance

MingQiang Ren, Silvia Pozzi, Gaia Bistulfi, Giulia Somenzi, Stefano Rossetti,and Nicoletta Sacchi*

Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263

Received 25 May 2005/Returned for modification 13 July 2005/Accepted 16 September 2005

Resistance to the growth-inhibitory action of retinoic acid (RA), the bioactive derivative of vitamin A, iscommon in human tumors. One form of RA resistance has been associated with silencing and hypermethyl-ation of the retinoic acid receptor �2 gene (RAR�2), an RA-regulated tumor suppressor gene. The presence ofan epigenetically silent RAR�2 correlates with lack of the RA receptor � (RAR�). Normally, RAR� regulatesRAR�2 transcription by mediating dynamic changes of RAR�2 chromatin in the presence and absence of RA.Here we show that interfering with RA signal through RAR� (which was achieved by use of a dominant-negative RAR�, by downregulation of RAR� by RNA interference, and by use of RAR� antagonists) induces anexacerbation of the repressed chromatin status of RAR�2 and leads to RAR�2 transcriptional silencing.Further, we demonstrate that RAR�2 silencing causes resistance to the growth-inhibitory effect of RA. Appar-ently, RAR�2 silencing can also occur in the absence of DNA methylation. Conversely, we demonstrate thatrestoration of RA signal at a silent RAR�2 through RAR� leads to RAR�2 reactivation. This report providesproof of principle that RAR�2 silencing and RA resistance are consequent to an impaired integration of RAsignal at RAR�2 chromatin.

Cells of different histotypes seem prone to lose the ability torespond to the growth-inhibitory action of retinoic acid (RA),the potent bioactive derivative of vitamin A. RA regulatesfundamental cellular processes, such as growth, differentiation,and apoptosis (7). Previously, we and others showed a corre-lation between a common form of RA resistance and repres-sive epigenetic changes (at both the histone and DNA levels)in the RA receptor �2 gene (RAR�2) (5, 33, 34, 40).

RAR�2 is an RA-regulated tumor suppressor gene (19, 26,32). Detection of aberrant RAR�2 methylation in tumors ofdifferent histotypes raised the question of whether this epige-netic change is critical for silencing this tumor suppressor gene.Previously, we proposed that aberrant RAR�2 inactivity mightinduce repressive epigenetic changes at RAR�2, leading toRAR�2 silencing and RA resistance (33, 34). RAR�2 transcrip-tion is normally regulated by dynamic histone changes in thepresence and absence of RA (9, 14, 29, 41). Therefore, wehypothesized that the impaired integration of RA signal atRAR�2 can create a state of exacerbated-protracted RAR�2 tran-scriptional inactivity and attract chromatin-repressive changes,including DNA methylation. The conversion of RAR�2 from astate permissive for transcription into a stable state nonper-missive for transcription would cause biological RA resistance.Our hypothesis hinges on the original supposition of Ng andBird (28) that chromatin inactivity, the prerequisite for epige-netic silencing of genes on chromosome X (18), could also leadto silencing of genes on other chromosomes. Thus, an aberrantinactive RAR�2 chromatin status would be the prerequisite forRAR�2 epigenetic silencing.

RAR�2 DNA methylation and silencing were shown to beinduced by active recruitment of repressor proteins by an on-cogenic fusion protein in leukemic cells (13). However, to ourknowledge, this oncoprotein has not been demonstrated inepithelial cancer cells and tumors of the breast, prostate, colon,lung, and head and neck, where RAR�2 has also been foundsilenced (33, 34). In contrast, cancer epithelial cells and tumorsappear to have either a low intracellular concentration of RAor a lack or derangement of proteins involved in either RAmetabolism and homeostasis or RAR�2 transcriptional regu-lation. Thus, RA resistance might be the consequence of anexacerbated-protracted RAR�2 transcriptional repressioncaused by a defective integration of RA signal at RAR�2,which might be induced by genetic, epigenetic, metabolic, andenvironmental factors capable of shutting off the “communi-cation” between RA and RAR�2 chromatin.

We identified and tested as a possible cause of aberrantRAR�2 inactivity the lack of functional RAR�, the upper reg-ulator of RAR�2 transcription. RAR� has the role of keepingthe chromatin of its direct target genes, such as RAR�2, poisedfor transcription yet inactive. Upon binding of RA to RAR�,the chromatin status of the target genes is converted from inactiveinto active because of the exchange of corepressor complexes withcoactivator complexes, which would rapidly induce histonechanges, chromatin remodeling, and transcription activation(14, 29).

In the course of our studies of RA-resistant breast andprostate cancer cell lines, we observed the following: (i) thepresence of low or negligible binding of RAR� at RAR�2 inRA-resistant breast and prostate cancer epithelial cells carry-ing RAR�2 nonpermissive alleles (we define as nonpermissivethe alleles that cannot be transcriptionally activated by RA andas permissive the alleles that are poised for transcription yetinactive in the absence of RA but capable of transcription in

* Corresponding author. Mailing address: Roswell Park CancerInstitute, Elm & Carlton Streets, C&V Bldg., RM 226, Buffalo, NY14263. Phone: (716) 845-1053. Fax: (716) 845-1741. E-mail: nicoletta.sacchi@roswellpark.org.

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the presence of RA); (ii) the presence of RAR�2 unmethylated(U), permissive alleles in RAR�-positive cells, which containmany other methylated (M) genes (20), pointing at RAR� as acritical factor that can spare RAR�2 chromatin from fallinginto a nonpermissive status; and (iii) the presence of a minimalstretch of methylated CpGs in the first RAR�2 exon—corre-sponding to exon 5 of the RAR� locus (38)—in methylatedalleles, suggesting that CpG methylation originates in a specificepicenter from unmethylated yet nonpermissive alleles.

In this study we simulated possible genetic, epigenetic, andmetabolic scenarios that could impair the flow of RA signal atRAR�2 chromatin via RAR�. Using three different strate-gies—a dominant-negative RAR� lacking the RA-binding do-main, downregulation of RAR� by RNA interference, and RAantagonists acting specifically at RAR�—we induced the con-version of RAR�2 permissive alleles into nonpermissive allelesin RA-sensitive human cells. The RAR�2 nonpermissive allelesdeveloped a significant load of repressive histone tail modifi-cations and failed to recruit RNA polymerase II at the regioncontaining the transcription start site. Only a percentage ofnonpermissive alleles developed CpG hypermethylation, thusshowing that aberrant hypermethylation is not an absoluterequirement for RAR�2 silencing. In this report we also dem-onstrate that restoring RA signal through RAR� at an epige-netically silent RAR�2 is critical to reestablishing a RAR�2status compatible with transcription. RAR�2 epigenetic silenc-ing has been described as being associated with an RA-resis-tant phenotype (26, 32). Here we prove that RA resistance isthe consequence of RAR�2 epigenetic silencing.

