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Cancer Research 67, 7731, August 15, 2007. doi: 10.1158/0008-5472.CAN-07-1476
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Epigenetic Modulation of Estrogen Receptor-{alpha} by pRb Family Proteins: A Novel Mechanism in Breast Cancer

Marcella Macaluso1,2,3, Micaela Montanari1,2, Paul Bart Noto1, Valter Gregorio1,3, Christian Bronner4 and Antonio Giordano1,2

1 Sbarro Institute for Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania; 2 Department of Human Pathology and Oncology, University of Siena, Siena, Italy; 3 Section of Oncology, Department of Oncology, University of Palermo, Palermo, Italy; and 4 Département Pharmacologie et Physicochimie, Faculté de Pharmacie, Institut Gilbert-Laustriat, UMR 7175-LC1 Centre National de la Recherche Scientifique/Université Louis Pasteur (Strasbourg I), Illkirch, France

Requests for reprints: Marcella Macaluso, Sbarro Institute for Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA 19122. Phone: 215-204-9523; Fax: 215-204-9522; E-mail: macaluso{at}temple.edu or Antonio Giordano, Sbarro Institute for Cancer Research and Molecular Medicine, Center of Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA 19122. Phone: 215-204-9520; Fax: 215-204-9522; E-mail: Giordano{at}temple.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogen receptor-{alpha} (ER-{alpha}) plays a crucial role in normal breast development and has also been linked to mammary carcinogenesis and clinical outcome in breast cancer patients. However, ER-{alpha} gene expression can change during the course of disease and, consequently, therapy resistance can occur. The molecular mechanism governing ER-{alpha} transcriptional activity and/or silencing is still unclear. Here, we showed that the presence of a specific pRb2/p130 multimolecular complex on the ER-{alpha} promoter strongly correlates with the methylation status of this gene. Furthermore, we suggested that pRb2/p130 could cooperate with ICBP90 (inverted CCAAT box binding protein of 90 kDa) and DNA methyltransferases in maintaining a specific methylation pattern of ER-{alpha} gene. The sequence of epigenetic events for establishing and maintaining the silenced state of ER-{alpha} gene can be locus- or pathway- specific, and the local remodeling of ER-{alpha} chromatin structure by pRb2/p130 multimolecular complexes may influence its susceptibility to specific DNA methylation. Our novel hypothesis could provide a basis for understanding how the complex pattern of ER-{alpha} methylation and transcriptional silencing is generated and for understanding the relationship between this pattern and its function during the neoplastic process. [Cancer Res 2007;67(16):7731–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The field of epigenetic is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without change in DNA sequence (1, 2). Epigenetic changes can influence a variety of cellular processes from regulation of gene transcription to proper chromosome segregation (3). DNA methylation is an important epigenetic mechanism of transcriptional control and plays an essential role in maintaining cellular functions. Moreover, changes in the status of DNA methylation represent one of the most common molecular alterations in human neoplasia, including breast cancer (1, 4). Aberrant methylation of DNA (global hypomethylation accompanied by region-specific hypermethylation) is frequently found in tumor cells and it has been associated with the inactivation of tumor suppressor genes and chromosomic instability (5).

The pRb family proteins (pRb1/p105, p107, and pRb2/p130), collectively referred to as pocket proteins, are believed to function primarily as regulators of the mammalian cell cycle progression and suppressors of cellular growth and proliferation (6, 7). In addition, different studies suggest that these pocket proteins are also involved in development and differentiation of various tissues (8, 9). More recently, a broad range of evidences indicates that pRb family proteins associate with a wide variety of transcription factors and chromatin remodeling enzymes forming transcriptional repressor complexes that control gene expression by inducing a repressive chromatin state around euchromatic promoters (10–14).

