![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, December 2005, p. 10591-10603, Vol. 25, No. 23
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.23.10591-10603.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(RAR). Normally, RAR regulates RARß2 transcription by mediating dynamic changes of RARß2 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 an exacerbation of the repressed chromatin status of RARß2 and leads to RARß2 transcriptional silencing. Further, we demonstrate that RARß2 silencing causes resistance to the growth-inhibitory effect of RA. Apparently, RARß2 silencing can also occur in the absence of DNA methylation. Conversely, we demonstrate that restoration of RA signal at a silent RARß2 through RAR leads to RARß2 reactivation. This report provides proof of principle that RARß2 silencing and RA resistance are consequent to an impaired integration of RA signal at RARß2 chromatin. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RARß2 is an RA-regulated tumor suppressor gene (19, 26, 32). Detection of aberrant RARß2 methylation in tumors of different histotypes raised the question of whether this epigenetic change is critical for silencing this tumor suppressor gene. Previously, we proposed that aberrant RARß2 inactivity might induce repressive epigenetic changes at RARß2, leading to RARß2 silencing and RA resistance (33, 34). RARß2 transcription is normally regulated by dynamic histone changes in the presence and absence of RA (9, 14, 29, 41). Therefore, we hypothesized that the impaired integration of RA signal at RARß2 can create a state of exacerbated-protracted RARß2 transcriptional inactivity and attract chromatin-repressive changes, including DNA methylation. The conversion of RARß2 from a state permissive for transcription into a stable state nonpermissive for transcription would cause biological RA resistance. Our hypothesis hinges on the original supposition of Ng and Bird (28) that chromatin inactivity, the prerequisite for epigenetic silencing of genes on chromosome X (18), could also lead to silencing of genes on other chromosomes. Thus, an aberrant inactive RARß2 chromatin status would be the prerequisite for RARß2 epigenetic silencing.
RARß2 DNA methylation and silencing were shown to be induced by active recruitment of repressor proteins by an oncogenic fusion protein in leukemic cells (13). However, to our knowledge, this oncoprotein has not been demonstrated in epithelial cancer cells and tumors of the breast, prostate, colon, lung, and head and neck, where RARß2 has also been found silenced (33, 34). In contrast, cancer epithelial cells and tumors appear to have either a low intracellular concentration of RA or a lack or derangement of proteins involved in either RA metabolism and homeostasis or RARß2 transcriptional regulation. Thus, RA resistance might be the consequence of an exacerbated-protracted RARß2 transcriptional repression caused by a defective integration of RA signal at RARß2, which might be induced by genetic, epigenetic, metabolic, and environmental factors capable of shutting off the "communication" between RA and RARß2 chromatin.
We identified and tested as a possible cause of aberrant RARß2 inactivity the lack of functional RAR, the upper regulator of RARß2 transcription. RAR has the role of keeping the chromatin of its direct target genes, such as RARß2, poised for transcription yet inactive. Upon binding of RA to RAR, the chromatin status of the target genes is converted from inactive into active because of the exchange of corepressor complexes with coactivator complexes, which would rapidly induce histone changes, chromatin remodeling, and transcription activation (14, 29).
In the course of our studies of RA-resistant breast and prostate cancer cell lines, we observed the following: (i) the presence of low or negligible binding of RAR at RARß2 in RA-resistant breast and prostate cancer epithelial cells carrying RARß2 nonpermissive alleles (we define as nonpermissive the alleles that cannot be transcriptionally activated by RA and as permissive the alleles that are poised for transcription yet inactive in the absence of RA but capable of transcription in the presence of RA); (ii) the presence of RARß2 unmethylated (U), permissive alleles in RAR-positive cells, which contain many other methylated (M) genes (20), pointing at RAR as a critical factor that can spare RARß2 chromatin from falling into a nonpermissive status; and (iii) the presence of a minimal stretch of methylated CpGs in the first RARß2 exon—corresponding to exon 5 of the RARß locus (38)—in methylated alleles, suggesting that CpG methylation originates in a specific epicenter from unmethylated yet nonpermissive alleles.
In this study we simulated possible genetic, epigenetic, and metabolic scenarios that could impair the flow of RA signal at RARß2 chromatin via RAR. Using three different strategies—a dominant-negative RAR lacking the RA-binding domain, downregulation of RAR by RNA interference, and RA antagonists acting specifically at RAR—we induced the conversion of RARß2 permissive alleles into nonpermissive alleles in RA-sensitive human cells. The RARß2 nonpermissive alleles developed a significant load of repressive histone tail modifications and failed to recruit RNA polymerase II at the region containing the transcription start site. Only a percentage of nonpermissive alleles developed CpG hypermethylation, thus showing that aberrant hypermethylation is not an absolute requirement for RARß2 silencing. In this report we also demonstrate that restoring RA signal through RAR at an epigenetically silent RARß2 is critical to reestablishing a RARß2 status compatible with transcription. RARß2 epigenetic silencing has been described as being associated with an RA-resistant phenotype (26, 32). Here we prove that RA resistance is the consequence of RARß2 epigenetic silencing.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 from Sigma (St. Louis, MO). These drugs were dissolved and stored as described previously (34). The RAR antagonist ER50891 was a kind gift of Kouichi Kikuchi, Discovery Research Laboratories, Ibaraki, Japan, and the RAR antagonist RO414253 was a kind gift from Salvatore Toma, National Cancer Institute, Genoa, Italy. Treatments with these drugs were all performed in medium supplemented with 5% charcoal-dextran-stripped serum.