MATERIALS AND METHODS

Cell lines. Breast and prostate cancer cell lines (ATCC, Manassas, VA) andCOS-1 cells (ATCC) were grown using standard protocols.

Drugs. All-trans-RA, 5-aza-2�-deoxycytidine (5-Aza), a demethylating agent(10), and trichostatin A (TSA), a histone deacetylase inhibitor (42), were all fromSigma (St. Louis, MO). These drugs were dissolved and stored as describedpreviously (34). The RAR� antagonist ER50891 was a kind gift of KouichiKikuchi, Discovery Research Laboratories, Ibaraki, Japan, and the RAR� an-tagonist RO414253 was a kind gift from Salvatore Toma, National Cancer In-stitute, Genoa, Italy. Treatments with these drugs were all performed in mediumsupplemented with 5% charcoal–dextran-stripped serum.

Colony formation assay. Exponentially growing cells were seeded at 5 � 102

cells/well in six-well plates in triplicate and allowed to attach to the substrate.Cells were left untreated or treated with drugs for 24 h, and then the medium wasreplaced with drug-free medium and the cells were grown for 14 to 21 days.Colonies were fixed with methanol, stained with Giemsa (Sigma), and counted toestablish the colony formation index as described previously (34). The statisticalsignificance was calculated by Student’s t test for three independent experiments.

Cell transfections. The RAR� dominant-negative mutant RAR�403 was sub-cloned by PCR into the FLAG-containing pCMV-tag vector (Stratagene, LaJolla, CA) with primers introducing EcoRI and XhoI restriction sites (sense,5�-TATGAATTCATGGCCAGCAACAGCAGCTC-3�; antisense, 5�-ATACTCGAGGGGATCTCCATCTTCAGCGT-3�), and the empty pCMV-tag vectorwas transfected in T47D by using Lipofectamine Plus (Invitrogen, Carlsbad, CA).The LNasRAR�2VI vector, which harbors six copies of RAR�2 antisense(asRAR�2) and the control empty vector LNSX (kindly provided by S. Y. Sun,University of Texas M.D. Anderson Cancer Center, Houston, TX) (37) were alsotransfected in T47D by Lipofectamine Plus. Stable clones were selected andmaintained with G418 (Invitrogen) at 0.8 mg/ml. Stable MDA-MB-231 clonesoverexpressing RAR� 1 were obtained by cotransfecting cells with pSG5-hRAR�1 vector (kindly provided by Fausto Andreola, National Cancer Institute, Be-thesda, MD) and G418-resistant pcDNA3.1(�) vector (Invitrogen) by use ofLipofectamine Plus. Control cells were cotransfected with the empty vectorpSG5 (Promega, Madison, WI) and pcDNA3.1(�). Stably transfected cells,selected with increasing concentrations of G418 (0.5 to 2.5 mg/ml), were tested

for expression of exogenous RAR� 1 by Western blotting with the C-20 anti-RAR� antibody (Santa Cruz Biotechnology).

Retroviral infections. Supernatants containing either the RAR� dominant-negative LXRAR�403SN or the empty LXSN retroviral particles (kindly pro-vided by Fausto Andreola, National Cancer Institute, Bethesda, MD) were usedto infect T47D cells in the presence of 4 mg/ml Polybrene (Sigma) as describedpreviously (39). Infected cells were selected with 0.8 mg/ml G418. Single cloneswere isolated after 14 to 21 days and screened for the presence of either theLXSN or the LXRAR�403SN construct by reverse transcription-PCR (RT-PCR). Positive clones were maintained in 0.8 mg/ml G418.

RAR� RNA interference. The 19-nucleotide sequence (AGCGCACCAGGAAACCTTC) corresponding to nucleotides 680 to 699 of RAR� exon 5 (GenBankaccession no. NM_000964) was inserted into pSUPER-retro (OligoEngine, Se-attle, WA) according to the manufacturer’s instructions. The silencing efficiencyof the resulting construct, pSUPER-RAR�, was first tested on exogenous RAR�by transiently cotransfecting COS-1 cells with pSG5-hRAR�1, encoding the hu-man RAR�1 and pSUPER-RAR� at different ratios. Exogenous RAR� levels,normalized to GAPD (glyceraldehyde-3-phosphate dehydrogenase) expressionlevels, were estimated by Western blotting with the C-20 anti-RAR� and anti-GAPD (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies and appropriatehorseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech-nology; Amersham, Piscataway, NJ). Stable transfections with pSUPER-RAR�and pSUPER-retro (control) were performed with Lipofectamine Plus. Trans-fected T47D clones were selected with 2 �g/ml puromycin (Sigma). Clonesdisplaying significant silencing of RAR� by real-time RT-PCR and Westernblotting were chosen for further analysis.

RAR� antagonist experiments. Cells (5 � 102) were seeded in six-well platesin triplicate in medium containing 5% charcoal–dextran-stripped serum, allowedto attach to the plastic substrate, and treated with ER50891 (10 �M) and RA (0.1�M), either alone or in combination, for 24 h. After this time, the medium wasreplaced by drug-free medium. Cells were grown until colonies became visible(14 to 21 days). Colonies were stained with Giemsa to establish the colonyformation index (34). Pools of colonies derived from cells that survived eachtreatment (125 colonies from one well with no drugs, 118 clones from one welltreated with ER50891 alone, and 115 clones from one well treated with ER50891plus RA) were used to isolate genomic DNA that was used for methylation-specific PCR (MSP) analysis with M4 primers and U4 primers. In a parallelreplica experiment, we instead used cloning cylinders to isolate six independentclones that survived the treatment with either ER50891 alone (which we desig-nated ER clones) or ER50891 in combination with RA (which we designatedER/RA clones). All clones from each group that were expanded in drug-freemedium did not show RA-induced RAR�2 transcription by real-time RT-PCRand were shown to be U or M by MSP. One clone from each of the groupsER-C6 and ER/RA-C5 was further expanded in drug-free medium.

Real-time RT-PCR. Total RNA was obtained using Trizol (Invitrogen),treated with DNase I (Ambion, Austin, TX), retrotranscribed with a SuperScriptfirst-strand synthesis system (Invitrogen), and amplified by real-time RT-PCR onan iCycler apparatus (Bio-Rad, Hercules, CA) by using iQ SYBR green Super-mix (Bio-Rad) and specific primers for RAR�2 (sense, 5�-GACTGTATGGATGTTCTGTCAG-3�; antisense, 5�-ATTTGTCCTGGCAGACGAAGCA-3�), RAR�(both isoforms 1 and 2) (sense, 5�-TGTGGACTTCGCCAAGCA-3�; antisense,5�-CGTGTACCGCGTGCAGA-3�), RAR� 1 (sense, 5�-GCCAGGCGCTCTGACCACTC-3�; antisense, 5�-CAGGCGCTGACCCCATAGTGGT-3�); and GAPD(sense, 5�-GAAGGTGAAGGTCGGAGTC-3�; antisense, 5�-GAAGATGGTGATGGGATTTC-3�), with the appropriate annealing temperature according to stan-dard protocols. The RAR� and RAR�2 transcription levels were normalized to theGAPD transcription level. The statistical significance was calculated using Student’st test for three independent determinations.