The pathogenesis of breast cancer is poorly understood, but epidemiologic, molecular, and clinical genetic studies have implicated factors and defined molecular markers that are associated with an increased risk of developing breast cancer (15, 16). The use of hormone therapy and chemotherapy has been aided by factors predicting the likelihood of therapeutic response, and one of the most important markers is the estrogen receptor-{alpha} (ER-{alpha}; refs. 17, 18). However, the ER-{alpha} status can change during the course of disease, and consequently, therapy resistance can occur. Therefore, insight in the mechanisms regulating the expression of ER-{alpha} during the neoplastic events is essential for optimal treatment.

The epigenetic process regulating the silencing of genes is complex and multifaceted. In this context, the molecular mechanisms governing ER-{alpha} transcriptional activity and/or silencing by chromatin remodeling in ER-{alpha}–positive and ER-{alpha}–negative breast cells are still being elucidated. In a previous study, we provided the first in vivo evidence that a specific fragment of ER-{alpha} promoter is bound by pRb2/p130-E2F4/5-histone deacetylase 1 (HDAC1)-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNA methyltransferase 1 (DNMT1) complexes in ER-{alpha}–positive and ER-{alpha}–negative breast cancer cells, respectively (14). Moreover, we suggested a link between pRb2/p130 and chromatin-modifying enzymes in governing ER-{alpha} gene expression by inducing chromatin remodeling.

Here, we show that the presence of a specific pRb2/p130 multimolecular complex strongly correlates with the methylation status of ER-{alpha} gene. Our data indicate that 5-Aza-2'-deoxycytidine (5-Aza-2dC) demethylating treatment induces reorganization of pRb2/p130 multimolecular complex on ER-{alpha} promoter in MDA-MB-231 ER-{alpha}–negative cells. On the contrary, we report that in MCF-7 cells 5-Aza-2dC treatment does not influence the composition of the pRb2/p130 multimolecular complex recruited to the ER-{alpha} promoter because this complex is identical to the complex that we previously showed to be bound to the ER-{alpha} promoter in untreated MCF-7 cells (14).

Furthermore, here we suggest that pRb2/p130 could cooperate with ICBP90 (inverted CCAAT box binding protein of 90 kDa) and DNMTs in maintaining a specific methylation pattern of ER-{alpha} gene in breast cancer cells. It has been reported that ICBP90 is overexpressed in several cancer cell lines and cancer tissues, including breast cancer (19, 20). In addition, it has been suggested that ICBP90 could play a role in tumorigenesis by altering the expression of genes know for playing a role in the neoplastic events (19, 21, 22). Interestingly, we have data showing that ICBP90 and DNMT1 may functionally interact and are present in a same macromolecular complex in Jurkat cell lines.5

Our hypothesis is that the sequence of epigenetic events for establishing and maintaining the silenced state of ER-{alpha} gene can be locus or pathway specific and that the remodeling of local chromatin structure of ER-{alpha} gene by pRb2/p130 multimolecular complexes may influence its susceptibility to specific DNA methylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and treatments. The breast cell lines MCF-7, MDA-MB-231, and MDA-MB-361 (Cell Applications, Inc.) were cultured according to the manufacturer's protocols.

For treatment, cells were seeded at a density of 5 x 105/100-mm tissue culture dishes. After 24 h of incubation, the culture medium was changed to medium containing 5-aza-2dC (2.5 µmol/L; Sigma) for up to 96 h.

Chromatin immunoprecipitation assay. Cross-link chromatin immunoprecipitation (XChIP) experiments were done using the ChIP Assay kit (Upstate Biotechnology).

Breast cell lines were cross-linked by adding formaldehyde (1% final concentration) directly to culture medium and incubated at 37°C. Immunoprecipitations were carried out using 1 to 2 µg of antibodies against pRb2/p130, p107, pRb1/p105, E2F4, E2F5, HDAC1, SUV39H1, DNMT1, p300 (Santa Cruz Biotechnology and Upstate Biotechnology), or ICBP90 (kindly provided by C. Bronner, Departement de Pharmacologie et Pharmacochimie, Faculte de Pharmacie, Illkirch, France). As negative controls, no-antibody immunoprecipitations and immunoprecipitations with an irrelevant antibody were done. The cross-link was reversed by incubating samples at 65°C overnight, and DNA was extracted with phenol/chloroform and ethanol precipitation. Primers spanning two specific regions of ER-{alpha} promoter were used in PCRs [region A: 5'-AGGAGCTGGCGGAGGGCGTTCG-3' (ER1) and 5'-AGCGCATGTCCCGCCGACACGC-3' (ER2); region B: 5'-CTGCGTATGCAACCGCAGACCCC-3' (ER3) and 5'-ACGGCCAGGGGGCGGGGGCG-3' (ER4); Genbank accession nos. X68051 and NM000125]. Total chromatin (0.5%, input) was used as positive control in PCRs.