Colony formation assay. Exponentially growing cells were seeded at 5 x 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 was replaced 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 to establish the colony formation index as described previously (34). The statistical significance was calculated by Student's t test for three independent experiments.
Cell transfections.
The RAR dominant-negative mutant RAR403 was subcloned by PCR into the FLAG-containing pCMV-tag vector (Stratagene, La Jolla, CA) with primers introducing EcoRI and XhoI restriction sites (sense, 5'-TATGAATTCATGGCCAGCAACAGCAGCTC-3'; antisense, 5'-ATACTCGAGGGGATCTCCATCTTCAGCGT-3'), and the empty pCMV-tag vector was transfected in T47D by using Lipofectamine Plus (Invitrogen, Carlsbad, CA). The LNasRARß2VI vector, which harbors six copies of RARß2 antisense (asRARß2) and the control empty vector LNSX (kindly provided by S. Y. Sun, University of Texas M.D. Anderson Cancer Center, Houston, TX) (37) were also transfected in T47D by Lipofectamine Plus. Stable clones were selected and maintained with G418 (Invitrogen) at 0.8 mg/ml. Stable MDA-MB-231 clones overexpressing RAR 1 were obtained by cotransfecting cells with pSG5-hRAR 1 vector (kindly provided by Fausto Andreola, National Cancer Institute, Bethesda, MD) and G418-resistant pcDNA3.1(+) vector (Invitrogen) by use of Lipofectamine Plus. Control cells were cotransfected with the empty vector pSG5 (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 LXRAR403SN or the empty LXSN retroviral particles (kindly provided by Fausto Andreola, National Cancer Institute, Bethesda, MD) were used to infect T47D cells in the presence of 4 mg/ml Polybrene (Sigma) as described previously (39). Infected cells were selected with 0.8 mg/ml G418. Single clones were isolated after 14 to 21 days and screened for the presence of either the LXSN or the LXRAR403SN 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 (GenBank accession no. NM_000964) was inserted into pSUPER-retro (OligoEngine, Seattle, WA) according to the manufacturer's instructions. The silencing efficiency of the resulting construct, pSUPER-RAR, was first tested on exogenous RAR by transiently cotransfecting COS-1 cells with pSG5-hRAR1, encoding the human RAR1 and pSUPER-RAR at different ratios. Exogenous RAR levels, normalized to GAPD (glyceraldehyde-3-phosphate dehydrogenase) expression levels, were estimated by Western blotting with the C-20 anti-RAR and anti-GAPD (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies and appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology; Amersham, Piscataway, NJ). Stable transfections with pSUPER-RAR and pSUPER-retro (control) were performed with Lipofectamine Plus. Transfected T47D clones were selected with 2 µg/ml puromycin (Sigma). Clones displaying significant silencing of RAR by real-time RT-PCR and Western blotting were chosen for further analysis.
RAR antagonist experiments.
Cells (5 x 102) were seeded in six-well plates in triplicate in medium containing 5% charcoal-dextran-stripped serum, allowed to attach to the plastic substrate, and treated with ER50891 (10 µM) and RA (0.1 µM), either alone or in combination, for 24 h. After this time, the medium was replaced by drug-free medium. Cells were grown until colonies became visible (14 to 21 days). Colonies were stained with Giemsa to establish the colony formation index (34). Pools of colonies derived from cells that survived each treatment (125 colonies from one well with no drugs, 118 clones from one well treated with ER50891 alone, and 115 clones from one well treated with ER50891 plus RA) were used to isolate genomic DNA that was used for methylation-specific PCR (MSP) analysis with M4 primers and U4 primers. In a parallel replica experiment, we instead used cloning cylinders to isolate six independent clones that survived the treatment with either ER50891 alone (which we designated ER clones) or ER50891 in combination with RA (which we designated ER/RA clones). All clones from each group that were expanded in drug-free medium did not show RA-induced RARß2 transcription by real-time RT-PCR and were shown to be U or M by MSP. One clone from each of the groups ER-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 SuperScript first-strand synthesis system (Invitrogen), and amplified by real-time RT-PCR on an iCycler apparatus (Bio-Rad, Hercules, CA) by using iQ SYBR green Supermix (Bio-Rad) and specific primers for RARß2 (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 standard protocols. The RAR and RARß2 transcription levels were normalized to the GAPD transcription level. The statistical significance was calculated using Student's t test for three independent determinations.
Luciferase assay. The luciferase reporter assay was performed essentially as described previously using the luciferase reporter vector RARß2-pGL2 (12) (kindly donated by K. Ozato, National Institutes of Health, Bethesda, MD) and the control vector pRL-TK (Promega). Vector DNAs were cotransfected with Lipofectamine Plus in cells grown in a 12-well plate; 24 h after transfection, the medium was replaced with medium with or without RA (1.0 µM). Luciferase activity 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 performed in triplicate.
ChIP.