Luciferase assay. The luciferase reporter assay was performed essentially asdescribed previously using the luciferase reporter vector RAR�2-pGL2 (12)(kindly donated by K. Ozato, National Institutes of Health, Bethesda, MD) andthe control vector pRL-TK (Promega). Vector DNAs were cotransfected withLipofectamine Plus in cells grown in a 12-well plate; 24 h after transfection, themedium was replaced with medium with or without RA (1.0 �M). Luciferaseactivity was measured by a dual luciferase reporter assay system (Promega)according to the manufacturer’s instructions. The values represent the averages(normalized to the control) of three independent experiments, each performedin triplicate.

ChIP. Quantitative chromatin immunoprecipitation (ChIP) was performedusing reagents purchased from Upstate (Lake Placid, NY) following the manu-facturer’s protocol. Chromatin was immunoprecipitated with antibodies againstacetyl-histone H4 (Ac-H4) (Upstate) (1:400), acetyl-lysine (K) 9 histone H3(H3-Ac-K9) (Upstate) (1:400), dimethyl-K4 histone H3 (H3-Me-K4) (Upstate)

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(1:400), trimethyl-K9 histone H3 (H3-Tri-Me-K9) (Upstate) (1:200), RAR� Cterminus (Santa Cruz Biotechnology) (1:60), RAR� N terminus (Biolegend, SanDiego, CA) (1:100), RAR� (Santa Cruz Biotechnology [1:70]; Active Motif,Carlsbad, CA [1:600]), RNA polymerase II (Upstate) (1:200), and FLAGepitope (Sigma) (1:350). Re-ChIP was performed with the anti-RAR� C-termi-nus antibody on chromatin immunoprecipitated with an anti-Ac-H4 antibodyafter elution with re-ChIP elution buffer (10 mM EDTA, 50 mM Tris-HCl [pH8.0], 0.7 M NaCl, 20 mM dithiothreitol). Control ChIPs were without the re-spective antibodies. The immunoprecipitated DNA was amplified by real-timePCR with either RAR�2 primers (sense, 5�-GGTTCACCGAAAGTTCACTCGCAT-3�; antisense, 5�-CAGGCTTGCTCGGCCAATCCA-3�) or the GAPDprimers (sense, 5�-GGTGCGTGCCCAGTTGAACCA-3�; antisense, 5�-AAAGAAGATGCGGCTGACTGTCGAA-3�). The RAR�2 DNA relative enrich-ment was calculated by normalizing the RAR�2 PCR signal to the PCR signalsobtained both from the input DNA (total chromatin fraction) and the GAPDDNA. Statistical significance was determined using the Student’s t test on threeindependent determinations.

DNA methylation analysis. Genomic DNA extracted with DNAzol (Invitro-gen) was modified with sodium bisulfite as described previously (33). ModifiedDNA was used for both MSP and sequencing analyses. For sequencing analysis,a 635-bp region, encompassing 27 RAR�2 CpG sites in the RAR�2-regulatoryregion, was amplified by nested PCR (40) using first the primer set RAR�2 sense1 (5�-GTATAGAGGAATTTAAAGTGTGGGTTGGG-3�) and RAR�2 anti-sense 1 (5�-CCTATAATTAATCCAAATAATCATTTACC-3�) and subse-quently the primer set RAR�2 sense 2 (5�-GTAGG(C/T)GGAATATTGTTTTTTAAGTTAAG-3�) and RAR�2 antisense 2 (5�-AATCATTTACCATTTTCCAAACTTACTC-3�). The PCR products were either directly sequenced or se-quenced after subcloning in the pCR4-TOPO plasmid vector (Invitrogen). In thelatter case, we sequenced a minimum of 20 to a maximum of 50 clones corre-sponding to the sample specified in Results. MSP was performed by amplifyingbisulfite-modified DNA with the first RAR�2 primer set (see above). Then, 2 �lof a 1:1,000 dilution of the first amplification product was reamplified with theU4 sense- and U4 antisense-specific primers or with the M4 sense- and M4antisense-specific primers (33) or with the different combinations of U4 and M4primers. RAR�2 alleles detected with both U4 primers were classified as U, andthe alleles amplified with either both M4 primers (these products are the onesdiscussed in Results) or the sense M4 primer and the U4 antisense primer wereclassified as M alleles. No product was amplified with the U4 sense primer andthe M4 antisense primer.

RESULTS

Lack of RAR� correlates with RAR�2 epigenetic silencing inRA-resistant cancer cells. Analysis of a panel of breast andprostate cell lines showed different RA-induced RAR�2 tran-scription levels in correlation with differential growth inhibi-tion by RA after 24 h of treatment (Fig. 1A, top and bottom,respectively). DNA methylation analysis using two comple-mentary techniques, bisulfite sequencing and MSP, identifiedcell lines (i) homozygous for RAR�2 M alleles (MCF7, MDA-MB-231, and LNCaP carry 100% of M alleles), (ii) homozy-gous for RAR�2 U alleles (T47D carries 100% of U alleles),and (iii) heterozygous for U and M alleles (DU145) (Fig. 1B).As determined on the basis of sequencing of 20 independentDU145 RAR�2 alleles, 30% were U alleles and 70% were Malleles. The distribution of methylated CpGs in the M alleles ofall cell lines involved at least a common stretch of CpGs in thefirst RAR�2 exon (Fig. 1B, bottom). Lack of RA-inducedRAR�2 transcription was also detected in cells (DU145) withU alleles, which must therefore be interpreted as nonpermis-sive. Moreover, RAR�2 was spared by methylation in T47D, acell line with several other hypermethylated genes (20).

Analysis of the chromatin associated with RAR�2 allelesenabled us to quantitate the level of a few histone modifica-tions—considered a hallmark of either repressive or activechromatin (4, 22)—in the RAR�2 region encompassing theRA-responsive element (RARE), the TATA box, the tran-

scription start site, and the common stretch of CpGs in the firstexon. We detected significant differences in the levels of acet-ylation of both histone H4 (Ac-H4) and histone H3 at lysine(K) 9 (H3-Ac-K9) in response to RA treatment for 24 h inT47D RAR�2 chromatin but not in DU145 chromatin (Fig. 1C,top left and bottom left). Moreover, both in the absence (base-line) and in the presence of RA, the level of methylation ofhistone H3 on K4 (H3-Me-K4)—a parameter of chromatinactivity—was higher in T47D than in DU145 chromatin (Fig.1C, bottom right), while the opposite was true for H3-K9methylation (H3-Tri-Me-K9)—a parameter of chromatin inac-tivity (Fig. 1C, top right). Thus, the overall quantity of histonemodifications, which we define as the “histone modificationthreshold” (Fig. 1C, dotted lines), as well as the quality ofhistone modifications, seems to be critical in order to “switchon” RAR�2 transcription in response to RA. RA induced bothqualitative and quantitative modifications in nonpermissive al-leles as well but, apparently, unless a critical accumulation ofhistone changes hit the threshold, the transcription remained“off.”