Immunoprecipitation assay. Cytoplasmic and nuclear proteins were extracted by using the Paris kit (Ambion). Efficient cytoplasmic and nuclear fractionation was confirmed by Western blotting analysis using anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody for cytoplasmic fraction and anti-Oct-1 antibody for nuclear fraction (Santa Cruz Biotechnology). Immunoprecipitation experiments were done from cytoplasmic and nuclear fractions and using anti-pRb2/p130 as immunoprecipitating antibody (Santa Cruz Biotechnology). The presence of ICBP90 in both pRb2/p130 nuclear and cytoplasmic precipitates was assessed with anti-ICBP90 antibody by Western blotting.

Protein extraction and Western blotting analysis. Cells were washed in PBS and lysed in a buffer containing 150 mmol/L NaCl, 50 mmol/L Tris (pH 7.5), 0.05% SDS, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, and 5 µg/mL leupeptin. After incubation on ice and centrifugation at 12,000 rpm for 10 min, the supernatants were collected, and the protein concentration was determined with the Protein Assay kit (Bio-Rad).

Whole lysate (40 µg) from MCF-7, MDA-MB-231, and MDA-MB-361 breast cells was fractioned on 6% or 8% SDS-PAGE gels for Western blotting. The blots were probed with antibodies specific for DNMT1, p300, ER-{alpha} (Santa Cruz Biotechnology), and ICBP90. The expression of HSP70 and ß-actin protein was assessed to normalize protein loading.

Multiplex reverse transcription-PCR. Total RNA was extracted from MCF-7 and MDA-MB-231 cells by using the RNeasy kit (Qiagen). Before further use, the total RNA was treated with DNase I, amplification b grade (1 unit DNase/1 mg total RNA; Life Technologies). Reverse transcription-PCR (RT-PCR) was done by using the Reverse Transcription System (Promega). Multiplex RT-PCR was carried out using 1/100 of cDNA and the following primers for each reaction: ER-{alpha} (exon 4), 5'-ATGTTGAAACACAAGCGCCAGA-3' (forward) and 5'-ATCATCGAAGCTTCACTGAA-3' (reverse); ß-actin, 5'-TGACGGGCTCACCCACACTGTGCCCA-3' (forward) and 5'-CTAGAAGCATTTGCGGTGGACGATGG-3' (reverse). The primer ratio for ß-actin and ER-{alpha} was 0.3:2.0, respectively. The amplified fragments were detected by 1.5% (w/w) agarose gel electrophoresis. Each band was quantified and the specific gene expression level was determined semiquantitatively by calculating the ratio of densitometric value from the ER-{alpha} band in relation to the internal standard represented by ß-actin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MCF-7 and MDA-MB-231 cells exhibit similar levels of DNMT1 and p300 proteins. In a previous study, we reported that pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-DNMT1-SUV39H1 multimolecular complexes bind to a specific region of ER-{alpha} gene in MCF-7 (ER-{alpha} positive) and MDA-MB-231 (ER-{alpha} negative) breast cancer cells, respectively (14). In addition, we suggested a functional interaction among pRb2/p130 retinoblastoma-related protein, E2F4 and E2F5 transcription factors, and chromatin-modifying enzymes DNMT1, HDAC1, histone methyltransferase (SUV39H1), and histone acetyltransferase (p300) in controlling ER-{alpha} gene expression.