Quantitative chromatin immunoprecipitation (ChIP) was performed using reagents purchased from Upstate (Lake Placid, NY) following the manufacturer's protocol. Chromatin was immunoprecipitated with antibodies against acetyl-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) (1:400), trimethyl-K9 histone H3 (H3-Tri-Me-K9) (Upstate) (1:200), RAR C terminus (Santa Cruz Biotechnology) (1:60), RAR N terminus (Biolegend, San Diego, CA) (1:100), RARß (Santa Cruz Biotechnology [1:70]; Active Motif, Carlsbad, CA [1:600]), RNA polymerase II (Upstate) (1:200), and FLAG epitope (Sigma) (1:350). Re-ChIP was performed with the anti-RAR C-terminus antibody on chromatin immunoprecipitated with an anti-Ac-H4 antibody after elution with re-ChIP elution buffer (10 mM EDTA, 50 mM Tris-HCl [pH 8.0], 0.7 M NaCl, 20 mM dithiothreitol). Control ChIPs were without the respective antibodies. The immunoprecipitated DNA was amplified by real-time PCR with either RARß2 primers (sense, 5'-GGTTCACCGAAAGTTCACTCGCAT-3'; antisense, 5'-CAGGCTTGCTCGGCCAATCCA-3') or the GAPD primers (sense, 5'-GGTGCGTGCCCAGTTGAACCA-3'; antisense, 5'-AAAGAAGATGCGGCTGACTGTCGAA-3'). The RARß2 DNA relative enrichment was calculated by normalizing the RARß2 PCR signal to the PCR signals obtained both from the input DNA (total chromatin fraction) and the GAPD DNA. Statistical significance was determined using the Student's t test on three independent determinations.
DNA methylation analysis. Genomic DNA extracted with DNAzol (Invitrogen) was modified with sodium bisulfite as described previously (33). Modified DNA was used for both MSP and sequencing analyses. For sequencing analysis, a 635-bp region, encompassing 27 RARß2 CpG sites in the RARß2-regulatory region, was amplified by nested PCR (40) using first the primer set RARß2 sense 1 (5'-GTATAGAGGAATTTAAAGTGTGGGTTGGG-3') and RARß2 antisense 1 (5'-CCTATAATTAATCCAAATAATCATTTACC-3') and subsequently the primer set RARß2 sense 2 (5'-GTAGG(C/T)GGAATATTGTTTTTTAAGTTAAG-3') and RARß2 antisense 2 (5'-AATCATTTACCATTTTCCAAACTTACTC-3'). The PCR products were either directly sequenced or sequenced after subcloning in the pCR4-TOPO plasmid vector (Invitrogen). In the latter case, we sequenced a minimum of 20 to a maximum of 50 clones corresponding to the sample specified in Results. MSP was performed by amplifying bisulfite-modified DNA with the first RARß2 primer set (see above). Then, 2 µl of a 1:1,000 dilution of the first amplification product was reamplified with the U4 sense- and U4 antisense-specific primers or with the M4 sense- and M4 antisense-specific primers (33) or with the different combinations of U4 and M4 primers. RARß2 alleles detected with both U4 primers were classified as U, and the alleles amplified with either both M4 primers (these products are the ones discussed in Results) or the sense M4 primer and the U4 antisense primer were classified as M alleles. No product was amplified with the U4 sense primer and the M4 antisense primer.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
correlates with RARß2 epigenetic silencing in RA-resistant cancer cells.
Analysis of a panel of breast and prostate cell lines showed different RA-induced RARß2 transcription levels in correlation with differential growth inhibition by RA after 24 h of treatment (Fig. 1A, top and bottom, respectively). DNA methylation analysis using two complementary techniques, bisulfite sequencing and MSP, identified cell lines (i) homozygous for RARß2 M alleles (MCF7, MDA-MB-231, and LNCaP carry 100% of M alleles), (ii) homozygous for RARß2 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 independent DU145 RARß2 alleles, 30% were U alleles and 70% were M alleles. The distribution of methylated CpGs in the M alleles of all cell lines involved at least a common stretch of CpGs in the first RARß2 exon (Fig. 1B, bottom). Lack of RA-induced RARß2 transcription was also detected in cells (DU145) with U alleles, which must therefore be interpreted as nonpermissive. Moreover, RARß2 was spared by methylation in T47D, a cell line with several other hypermethylated genes (20).
|
Normally, RAR bound to RARß2 keeps the chromatin poised for transcription (yet inactive) (14) (Fig. 1D, top left) and ready for activation by RA (Fig. 1D, top right). RA binding to RAR would induce histone modifications capable of activating RARß2 very rapidly (29). Once RARß2 is induced, it could regulate its own transcription (8). We observed that the presence of RARß2 nonpermissive alleles (regardless of their DNA methylation status) always correlated with a lower level of RAR transcription (Fig. 1D, bottom left). Consistent with this observation, quantitative ChIP with anti-RAR antibodies, followed by RARß2 DNA amplification of the region encompassing the RARE (schematic diagram in Fig. 1B), detected significantly more RAR associated with RARß2 in T47D cells than in MDA-MB-231 and DU145 cells (Fig. 1D, bottom right). Of note, by ChIP with anti-RARß antibodies we found RARß2 binding only at the T47D RARß2 promoter (data not shown).
Altogether, these observations made us hypothesize that a defective integration of RA signal at RARß2 due to lack of functional RAR can convert RARß2 into inactivity, marked by repressive epigenetic changes at histone and DNA level, and RA resistance.
Induction of RARß2 epigenetic silencing by a dominant-negative RAR in RA-sensitive cells.