Normally, RAR� bound to RAR�2 keeps the chromatinpoised for transcription (yet inactive) (14) (Fig. 1D, top left)and ready for activation by RA (Fig. 1D, top right). RA bind-ing to RAR� would induce histone modifications capable ofactivating RAR�2 very rapidly (29). Once RAR�2 is induced, itcould regulate its own transcription (8). We observed that thepresence of RAR�2 nonpermissive alleles (regardless of theirDNA methylation status) always correlated with a lower levelof RAR� transcription (Fig. 1D, bottom left). Consistent withthis observation, quantitative ChIP with anti-RAR� antibod-ies, followed by RAR�2 DNA amplification of the region en-compassing the RARE (schematic diagram in Fig. 1B), de-tected significantly more RAR� associated with RAR�2 inT47D cells than in MDA-MB-231 and DU145 cells (Fig. 1D,bottom right). Of note, by ChIP with anti-RAR� antibodies wefound RAR�2 binding only at the T47D RAR�2 promoter(data not shown).

Altogether, these observations made us hypothesize that adefective integration of RA signal at RAR�2 due to lack offunctional RAR� can convert RAR�2 into inactivity, markedby repressive epigenetic changes at histone and DNA level,and RA resistance.

Induction of RAR�2 epigenetic silencing by a dominant-negative RAR� in RA-sensitive cells. First, we simulated agenetic scenario whereby an RAR� mutation with dominant-negative features occurs in RA-sensitive, RAR�-positive cellshomozygous for RAR�2-permissive alleles. We used a well-characterized dominant-negative RAR� mutant, RAR�403,which lacks the C-terminal RA-binding domain but retains thecapacity to heterodimerize with the retinoid X receptor andbind to the RARE regions (Fig. 2A) (11, 15, 39). RAR� 403should compete with wild-type RAR� in the heterodimeriza-tion with retinoid X receptor (23). It might also cause animpaired turnover of corepressor complexes at RAR� targetgenes, because it lacks the C terminus (27, 29).

At 24 h after transient transfection with a FLAG-RAR�403construct, T47D breast cancer cells (which express mainly en-dogenous RAR�1; data not shown) showed the presence of thedominant-negative mutant at the RAR�2 promoter (ChIP; Fig.2B, bottom left), concomitant with significant RAR�2 tran-

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scriptional downregulation in response to the presence of RA(Fig. 2B, bottom middle). Thus, RAR�2 silencing seems to beinitiated when RAR�403 resides at the RAR�2 promoter re-gion. The dominant-negative RAR� was also found at theRAR�2 of a T47D clone expressing the dominant-negativeprotein (DN C8). This was deduced on the basis of a ChIPexperiment with an antibody directed against the RAR� Cterminus; this antibody detected remarkably less wild-typeRAR� at RAR�2 than an antibody directed against the RAR�N terminus in DN C8 relative to LX C5 (Fig. 2B, bottom right).RA treatment (24 h) failed to induce luciferase transcription

from a transiently transfected RAR�2 promoter-luciferase con-struct (Fig. 2C, left), thus proving the dominant-negative effectof RAR�403 on endogenous RAR�. Finally, in response tothe presence of RA, DN C8 did not show endogenous RAR�2transcription (real-time RT-PCR; Fig. 2C, middle). This find-ing was mirrored by a lack of RNA polymerase II at RAR�2(Fig. 2C, right). The repressed transcriptional status in the DNC8 clone was paralleled by repressive quantitative and quali-tative histone modifications, which, in response to RA treat-ment, did not reach the threshold necessary for “switching on”RAR�2 transcription (Fig. 2D; dotted line). Interestingly, MSP

FIG. 1. Lack of RAR� correlates with RAR�2 epigenetic silencing in RA-resistant cells. (A) Real-time RT-PCR (top) and colony formationassay (bottom) on a panel of breast and prostate cell lines showed a positive correlation between RA-induced RAR�2 transcription andRA-induced growth inhibition. (B) Methylation-specific PCR (MSP, top) and bisulfite sequencing (bottom) show the presence of cells homozygousfor RAR�2 M alleles (MCF-7, LNCaP, and MDA-MB-231), heterozygous for U and M alleles (DU145), and homozygous for U alleles (T47D)and the localization of methylated CpGs in M alleles. (C) ChIP analysis shows a significant increase in the levels of both histone Ac-H4 (P � 0.05)and H3-Ac-K9 (P � 0.01) in T47D RAR�2 chromatin but not in DU145 in response to RA. The levels of H3-Tri-Me-K9 and H3-Me-K4 weresignificantly higher (P � 0.01) and lower (P � 0.01), respectively, in DU145 relative to T47D. The dotted lines represent what we defined as “thehistone modification threshold” required for RAR�2 transcription. (D) Schematic diagram showing that RAR� binds the RAR�2 RARE regionin the absence of RA and that transcription is induced in the presence of RA (top) (14). RAR� transcription (bottom left) and binding of RAR�protein at RAR�2 (bottom right) are significantly higher (P � 0.05) in cells with RAR�2-permissive alleles (T47D) than in cells with RAR�2nonpermissive alleles (DU145 and MDA-MB-231).

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analysis of DN C8 RAR�2 DNA with M4 and U4 primers (seeMaterials and Methods) showed U and M alleles. After bisul-fite sequencing of 50 alleles, we found that DN C8 contained60% U and 40% M alleles with a few methylated CpGs (Fig.2E). In contrast to what was reported for cells carrying anotherdominant-negative RAR� with an intact RA-binding domain(13), RA treatment (1 �M, 72 h) did not reverse RAR�2 DNAmethylation. Only treatment with 5-Aza (0.8 �M for 72 h) ledto demethylation and RA-induced RAR�2 transcription in DNC8 (data not shown).