We hypothesize that pRb2/p130 can actively modulate ER-{alpha} transcription by recruiting chromatin-modifying activity to the ER-{alpha} promoter and altering chromatin conformation. The specificity of the recruited enzymes may be a key element in determining a specific pattern of local chromatin remodeling that may dictate different "transcription modulation environments" closely correlated to ER-{alpha} expression in ER-{alpha}–positive and ER-{alpha}–negative breast cancer cells. In our context, DNMT1 and p300 could represent key enzymes. For example, the expression level of DNMT1 and p300 may differ between MCF-7 and MDA-MB-231 cells.

Here, we investigated whether the presence of DNMT1 binding only the ER-{alpha} promoter fragment immunoprecipitated from MDA-MB-231 cells, as well as the absence of p300 binding, may stem from unrelated differences between MDA-MB-231 and MCF-7 cells in the expression of endogenous level of DNMT1 and p300 proteins. As shown in Fig. 1 , the protein expression level of DNMT1 and p300, which we found to be differentially associated with pRb2/p130 on the ER-{alpha} promoter, is similar in MDA-MB-231 and MCF-7 cells.


Figure 1
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  		Figure 1. Protein expression levels of DNMT1 and p300 in MDA-MB-231 and MCF-7 cells. The relative protein levels were detected by Western blotting using 40 µg of whole lysate from MDA-MB-231 and MCF-7 cells. The expression of HSP70 protein was assessed to normalize protein loading.

 
In the light of these results, we believe that the presence of specific enzymes in pRb2/p130 complexes on ER-{alpha} promoter is not due to stochastic events triggered by a different protein levels of endogenous DNMT1 and p300 proteins but can be dictated by altered physiologic molecular pathways, such as genomic and/or nongenomic estrogen signaling, leading to specific protein-DNA interactions. In an "altered context," p300 and DNMT1 may be sequestered by other proteins and be unavailable to the pRb2/p130 complex in a particular cell. Moreover, it is possible that pRb2/p130-associated complexes may contain additional components that are not probed for here and that influence the recruitment of p300 and DNMT1.

5-Aza-2dC treatment induces reorganization of pRb2/p130 complex on MDA-MB-231 cells. We tested whether the demethylating agent 5-Aza-2dC can affect the organization of proteins in the pRb2/p130 complexes bound to a specific ER-{alpha} promoter region in MCF-7 (ER-{alpha} positive) and MDA-MB-231 (ER-{alpha} negative) cells.

The results clearly indicated that 5-Aza-2dC treatment significantly enhances the expression levels of ER-{alpha} RNA and protein, which is especially evident at longer time points (48–96 h) in MDA-MB-231 cells (Fig. 2A and B ). On the contrary, 5-Aza-2dC treatment did not significantly influence the expression of ER-{alpha} RNA or protein levels in MCF-7 cells (data not shown).


Figure 2
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  		Figure 2. Effects of 5-Aza-2dC on ER-{alpha} RNA and protein expression in MDA-MB-231 cells. A, MDA-MB-231 cells grown in DMEM at the density of 5 x 105/100-mm plate were treated with 2.5 µmol/L DNMT inhibitor (5-Aza-2dC) for 24, 36, 48, 72, and 96 h or were left untreated. ER-{alpha} RNA was detected with RT-PCR using total RNA. ß-Actin RNA expression was determined by RT-PCR to normalize RNA loading. B, ER-{alpha} protein was detected by Western blotting using whole lysates from MDA-MB-231 cells untreated or treated with 2.5 µmol/L 5-Aza-2dC for 24, 36, 48, 72, and 96 h. The expression of ß-actin protein was assessed to normalize protein loading. C, in vivo ER-{alpha} promoter occupancy by pRb2/p130-E2F4-HDAC1-SUV39H1-DNMT1-p300 in MDA-MB-231 breast cancer cells treated with 5-Aza-2dC. The recruitment of pRb2/p130 multimolecular complexes on the ER-{alpha} promoter was determined by XChIP. The cells were treated with 5-Aza-2dC for 72 h and cross-linked with formaldehyde. Soluble chromatin was immunoprecipitated with specific antibodies recognizing pRb2/p130, E2F4, HDAC1, SUV39H1, DNMT1, and p300. Irrelevant antisera have been used as negative controls as well as multiple antibodies both for pRb2/p130 and aforementioned enzymes to further validate the results. The presence of ER-{alpha} promoter regions in the immunoprecipitates was tested by PCR using primers spanning a specific fragment (region A) of ER-{alpha} promoter. Total chromatin (0.5%, inputs) was used as positive control in PCRs. D, schematic representation of region A on the ER-{alpha} promoter recognized by ER1/ER2 primers (Genbank accession no. NM000125).