First, we simulated a genetic scenario whereby an RAR mutation with dominant-negative features occurs in RA-sensitive, RAR-positive cells homozygous for RARß2-permissive alleles. We used a well-characterized dominant-negative RAR mutant, RAR403, which lacks the C-terminal RA-binding domain but retains the capacity to heterodimerize with the retinoid X receptor and bind to the RARE regions (Fig. 2A) (11, 15, 39). RAR 403 should compete with wild-type RAR in the heterodimerization with retinoid X receptor (23). It might also cause an impaired turnover of corepressor complexes at RAR target genes, because it lacks the C terminus (27, 29).
|
403 construct, T47D breast cancer cells (which express mainly endogenous RAR1; data not shown) showed the presence of the dominant-negative mutant at the RARß2 promoter (ChIP; Fig. 2B, bottom left), concomitant with significant RARß2 transcriptional downregulation in response to the presence of RA (Fig. 2B, bottom middle). Thus, RARß2 silencing seems to be initiated when RAR403 resides at the RARß2 promoter region. The dominant-negative RAR was also found at the RARß2 of a T47D clone expressing the dominant-negative protein (DN C8). This was deduced on the basis of a ChIP experiment with an antibody directed against the RAR C terminus; this antibody detected remarkably less wild-type RAR at RARß2 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 RARß2 promoter-luciferase construct (Fig. 2C, left), thus proving the dominant-negative effect of RAR403 on endogenous RAR. Finally, in response to the presence of RA, DN C8 did not show endogenous RARß2 transcription (real-time RT-PCR; Fig. 2C, middle). This finding was mirrored by a lack of RNA polymerase II at RARß2 (Fig. 2C, right). The repressed transcriptional status in the DN C8 clone was paralleled by repressive quantitative and qualitative histone modifications, which, in response to RA treatment, did not reach the threshold necessary for "switching on" RARß2 transcription (Fig. 2D; dotted line). Interestingly, MSP analysis of DN C8 RARß2 DNA with M4 and U4 primers (see Materials and Methods) showed U and M alleles. After bisulfite sequencing of 50 alleles, we found that DN C8 contained 60% U and 40% M alleles with a few methylated CpGs (Fig. 2E). In contrast to what was reported for cells carrying another dominant-negative RAR with an intact RA-binding domain (13), RA treatment (1 µM, 72 h) did not reverse RARß2 DNA methylation. Only treatment with 5-Aza (0.8 µM for 72 h) led to demethylation and RA-induced RARß2 transcription in DN C8 (data not shown).
RARß2 epigenetic silencing confers an RA-resistant phenotype.
Concomitant with RARß2 epigenetic silencing, DN C8 cells developed stable resistance to the growth-inhibitory effect of RA, as assessed by colony formation (Fig. 3A). Because RAR controls several RA-responsive target genes, we tested whether the observed RA resistance phenotype was indeed caused by RARß2 silencing. To this end we targeted T47D RARß2 transcription with an antisense RARß2 (37). Stable T47D clones transfected with LNasRARß2VI that carry multiple copies of the RARß2 antisense, like the As-C3 and As-C4 clones (where "As" represents "antisense") (Fig. 3B, left), were significantly less sensitive to RA-induced growth inhibition than the cognate control clone EV-C3 carrying the empty vector LNSX (Fig. 3B, right). Thus, it is conceivable that the RA-resistant phenotype developed by DN C8 cells is the consequence of RARß2 epigenetic silencing.
|
|
by RNA interference also triggers RARß2 epigenetic silencing and RA resistance.
It can be argued that RARß2 silencing in DN C8 cells is due to the recruitment of histone-modifying enzymes or DNA methyltransferases (DNMTs) at RARß2 by the RAR403 protein, as shown for the PML-RAR protein (13). While nonrandom dominant-negative RAR mutations were never to our knowledge reported in cancer epithelial cells, RAR expression was reported to be low or absent in these cells (30). This could be due to loss of heterozygosity or RAR epigenetic silencing or both. Thus, we tested the effect of RAR knockdown on RARß2 transcription by using RAR-specific RNA interference in T47D cells.
Stable expression in T47D cells of a short hairpin RNA (targeting a sequence common to both RAR1 and RAR2; Fig. 5A, top left), selected for efficient knockdown of exogenous RAR1 overexpressed into COS cells (Fig. 5A, bottom left), silenced the endogenous RAR in prototypic clones such as SI C7 (Fig. 5A, top right). As a result, RA-induced RARß2 transcription (Fig. 5A, bottom right) was abrogated in association with development of a RARß2 chromatin status unable to integrate the RA signal (Fig. 5B). Indeed, RA failed to induce the level of both Ac-H4 and H3-Ac-K9 up to the threshold (Fig. 5B, left and middle; dotted line) associated with RARß2 transcription. Moreover, SI C7 chromatin showed a significantly higher level of H3-Tri-Me-K9 than the control pSC2 chromatin (Fig. 5B, right). MSP analysis showed the presence of M alleles. Bisulfite sequencing of 25 alleles evidenced, as it did in DN C8, either U alleles or M alleles methylated in just a few CpGs (Fig. 5C). Surprisingly, profound RAR downregulation seems sufficient to create a stable nonpermissive RARß2 status, apparently marked by an accumulation of histone-repressive changes but not always by CpG methylation. We do not know yet how lack of RAR makes the RARß2-chromatin a "prey" of repressor proteins. Also, in this case, when RARß2 falls into silencing, cells apparently acquire resistance to RA-induced growth inhibition (Fig. 5D).
|
antagonists.