RAR�2 epigenetic silencing confers an RA-resistant pheno-type. Concomitant with RAR�2 epigenetic silencing, DN C8

cells developed stable resistance to the growth-inhibitory effectof RA, as assessed by colony formation (Fig. 3A). BecauseRAR� controls several RA-responsive target genes, we testedwhether the observed RA resistance phenotype was indeedcaused by RAR�2 silencing. To this end we targeted T47DRAR�2 transcription with an antisense RAR�2 (37). StableT47D clones transfected with LNasRAR�2VI that carry mul-tiple copies of the RAR�2 antisense, like the As-C3 and As-C4clones (where “As” represents “antisense”) (Fig. 3B, left),were significantly less sensitive to RA-induced growth inhibi-tion than the cognate control clone EV-C3 carrying the emptyvector LNSX (Fig. 3B, right). Thus, it is conceivable that the

FIG. 2. Functional RAR� inhibition by a dominant-negative RAR� in RA-sensitive cells leads to RAR�2 silencing marked by repressiveepigenetic changes. (A) Schematic diagram representing the structure of the RAR�-dominant-negative RAR�403 (bottom) missing part of theRA-binding domain. (B) The FLAG-tagged RAR�403, transiently transfected into T47D cells, binds the RAR�2 promoter concomitant with asignificant (P � 0.05) decrease of RA-induced RAR�2 transcription (middle). ChIP experiments with antibodies (Ab) directed against the RAR�C and N termini, showing that the C-Ab, but not the N-Ab, detects remarkably less wild-type RAR� at RAR�2 in DN C8 cells, carrying both thedominant-negative and wild-type RAR� isoforms, than LX C5 cells carrying only wild-type RAR� (right). (C) In contrast to LX C5, DN C8 doesnot significantly express both luciferase from an exogenous RAR�2 promoter (P � 0.05) (left) and endogenous RAR�2 (P � 0.05) (middle) inresponse to the presence of RA, consistent with lack of recruitment of RNA polymerase II at the promoter (right). (D) RAR�2 ChIP analysisshowing that RA significantly (P � 0.01) increases the level of activating histone modifications (Ac-H4, H3-K9-Ac, and H3-Me-K4) to the“threshold” (dotted line) associated with transcription activation (on) only in LX C5 chromatin and not in DN C8 chromatin. Consistently, the levelof H3-Tri-Me-K9 is higher in DN C8 chromatin than in LX C5 chromatin. (E) MSP (left) and bisulfite sequencing (right) show that DN C8 isheterozygous for U and M RAR�2 alleles, with the methylated CpGs mostly localized in exon 1.

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RA-resistant phenotype developed by DN C8 cells is the con-sequence of RAR�2 epigenetic silencing.

RAR�2 DNA methylation is not necessary to confer a non-permissive transcriptional status. Because RA did not inducetranscription from both RAR�2-U and RAR�2-M nonpermis-sive alleles in DN C8 cells, we hypothesized the existence ofRA-resistant DN C8 cells homozygous for nonpermissive un-methylated alleles. We isolated 20 independent DN C8 sub-clones which were resistant to RA and unable to reexpress theRAR�2 transcript in response to RA. Screening for the pres-ence of methylation in the first exon by MSP identified 18clones heterozygous for both U and M alleles and 2 cloneshomozygous for U alleles (Fig. 4A). The CpG methylation-freestatus of the U alleles was confirmed by bisulfite sequencing

(20 alleles of one of the two subclones are shown in Fig. 4B).The level of Ac-H4 did not increase significantly in response toRA in the RAR�2 chromatin of this subclone as it did in theRAR�2 chromatin of LX C5 (Fig. 4C, top) that is insteadhomozygous for RAR�2 U alleles that are permissive (Fig. 2E,top). The level of H3-Tri-Me-K9 in the DN C8 subclone chro-matin was in the range observed in the parental DN C8 clone(Fig. 4C, bottom). Apparently, RAR�2 silencing also can beimposed in the absence of DNA methylation.

Knocking down RAR� by RNA interference also triggersRAR�2 epigenetic silencing and RA resistance. It can be ar-gued that RAR�2 silencing in DN C8 cells is due to the re-cruitment of histone-modifying enzymes or DNA methyltrans-ferases (DNMTs) at RAR�2 by the RAR�403 protein, as

FIG. 3. RAR�2 epigenetic silencing confers an RA-resistant phenotype. (A) Colony formation assay showing that DN C8 cells, but not LX C5cells, display RA resistance. (B) The RAR�2 antisense construct LNasRAR�2VI (top left) is detected in the T47D As-C3 and As-C4 clones, asshown by PCR (bottom left). Colony formation assay showing that As-C3 and As-C4, but not the control EV-C3 clone carrying the empty vector,display RA resistance (right).

FIG. 4. RAR�2 DNA methylation is not necessary to confer a nonpermissive RAR�2 transcriptional status. (A) RAR�2 methylation andtranscription in 20 DN C8 subclones shows that 2 clones do not present CpG methylation and RA-induced RAR�2 transcription. (B) MSP andbisulfite sequencing analysis of one of the two DN C8 subclones homozygous for U alleles. (C) ChIP analysis shows that the chromatin of the DNC8 subclone homozygous for U alleles and the parental DN C8 is marked by comparable levels of Ac-H4 (top) and H3-Tri-Me-K9 (bottom) inthe presence and absence of RA.

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shown for the PML-RAR� protein (13). While nonrandomdominant-negative RAR� mutations were never to our knowl-edge reported in cancer epithelial cells, RAR� expression wasreported to be low or absent in these cells (30). This could bedue to loss of heterozygosity or RAR� epigenetic silencing orboth. Thus, we tested the effect of RAR� knockdown onRAR�2 transcription by using RAR�-specific RNA interfer-ence in T47D cells.

Stable expression in T47D cells of a short hairpin RNA(targeting a sequence common to both RAR�1 and RAR�2;Fig. 5A, top left), selected for efficient knockdown of exoge-nous RAR�1 overexpressed into COS cells (Fig. 5A, bottomleft), silenced the endogenous RAR� in prototypic clones suchas SI� C7 (Fig. 5A, top right). As a result, RA-induced RAR�2transcription (Fig. 5A, bottom right) was abrogated in associ-ation with development of a RAR�2 chromatin status unable tointegrate the RA signal (Fig. 5B). Indeed, RA failed to inducethe level of both Ac-H4 and H3-Ac-K9 up to the threshold(Fig. 5B, left and middle; dotted line) associated with RAR�2transcription. Moreover, SI� C7 chromatin showed a signifi-cantly higher level of H3-Tri-Me-K9 than the control pSC2chromatin (Fig. 5B, right). MSP analysis showed the presenceof M alleles. Bisulfite sequencing of 25 alleles evidenced, as itdid in DN C8, either U alleles or M alleles methylated in justa few CpGs (Fig. 5C). Surprisingly, profound RAR� downregu-lation seems sufficient to create a stable nonpermissive RAR�2

status, apparently marked by an accumulation of histone-re-pressive changes but not always by CpG methylation. We donot know yet how lack of RAR� makes the RAR�2-chromatina “prey” of repressor proteins. Also, in this case, when RAR�2falls into silencing, cells apparently acquire resistance to RA-induced growth inhibition (Fig. 5D).

Induction of RAR�2 epigenetic silencing by RAR� antago-nists. It was reported that the intracellular RA level is lower incancer epithelial cells than in their normal counterparts (17).Thus, we set out to test whether interfering with RA availabil-ity at endogenous RAR� with RAR� antagonists can forceRAR�2 into a deep state of inactivity.

First we tested that the RAR�-specific antagonist ER50891(25) (Fig. 6A, top) was able to significantly inhibit the induc-tion of RAR�2 transcription in response to RA in a time courseexperiment in which T47D cells were grown in the presence ofER50891 (10 �M) alone, RA (0.1 �M) alone, or ER50891 andRA in combination for 4, 8, and 24 h (Fig. 6A, bottom). Byusing ChIP analysis with a RAR�-specific antibody we ob-served that the levels of endogenous RAR� occupancy of theRAR�2 region containing the RARE did not differ significantlyduring the 24 h in cells grown in the presence of the antagonist(Fig. 6B, top). In contrast, we observed that in the same cellsamples the levels of H3-Tri-Me-K9 (hallmark of repressivechromatin) and H3-Ac-K9 (hallmark of active chromatin) as-sociated with RAR�2 DNA increased and decreased, respec-

FIG. 5. Stable RNA interference of RAR� triggers RAR�2 epigenetic silencing and RA resistance. (A) The RAR� targeting sequence (top left),cloned in the pSUPER-retro vector and selected for efficient knockdown of exogenous RAR� (encoded by pSG-hRAR� 1) in COS cells (bottomleft), efficiently knocks down the endogenous RAR� protein in the T47D clone SI� C7 (top right) consistent with significant transcriptionaldownregulation (P � 0.01) (bottom right). (B) Quantitative ChIP analysis showing that RA induces in SI� C7 RAR�2 chromatin significant lowerlevels of both Ac-H4 (P � 0.05) (left) and H3-Ac-K9 (P � 0.05) (middle) than in pS C2 chromatin, well below the threshold (dotted line) requiredfor transcription. The level of H3-Tri-Me-K9 (right) is also significantly higher (P � 0.05) in SI� C7 RAR�2 chromatin than in pS C2 chromatin.(C) MSP (right) and bisulfite sequencing (left) show CpG hypermethylation in a few SI� C7 RAR�2 alleles and the localization of the CpGs.(D) Colony formation assay showing that SI� C7 cells are RA resistant.

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tively (Fig. 6B, bottom two panels). We also detected by bothMSP analysis and bisulfite sequencing the appearance of Malleles (Fig. 6C, top), with methylation emerging once again ina few CpGs in the first exon (Fig. 6C). This experiment sug-gests that upon the RAR� antagonist binding to endogenousRAR�, a few histone and DNA-modifying enzymes might berapidly recruited at RAR�2 to impose repressive changes. Oursupposition is corroborated also by the observation that T47Dcells treated up to 96 h with another RAR� antagonist,RO415253 (10 �M) (1), developed DNA methylation withinthe first 4 h (Fig. 6D). This is consistent with previous reportsshowing that a dominant-negative RAR� mutant transfected

in cells carrying an unmethylated RAR�2 triggered the appear-ance of RAR�2 DNA methylation within a few hours of trans-fection (13). Thus, aberrant RAR�2-repressive changes seemto occur as rapidly as normal chromatin changes (29). How-ever, we do not know yet which chromatin repressor proteinsare recruited and in which order at RAR�2 in response to theRAR� antagonist.

It can be argued that RAR�2 epigenetic silencing was ob-served because of the constant presence in the cell of inductivefactors (such as the dominant-negative RAR�, the RAR�-RNA interference construct, and the RAR� antagonists).

Here we show that RAR�2-repressive chromatin changes are

FIG. 6. Induction of RAR�2 epigenetic silencing by RAR� antagonists. (A and B) (A) Time course analysis showing that the RAR� antagonistER50891 (top) abrogates RA-induced RAR�2 transcription in T47D cells (bottom) concomitant with (B) induction of a significant (P � 0.05)increase of repressive H3-Tri-Me-K9 (middle) and a significant (P � 0.05) decrease of activating H3-Ac-K9 (bottom), while RAR� resides atRAR�2 (top). (C) MSP (top) and bisulfite sequencing (bottom) show CpG methylation already at 4 h after treatment with ER50891. (D) Timecourse analysis of RAR�2 methylation with the RAR� antagonist RO415253, showing rapid occurrence of CpG methylation (MSP, middle;bisulfite sequencing, bottom). (E) Colony formation assay showing that ER50891 can rescue T47D cells from RA-induced growth inhibition (top).MSP analysis of pools of clones derived from cells that survived treatment with ER50891 alone or in combination with RA detected both U andM alleles (bottom). (F) Stability of RAR�2 epigenetic silencing in ER-C6 and ER/RA-C5, showing heterozygosity for U and M alleles (top left),RA resistance (bottom left), lack of RA-induced RAR�2 transcription (top right), and histone H4 hypoacetylation (bottom right).

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retained by T47D cells after removal of the RAR� antagonist.T47D cells treated for up to 24 h with ER50891 (10 �M), aloneor in combination with RA (0.1 �M), were grown in drug-freemedium until we observed the appearance of discrete colonies.A clonogenicity assay showed that ER50891 could rescue cellsfrom RA-induced growth inhibition (Fig. 6E, top). DNA wasextracted by the entire pool of clones that survived treatmentwith ER50891 alone or in combination with RA (for details,see Materials and Methods). MSP showed the presence in bothpools of clones of RAR�2 alleles with and without CpG meth-ylation (Fig. 6E, bottom). MSP analysis of two independentclones that survived ER50891 treatment (clone ER-C6) orcombined ER50891 and RA treatment (clone ER/RA-C5)showed the presence of both U and M alleles (Fig. 6F, topleft). The chromatin associated with the silent RAR�2 alleles inthese clones was marked by histone H4 hypoacetylation (Fig. 6F,bottom right), consistent with lack of RA inducibility of RAR�2transcription (Fig. 6F, top right) and RA resistance (Fig. 6F,bottom left). Apparently, RAR� was no longer bound at RAR�2(S. Pozzi, unpublished observations). We conclude that after re-moval of the inductive factor (RAR� antagonist), the RAR�2-repressive epigenetic changes remain stable in at least some of thecells that were originally exposed to the inductive factor.

RAR�2 reactivation requires restoration of RA signal at asilent RAR�2 through RAR�. Our work, as well as the work ofothers, has shown that treatment of cells carrying a silentRAR�2 with either histone deacetylase inhibitors or demethy-lating agents, alone or in combination, can resensitize an epi-genetically silent RAR�2 to RA (6, 24, 34). A few preliminaryobservations pointed at RAR� as a critical factor for RA-induced RAR�2 reactivation from an epigenetically silent pro-moter. We observed specifically that (i) the occurrence ofreactivation of RA-induced RAR�2 transcription in a RAR�-negative SG5C1 clone (derived from MDA-MB-231) treatedwith TSA (330 nM) and/or 5-Aza (0.8 �M), alone or in com-bination (Fig. 7A, bottom left), was concomitant with reacti-vation of endogenous RAR� (Fig. 7A, top left), (ii) the drug-induced endogenous RAR� was found at the RAR�2 promoterin response to the presence of RA (Fig. 7A, middle), and (iii)RA-induced RAR�2 transcription after treatment with the dif-ferent drugs was abrogated by the RAR� antagonist ER50891(Fig. 7A, right).