It was reported that the intracellular RA level is lower in cancer epithelial cells than in their normal counterparts (17). Thus, we set out to test whether interfering with RA availability at endogenous RAR with RAR antagonists can force RARß2 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 induction of RARß2 transcription in response to RA in a time course experiment in which T47D cells were grown in the presence of ER50891 (10 µM) alone, RA (0.1 µM) alone, or ER50891 and RA in combination for 4, 8, and 24 h (Fig. 6A, bottom). By using ChIP analysis with a RAR-specific antibody we observed that the levels of endogenous RAR occupancy of the RARß2 region containing the RARE did not differ significantly during the 24 h in cells grown in the presence of the antagonist (Fig. 6B, top). In contrast, we observed that in the same cell samples the levels of H3-Tri-Me-K9 (hallmark of repressive chromatin) and H3-Ac-K9 (hallmark of active chromatin) associated with RARß2 DNA increased and decreased, respectively (Fig. 6B, bottom two panels). We also detected by both MSP analysis and bisulfite sequencing the appearance of M alleles (Fig. 6C, top), with methylation emerging once again in a few CpGs in the first exon (Fig. 6C). This experiment suggests that upon the RAR antagonist binding to endogenous RAR, a few histone and DNA-modifying enzymes might be rapidly recruited at RARß2 to impose repressive changes. Our supposition is corroborated also by the observation that T47D cells treated up to 96 h with another RAR antagonist, RO415253 (10 µM) (1), developed DNA methylation within the first 4 h (Fig. 6D). This is consistent with previous reports showing that a dominant-negative RAR mutant transfected in cells carrying an unmethylated RARß2 triggered the appearance of RARß2 DNA methylation within a few hours of transfection (13). Thus, aberrant RARß2-repressive changes seem to occur as rapidly as normal chromatin changes (29). However, we do not know yet which chromatin repressor proteins are recruited and in which order at RARß2 in response to the RAR antagonist.
|
, the RAR-RNA interference construct, and the RAR antagonists).
Here we show that RARß2-repressive chromatin changes are retained by T47D cells after removal of the RAR antagonist. T47D cells treated for up to 24 h with ER50891 (10 µM), alone or in combination with RA (0.1 µM), were grown in drug-free medium until we observed the appearance of discrete colonies. A clonogenicity assay showed that ER50891 could rescue cells from RA-induced growth inhibition (Fig. 6E, top). DNA was extracted by the entire pool of clones that survived treatment with ER50891 alone or in combination with RA (for details, see Materials and Methods). MSP showed the presence in both pools of clones of RARß2 alleles with and without CpG methylation (Fig. 6E, bottom). MSP analysis of two independent clones that survived ER50891 treatment (clone ER-C6) or combined ER50891 and RA treatment (clone ER/RA-C5) showed the presence of both U and M alleles (Fig. 6F, top left). The chromatin associated with the silent RARß2 alleles in these clones was marked by histone H4 hypoacetylation (Fig. 6F, bottom right), consistent with lack of RA inducibility of RARß2 transcription (Fig. 6F, top right) and RA resistance (Fig. 6F, bottom left). Apparently, RAR was no longer bound at RARß2 (S. Pozzi, unpublished observations). We conclude that after removal of the inductive factor (RAR antagonist), the RARß2-repressive epigenetic changes remain stable in at least some of the cells that were originally exposed to the inductive factor.
RARß2 reactivation requires restoration of RA signal at a silent RARß2 through RAR.
Our work, as well as the work of others, has shown that treatment of cells carrying a silent RARß2 with either histone deacetylase inhibitors or demethylating agents, alone or in combination, can resensitize an epigenetically silent RARß2 to RA (6, 24, 34). A few preliminary observations pointed at RAR as a critical factor for RA-induced RARß2 reactivation from an epigenetically silent promoter. We observed specifically that (i) the occurrence of reactivation of RA-induced RARß2 transcription in a RAR-negative SG5C1 clone (derived from MDA-MB-231) treated with TSA (330 nM) and/or 5-Aza (0.8 µM), alone or in combination (Fig. 7A, bottom left), was concomitant with reactivation of endogenous RAR (Fig. 7A, top left), (ii) the drug-induced endogenous RAR was found at the RARß2 promoter in response to the presence of RA (Fig. 7A, middle), and (iii) RA-induced RARß2 transcription after treatment with the different drugs was abrogated by the RAR antagonist ER50891 (Fig. 7A, right).
|
in the reactivation of transcription from a silent RARß2 in an MDA-MB-231 clone expressing exogenous RAR (RAR C21) (Fig. 7B, top left). We observed that RARß2 transcriptional reactivation (Fig. 7B, top middle) and recruitment of RNA polymerase II at RARß2 (Fig. 7B, top right) in response to the presence of RA (1 µM, 24 h) occurred with significant reacetylation of histone H4 (Fig. 7B, bottom left). By immunoprecipitating with anti-RAR antibodies the reacetylated chromatin, we found a significantly higher level of RAR bound at RARß2 in response to RA (see Re-ChIP panel, Fig. 7B, bottom middle). MSP analysis of the RARß2 DNA immunoprecipitated by RAR showed persistence of RARß2 CpG methylation (Fig. 7B, bottom right). Apparently, reestablishing the RA signal at RARß2 via exogenous RAR by inducing RARß2 chromatin reacetylation enabled transcription from the methylated RARß2.
Interestingly, the level of RA-induced RARß2 transcription obtained with exogenous RAR was comparable to the level of RA-induced transcription obtained with the two drugs in SG5C1 (Fig. 7C, top left, arrows) and correlated with the increase of histone H4 reacetylation at RARß2 up to the threshold enabling transcription (Fig. 7C, middle left, arrows) rather than with demethylation of RARß2 DNA (Fig. 7C, bottom left). The increment in the level of drug-induced endogenous RAR in SG5C1 and drug-induced endogenous plus exogenous RAR in RARC21 at RARß2 (Fig. 7C, top right) was mirrored by an increment of RNA polymerase II recruitment at RARß2 (Fig. 7C, bottom right). This latter increment, paralleled by the increment in RARß2 transcription, might reflect an involvement of RARß2 itself, which, once induced by RA, would contribute to its own transcription by a positive-feedback autoregulatory loop (8, 21, 36). However, we were not able to prove it by ChIP analysis with anti-RARß2 antibodies as we did instead in control T47D cells (data not shown).
Based on the overall findings that we summarized in the schematic diagram in Fig. 7D, RAR appears to be critical in the restoration of a permissive transcriptional status at a silent RARß2.
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the first part of this report we prove that impairing the integration of RA signal through RAR at RARß2 leads to RARß2 epigenetic silencing. Conversely, we show that reintegration of RA-RAR signaling at a silent RARß2 leads to transcriptional reactivation. RAR, the upper regulator of RARß2 transcription, is expressed in RA-sensitive cells where RARß2 can be induced by RA and is homozygous for unmethylated RARß2 alleles (Fig. 1). When we interfered with RA signaling at RARß2 by three alternative strategies, namely, a dominant-negative RAR lacking the RA-binding domain (Fig. 2), RAR-RNA interference (Fig. 5), and RAR antagonists (Fig. 6) in RA-sensitive cells, we always induced the conversion of RARß2 alleles permissive for transcription into a nonpermissive (unresponsive to RA) status. The chromatin of nonpermissive alleles was marked by repressive histone modifications (H3-Tri-Me-K9, hypoacetylation of histone H4, and H3-K9), and also by CpG methylation, but in only a fraction of alleles, which remained unresponsive to RA. RA could not influence the level of critical histone modifications to reach what we define as the "threshold" of histone modifications required for transcription (as it happens instead in cognate control clones) even when used at a pharmacological concentration (1 µM). Remarkably, a nonpermissive RARß2 status can be conferred without CpG methylation. Consistently, we identified RA-resistant cells, which were homozygous for RARß2 unmethylated alleles yet nonpermissive for transcription in response to RA (Fig. 4).
We further demonstrated that the RARß2 nonpermissive (nonresponsive to RA) status was stable also in the absence of the inductive factor. Specifically, we demonstrated that this is the case by attenuating the RA signal at RARß2 with a RAR antagonist and showing that the RARß2 nonpermissive status—marked by repressive histone H4 hypoacetylation and, in some alleles, also CpG methylation—was maintained for a long time after the RAR antagonist was removed (Fig. 6E and 6F).
It was beyond the scope of this study to show which repressor proteins (including histone-modifying enzymes and DNA methyltransferases) initiated the exacerbation of the RARß2-repressed status in response to different inductive factors. However, from the initial repressive events induced by either the dominant-negative RAR or the RAR antagonist ER50891 we speculate that both histone and DNA-modifying enzymes might have been recruited at RARß2 while there was persisting occupancy of the promoter by either the dominant-negative (Fig. 2B) or endogenous wild-type RAR (Fig. 6B). The order in which repressive changes at histone and DNA level accumulate at gene promoters has been addressed in a few studies (2, 35). In the case of RARß2, the accumulation of repressive histone modifications appears to precede CpG methylation. We inferred that this might be the case because we found that only a fraction of nonpermissive alleles developed CpG methylation and in only a few CpGs in the first exon (Fig. 2, 5, and 6). It is conceivable that this region is the epicenter of CpG methylation. These findings suggest indirectly that DNMTs are recruited after other repressive critical proteins. This is because the nonpermissive status can also be achieved in the absence of CpG methylation.
We also demonstrated that induction of a silent, nonpermissive RARß2 status is indeed the cause of biological RA resistance. Directly targeting RARß2 transcription with a RARß2 antisense in RA-sensitive T47D cells led to the same RA-resistant phenotype (Fig. 3) observed after induction of RARß2 epigenetic silencing with any of the three strategies used to functionally inactivate RAR. In summary, impairment of RA signal at RARß2 through RAR in RA-sensitive cells appears to lead to an exacerbation of the RARß2 chromatin status, stable silencing, and, ultimately, RA resistance (Fig. 8).