Next, we mechanistically tested the involvement of RAR� inthe reactivation of transcription from a silent RAR�2 in anMDA-MB-231 clone expressing exogenous RAR� (RAR�C21) (Fig. 7B, top left). We observed that RAR�2 transcrip-tional reactivation (Fig. 7B, top middle) and recruitment ofRNA polymerase II at RAR�2 (Fig. 7B, top right) in responseto the presence of RA (1 �M, 24 h) occurred with significantreacetylation of histone H4 (Fig. 7B, bottom left). By immu-noprecipitating with anti-RAR� antibodies the reacetylatedchromatin, we found a significantly higher level of RAR�bound at RAR�2 in response to RA (see Re-ChIP panel, Fig.7B, bottom middle). MSP analysis of the RAR�2 DNA immu-noprecipitated by RAR� showed persistence of RAR�2 CpGmethylation (Fig. 7B, bottom right). Apparently, reestablishingthe RA signal at RAR�2 via exogenous RAR� by inducingRAR�2 chromatin reacetylation enabled transcription from themethylated RAR�2.

Interestingly, the level of RA-induced RAR�2 transcription

obtained with exogenous RAR� was comparable to the level ofRA-induced transcription obtained with the two drugs inSG5C1 (Fig. 7C, top left, arrows) and correlated with theincrease of histone H4 reacetylation at RAR�2 up to thethreshold enabling transcription (Fig. 7C, middle left, arrows)rather than with demethylation of RAR�2 DNA (Fig. 7C, bot-tom left). The increment in the level of drug-induced endog-enous RAR� in SG5C1 and drug-induced endogenous plusexogenous RAR� in RAR�C21 at RAR�2 (Fig. 7C, top right)was mirrored by an increment of RNA polymerase II recruit-ment at RAR�2 (Fig. 7C, bottom right). This latter increment,paralleled by the increment in RAR�2 transcription, mightreflect an involvement of RAR�2 itself, which, once inducedby RA, would contribute to its own transcription by a positive-feedback autoregulatory loop (8, 21, 36). However, we werenot able to prove it by ChIP analysis with anti-RAR�2 anti-bodies as we did instead in control T47D cells (data notshown).

Based on the overall findings that we summarized in theschematic diagram in Fig. 7D, RAR� appears to be critical inthe restoration of a permissive transcriptional status at asilent RAR�2.

DISCUSSION

We proposed previously that hormone-regulated genes,whose transcription is normally regulated by dynamic changesof the chromatin status in the presence and absence of thespecific hormone (9, 14, 29, 41), might be ideal models fortesting the etiology of aberrant epigenetic silencing in cancerand aging (33). In particular, we hypothesized that an aberrantchromatin-repressive status, consequent to lack of either RAor a crucial component of the machinery necessary to mediatethe integration of RA signal at RAR�2, could explain whyRAR�2 is so frequently epigenetically silenced in RA-resistantcancer cells (33) and RA-resistant tumors (34).

In the first part of this report we prove that impairing theintegration of RA signal through RAR� at RAR�2 leads toRAR�2 epigenetic silencing. Conversely, we show that reinte-gration of RA-RAR� signaling at a silent RAR�2 leads totranscriptional reactivation. RAR�, the upper regulator ofRAR�2 transcription, is expressed in RA-sensitive cells whereRAR�2 can be induced by RA and is homozygous for unmeth-ylated RAR�2 alleles (Fig. 1). When we interfered with RAsignaling at RAR�2 by three alternative strategies, namely, adominant-negative RAR� lacking the RA-binding domain(Fig. 2), RAR�-RNA interference (Fig. 5), and RAR� antag-onists (Fig. 6) in RA-sensitive cells, we always induced theconversion of RAR�2 alleles permissive for transcription into anonpermissive (unresponsive to RA) status. The chromatin ofnonpermissive alleles was marked by repressive histone mod-ifications (H3-Tri-Me-K9, hypoacetylation of histone H4, andH3-K9), and also by CpG methylation, but in only a fraction ofalleles, which remained unresponsive to RA. RA could notinfluence the level of critical histone modifications to reachwhat we define as the “threshold” of histone modificationsrequired for transcription (as it happens instead in cognatecontrol clones) even when used at a pharmacological concen-tration (1 �M). Remarkably, a nonpermissive RAR�2 statuscan be conferred without CpG methylation. Consistently, we

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identified RA-resistant cells, which were homozygous forRAR�2 unmethylated alleles yet nonpermissive for transcrip-tion in response to RA (Fig. 4).

We further demonstrated that the RAR�2 nonpermissive(nonresponsive to RA) status was stable also in the absence ofthe inductive factor. Specifically, we demonstrated that this isthe case by attenuating the RA signal at RAR�2 with a RAR�antagonist and showing that the RAR�2 nonpermissive sta-tus—marked by repressive histone H4 hypoacetylation and, insome alleles, also CpG methylation—was maintained for along time after the RAR� antagonist was removed (Fig. 6Eand 6F).

It was beyond the scope of this study to show which repres-sor proteins (including histone-modifying enzymes and DNAmethyltransferases) initiated the exacerbation of the RAR�2-repressed status in response to different inductive factors.However, from the initial repressive events induced by either thedominant-negative RAR� or the RAR� antagonist ER50891 wespeculate that both histone and DNA-modifying enzymesmight have been recruited at RAR�2 while there was persistingoccupancy of the promoter by either the dominant-negative

(Fig. 2B) or endogenous wild-type RAR� (Fig. 6B). The orderin which repressive changes at histone and DNA level accu-mulate at gene promoters has been addressed in a few studies(2, 35). In the case of RAR�2, the accumulation of repressivehistone modifications appears to precede CpG methylation.We inferred that this might be the case because we found thatonly a fraction of nonpermissive alleles developed CpG meth-ylation and in only a few CpGs in the first exon (Fig. 2, 5, and6). It is conceivable that this region is the epicenter of CpGmethylation. These findings suggest indirectly that DNMTs arerecruited after other repressive critical proteins. This is be-cause the nonpermissive status can also be achieved in theabsence of CpG methylation.