|
at a silent, heavily hypermethylated RARß2 in RA-resistant cells results in transcriptional reactivation concomitant with RARß2 chromatin histone H4 reacetylation but not demethylation (Fig. 7C, left). Interestingly, the level of RA-induced RARß2 reactivation in the presence of exogenous RAR was comparable to the level induced with TSA in the presence of endogenous RAR. Also, in this case RARß2 reactivation was achieved without demethylation. Apparently, RA binding to RAR, which is known to actively recruit coactivator complexes with histone acetyltransferase activity (29), is sufficient to convert RARß2 from a silent to a permissive state, as we show in the schematic diagram in Fig. 7D. A consequence of restoring RA-RAR signaling at RARß2 likely is the reactivation of the RARß2 receptor itself, which is expected to sustain its own transcription (8, 21, 36). Because we did not detect RA-induced RARß2 reactivation after treatment with TSA and 5-Aza alone or in combination when we abrogated RAR function with the RAR antagonist (Fig. 7A, right), we conclude that RARß2 can play a role in its own transcription but only after its transcription is triggered by RAR, as we show in the schematic diagram in Fig. 7D. In summary, reintegration of RA signal at an epigenetically silent RARß2 through RAR in RA-resistant cells would restore a chromatin status enabling transcription of the tumor suppressor 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 RARß2 through RAR might occur in vivo. RAR is not expressed in a high percentage of tumors (31). This leads to the hypothesis that in vivo RARß2 silencing can be a consequence of RAR loss or silencing. In support of this hypothesis, we observed that reactivation of RARß2 by TSA and 5-Aza also restored RAR (Fig. 7A, left). Interestingly, estrogen receptor alpha (ER) is often epigenetically silenced in RAR-negative tumors (16). RAR is regulated, at least in part, by ER (30). This makes us speculate that epigenetic silencing of a few hormonally regulated genes could occur in a "domino" fashion. We continue to assert (33, 34) that lack of intracellular RA availability at RAR could also trigger a repressive epigenetic domino effect. Recently we found that RARß2 silencing leads to the silencing of downstream genes involved in RA metabolism, thus suggesting the existence of epigenetic networks (unpublished observations).
A few translational considerations come to mind at the end of this report. Detection of RARß2 hypermethylation, a test for early breast cancer (3) and other cancers, apparently underestimates epigenetic RA resistance, because it may miss cells homozygous for silent, yet unmethylated, RARß2 alleles (Fig. 4). Therapeutic strategies aimed at resensitizing RA-resistant cells to RA by restoring RARß2 would require drugs powerful enough to reinduce simultaneously both RARß2 and critical RARß2 regulators. But more than anything else, this report highlights the importance of identifying the intrinsic and extrinsic factors that in vivo might force genes like RARß2 into aberrant or protracted inactivity. Identification of these factors can lead to novel cancer prevention strategies.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by an AIRC grant (Italy) (N.S.), U.S. Army grant DAMD17-02-01-0432 (N.S.), a Roswell Park Cancer Institute Alliance grant (N.S.), the Graduate Program of Molecular Medicine, University of Milan (S.P., G.B., and G.S.), and the CISI Center of Excellence, University of Milan (S.R.).
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Bachman, K. E., B. H. Park, I. Rhee, H. Rajagopalan, J. G. Herman, S. B. Baylin, K. W. Kinzler, and B. Vogelstein. 2003. Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene. Cancer Cell 3:89-95.[CrossRef][Medline]
3. Bean, G. R., V. Scott, L. Yee, B. Ratliff-Daniel, M. M. Troch, P. Seo, M. L. Bowie, P. K. Marcom, J. Slade, B. F. Kimler, C. J. Fabian, C. M. Zalles, G. Broadwater, J. C. Baker, Jr., L. G. Wilke, and V. L. Seewaldt. 2005. Retinoic acid receptor-beta2 promoter methylation in random periareolar fine needle aspiration. Cancer Epidemiol. Biomarkers Prev. 14:790-798.
4. Berger, S. L. 2002. Histone modifications in transcriptional regulation. Curr. Opin. Genet. Dev. 12:142-148.[CrossRef][Medline]
5. Bovenzi, V., N. L. Le, S. Cote, D. Sinnett, L. F. Momparler, and R. L. Momparler. 1999. DNA methylation of retinoic acid receptor beta in breast cancer and possible therapeutic role of 5-aza-2'-deoxycytidine. Anticancer Drugs 10:471-476.[Medline]
6. Cameron, E. E., K. E. Bachman, S. Myohanen, J. G. Herman, and S. B. Baylin. 1999. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21:103-107.[CrossRef][Medline]
7. Chambon, P. 1996. A decade of molecular biology of retinoic acid receptors. FASEB J. 10:940-954.[Abstract]
8. Chiba, H., J. Clifford, D. Metzger, and P. Chambon. 1997. Distinct retinoid X receptor-retinoic acid receptor heterodimers are differentially involved in the control of expression of retinoid target genes in F9 embryonal carcinoma cells. Mol. Cell. Biol. 17:3013-3020.[Abstract]
9. Collingwood, T. N., F. D. Urnov, and A. P. Wolffe. 1999. Nuclear receptors: coactivators, corepressors and chromatin remodeling in the control of transcription. J. Mol. Endocrinol. 23:255-275.[Abstract]
10. Creusot, F., G. Acs, and J. K. Christman. 1982. Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2'-deoxycytidine. J. Biol. Chem. 257:2041-2048.[Medline]
11. Damm, K., R. A. Heyman, K. Umesono, and R. M. Evans. 1993. Functional inhibition of retinoic acid response by dominant negative retinoic acid receptor mutants. Proc. Natl. Acad. Sci. USA 90:2989-2993.
12. Dey, A., S. Minucci, and K. Ozato. 1994. Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo. Mol. Cell. Biol. 14:8191-8201.