We also demonstrated that induction of a silent, nonpermis-sive RAR�2 status is indeed the cause of biological RA resis-tance. Directly targeting RAR�2 transcription with a RAR�2antisense in RA-sensitive T47D cells led to the same RA-resistant phenotype (Fig. 3) observed after induction of RAR�2epigenetic silencing with any of the three strategies used tofunctionally inactivate RAR�. In summary, impairment of RAsignal at RAR�2 through RAR� in RA-sensitive cells appears

FIG. 7. Restoring RA signal at a silent RAR�2 through RAR� leads to RAR�2 reactivation. (A) Concomitant reactivation of RAR� (top left)and RAR�2 (bottom left) with TSA and 5-Aza alone or in combination in MDA-MB-231 SG5C1 leads to a significant increase of RAR� bindingat RAR�2 (middle). The RAR� antagonist ER50891 significantly abrogates RA-induced RAR�2 transcription after TSA and 5-Aza treatmentalone (P � 0.05) or in combination (P � 0.001) (right). Ab, antibody. (B) Exogenous expression of RAR� in MDA-MB-231 RAR�C21 (top left)leads to significantly (P � 0.001) higher RA-induced RAR�2 transcription (top middle) concomitant with significant recruitment (P � 0.05) ofRNA polymerase II at RAR�2 (top right) and a significant increase of Ac-H4 (P � 0.05) in the RAR�2 chromatin (bottom left). Apparently RAR�associates significantly (P � 0.05) more with the reacetylated chromatin in the presence of RA (middle bottom). RAR�2 DNA associated withRAR� is methylated (bottom right). (C) Real-time RT-PCR shows that the level of RA-induced RAR�2 transcription after treatment with TSAis significantly (P � 0.01) higher in RAR�C21 than in SG5C1 (top left), and paralleled by a significant (P � 0.05) increase in Ac-H4 in the RAR�2chromatin (middle left), as well as binding of both RAR� and RNA polymerase II at RAR�2 (top right and bottom right, respectively), in theabsence of demethylation of RAR�2 DNA (bottom left). Demethylation was observed only in the samples treated with 5-Aza (bottom left). (D).Schematic diagram based on the findings presented in panels A, B, and C showing that RAR�—either drug-induced endogenous (en-RAR�) orexogenous (ex-RAR�)—plays an active role in reestablishing a RAR�2 status, which is permissive for transcription.

FIG. 8. The critical role of RA-RAR� signaling in RAR�2 epigenetic silencing and RA resistance. (A) Normally, in the absence of RA, RAR�keeps RAR�2 chromatin poised for transcription; upon RA binding at RAR� the chromatin is converted to an active state (14). RAR�2 in turnenhances its own transcription (8, 21, 36). Induction of the RAR�2 tumor suppressor results in cell growth inhibition. (B) Impaired RA signalthrough RAR� at RAR�2 apparently converts RAR�2 chromatin from a poised to a severely repressed, silenced state no longer compatible withtranscription. This state is characterized by epigenetic repressive modifications at the histone and DNA level. The biological consequence of theconversion of RAR�2 into a silent state is the resistance to the growth-inhibitory action of RA. (C) Conversely, reconnecting RA signal at a silentRAR�2 chromatin through RAR� restores a chromatin state again compatible with transcription. RAR�2 reactivation results in the reestablish-ment of responsiveness to the growth-inhibitory action of RA.

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to lead to an exacerbation of the RAR�2 chromatin status,stable silencing, and, ultimately, RA resistance (Fig. 8).

Conversely, we prove that reconnecting RA signal throughan exogenous RAR� at a silent, heavily hypermethylatedRAR�2 in RA-resistant cells results in transcriptional reacti-vation concomitant with RAR�2 chromatin histone H4 reacety-lation but not demethylation (Fig. 7C, left). Interestingly, thelevel of RA-induced RAR�2 reactivation in the presence ofexogenous RAR� was comparable to the level induced withTSA in the presence of endogenous RAR�. Also, in this caseRAR�2 reactivation was achieved without demethylation. Ap-parently, RA binding to RAR�, which is known to activelyrecruit coactivator complexes with histone acetyltransferaseactivity (29), is sufficient to convert RAR�2 from a silent to apermissive state, as we show in the schematic diagram in Fig.7D. A consequence of restoring RA-RAR� signaling atRAR�2 likely is the reactivation of the RAR�2 receptor itself,which is expected to sustain its own transcription (8, 21, 36).Because we did not detect RA-induced RAR�2 reactivationafter treatment with TSA and 5-Aza alone or in combinationwhen we abrogated RAR� function with the RAR� antagonist(Fig. 7A, right), we conclude that RAR�2 can play a role in itsown transcription but only after its transcription is triggered byRAR�, as we show in the schematic diagram in Fig. 7D. Insummary, reintegration of RA signal at an epigenetically silentRAR�2 through RAR� in RA-resistant cells would restore achromatin status enabling transcription of the tumor suppres-sor gene in response to the presence of RA and, consequently,sensitivity to the growth-inhibitory action of RA (Fig. 8).

Lack of functional integration of RA signal at RAR�2through RAR� might occur in vivo. RAR� is not expressed ina high percentage of tumors (31). This leads to the hypothesisthat in vivo RAR�2 silencing can be a consequence of RAR�loss or silencing. In support of this hypothesis, we observedthat reactivation of RAR�2 by TSA and 5-Aza also restoredRAR� (Fig. 7A, left). Interestingly, estrogen receptor alpha(ER�) is often epigenetically silenced in RAR�-negative tu-mors (16). RAR� is regulated, at least in part, by ER� (30).This makes us speculate that epigenetic silencing of a fewhormonally regulated genes could occur in a “domino” fash-ion. We continue to assert (33, 34) that lack of intracellular RAavailability at RAR� could also trigger a repressive epigeneticdomino effect. Recently we found that RAR�2 silencing leadsto the silencing of downstream genes involved in RA metabo-lism, thus suggesting the existence of epigenetic networks (un-published observations).

A few translational considerations come to mind at the endof this report. Detection of RAR�2 hypermethylation, a test forearly breast cancer (3) and other cancers, apparently underes-timates epigenetic RA resistance, because it may miss cellshomozygous for silent, yet unmethylated, RAR�2 alleles (Fig.4). Therapeutic strategies aimed at resensitizing RA-resistantcells to RA by restoring RAR�2 would require drugs powerfulenough to reinduce simultaneously both RAR�2 and criticalRAR�2 regulators. But more than anything else, this reporthighlights the importance of identifying the intrinsic and ex-trinsic factors that in vivo might force genes like RAR�2 intoaberrant or protracted inactivity. Identification of these factorscan lead to novel cancer prevention strategies.

ACKNOWLEDGMENTS

We thank James Herman, David Kowalski, Andre Hoogeveen, andthe anonymous reviewers for helpful suggestions and constructive crit-icism and Ellen Sanders for manuscript editing.

This work was supported by an AIRC grant (Italy) (N.S.), U.S. Armygrant DAMD17-02-01-0432 (N.S.), a Roswell Park Cancer InstituteAlliance grant (N.S.), the Graduate Program of Molecular Medicine,University of Milan (S.P., G.B., and G.S.), and the CISI Center ofExcellence, University of Milan (S.R.).

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