13. Di Croce, L., V. A. Raker, M. Corsaro, F. Fazi, M. Fanelli, M. Faretta, F. Fuks, C. F. Lo, T. Kouzarides, C. Nervi, S. Minucci, and P. G. Pelicci. 2002. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 295:1079-1082.
14. Dilworth, F. J., and P. Chambon. 2001. Nuclear receptors coordinate the activities of chromatin remodeling complexes and coactivators to facilitate initiation of transcription. Oncogene 20:3047-3054.[CrossRef][Medline]
15. Durand, B., M. Saunders, C. Gaudon, B. Roy, R. Losson, and P. Chambon. 1994. Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO J. 13:5370-5382.[Medline]
16. Ferguson, A. T., R. G. Lapidus, S. B. Baylin, and N. E. Davidson. 1995. Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. Cancer Res. 55:2279-2283.
17. Guo, X., A. Ruiz, R. R. Rando, D. Bok, and L. J. Gudas. 2000. Esterification of all-trans-retinol in normal human epithelial cell strains and carcinoma lines from oral cavity, skin and breast: reduced expression of lecithin:retinol acyltransferase in carcinoma lines. Carcinogenesis 21:1925-1933.
18. Heard, E., P. Clerc, and P. Avner. 1997. X-chromosome inactivation in mammals. Annu. Rev. Genet. 31:571-610.[CrossRef][Medline]
19. Houle, B., C. Rochette-Egly, and W. E. Bradley. 1993. Tumor-suppressive effect of the retinoic acid receptor beta in human epidermoid lung cancer cells. Proc. Natl. Acad. Sci. USA 90:985-989.
20. Huang, T. H., M. R. Perry, and D. E. Laux. 1999. Methylation profiling of CpG islands in human breast cancer cells. Hum. Mol. Genet. 8:459-470.
21. Husmann, M., J. Lehmann, B. Hoffmann, T. Hermann, M. Tzukerman, and M. Pfahl. 1991. Antagonism between retinoic acid receptors. Mol. Cell. Biol. 11:4097-4103.
22. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science 293:1074-1080.
23. Johnson, B. S., R. A. Chandraratna, R. A. Heyman, E. A. Allegretto, L. Mueller, and S. J. Collins. 1999. Retinoid X receptor (RXR) agonist-induced activation of dominant-negative RXR-retinoic acid receptor 403 heterodimers is developmentally regulated during myeloid differentiation. Mol. Cell. Biol. 19:3372-3382.
24. Jones, P. A., and S. B. Baylin. 2002. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3:415-428.[Medline]
25. Kikuchi, K., K. Tagami, S. Hibi, H. Yoshimura, N. Tokuhara, K. Tai, T. Hida, T. Yamauchi, and M. Nagai. 2001. Syntheses and evaluation of quinoline derivatives as novel retinoic acid receptor alpha antagonists. Bioorg. Med. Chem. Lett. 11:1215-1218.[CrossRef][Medline]
26. Liu, Y., M. O. Lee, H. G. Wang, Y. Li, Y. Hashimoto, M. Klaus, J. C. Reed, and X. Zhang. 1996. Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells. Mol. Cell. Biol. 16:1138-1149.[Abstract]
27. Molinari, E., M. Gilman, and S. Natesan. 1999. Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo. EMBO J. 18:6439-6447.[CrossRef][Medline]
28. Ng, H. H., and A. Bird. 1999. DNA methylation and chromatin modification. Curr. Opin. Genet. Dev. 9:158-163.[CrossRef][Medline]
29. Perissi, V., A. Aggarwal, C. K. Glass, D. W. Rose, and M. G. Rosenfeld. 2004. A corepressor/coactivator exchange complex required for transcriptional activation by nuclear receptors and other regulated transcription factors. Cell 116:511-526.[CrossRef][Medline]
30. Rishi, A. K., Z. M. Shao, R. G. Baumann, X. S. Li, M. S. Sheikh, S. Kimura, N. Bashirelahi, and J. A. Fontana. 1995. Estradiol regulation of the human retinoic acid receptor alpha gene in human breast carcinoma cells is mediated via an imperfect half-palindromic estrogen response element and Sp1 motifs. Cancer Res. 55:4999-5006.
31. Roman, S. D., C. J. Ormandy, D. L. Manning, R. W. Blamey, R. I. Nicholson, R. L. Sutherland, and C. L. Clarke. 1993. Estradiol induction of retinoic acid receptors in human breast cancer cells. Cancer Res. 53:5940-5945.
32. Seewaldt, V. L., B. S. Johnson, M. B. Parker, S. J. Collins, and K. Swisshelm. 1995. Expression of retinoic acid receptor beta mediates retinoic acid-induced growth arrest and apoptosis in breast cancer cells. Cell Growth Differ. 6:1077-1088.[Abstract]
33. Sirchia, S. M., A. T. Ferguson, E. Sironi, S. Subramanyan, R. Orlandi, S. Sukumar, and N. Sacchi. 2000. Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor beta2 promoter in breast cancer cells. Oncogene 19:1556-1563.[CrossRef][Medline]
34. Sirchia, S. M., M. Ren, R. Pili, E. Sironi, G. Somenzi, R. Ghidoni, S. Toma, G. Nicolo, and N. Sacchi. 2002. Endogenous reactivation of the RARbeta2 tumor suppressor gene epigenetically silenced in breast cancer. Cancer Res. 62:2455-2461.