 
Interestingly, results from XChIP experiments showed that, at the time of maximal ER-{alpha} RNA expression (72 h) in 5-Aza-2dC–treated MDA-MB-231 cells, a specific pRb2/p130 multimolecular complex is recruited on the ER-{alpha} promoter fragment investigated (region A) and this complex contains pRb2/p130, E2F4, SUV39H1, p300, and HDAC1 but does not contain DNMT1 (Fig. 2C and D). This complex is identical to the complex that we previously showed to be associated with the ER-{alpha} promoter in untreated MCF-7 cells (14). On the contrary, we found that in MCF-7 cells the 5-Aza-2dC treatment did not influence the composition of pRb2/p130 multimolecular complex recruited to the ER-{alpha} promoter because this complex is identical to the complex that we previously showed to be bound to the ER-{alpha} promoter in untreated MCF-7 cells (data not shown; see ref. 14).

Taken together, these results indicate a strong correlation between the presence of specific pRb2/p130 complexes on the ER-{alpha} promoter and its methylation status.

In vivo binding of pRb1/p105, p107, and pRb2/p130 to ER-{alpha} promoter. We studied whether pRb1/p105 and p107 bind in vivo to ER-{alpha} promoter. We did XChIP experiments and, as shown in Fig. 3 , found that in cycling MCF-7 and MDA-MD-231 cells pRb/p105 and p107 bind to ER-{alpha} promoter in both the regions investigated: the region A, which we previously showed to be bound by pRb2/p130 complexes, and the region B, which is located 200 bp upstream from region A. Interestingly, we found that pRb2/p130 was not able to bind the region B too. These results may suggest that (a) pRb1/p105 and p107 could bind to ER-{alpha} promoter and do the same function of pRb2/p130 perhaps during a specific time of cell cycle and/or (b) pRb1/p105 and p107 could participate synergistically to regulate ER-{alpha} transcription by recruiting chromatin remodeling proteins over other adjacent nucleosomes. Actually, we are investigating our hypothesis that the binding of pRb1/p105, p107, and pRb2/p130 proteins on ER-{alpha} promoter may be directly correlated with a specific ER-{alpha} transcriptional environment.


Figure 3
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  		Figure 3. In vivo binding of pRb1/p105, p107, and pRb2/p130 to ER-{alpha} promoter. A, XChIPs were done in MCF-7 and MDA-MD-231 cells using pRb1/p105, p107, or pRb2/p130 (region B) as immunoprecipitating antibodies. The presence of ER-{alpha} promoter regions in the immunoprecipitates was tested by PCR using specific primers spanning the region A (ER1/ER2) and region B (ER3/ER4) of ER-{alpha} promoter. B, schematic representation of regions A and B on the ER-{alpha} promoter recognized by ER1/ER2 and ER3/ER4 primers, respectively (Genbank accession nos. X68051 and NM000125).

 
pRb2/p130 and ICBP90: a novel interaction. To address the importance of other proteins that might be involved in pRb2/p130-mediated methylation, we included in our study ICBP90, a transcription regulator of the topoisomerase II{alpha} gene (23), which has been found overexpressed in several cancer cell lines and cancer tissues, including breast cancer, lung cancer, prostate cancer, gliomas, and pancreatic adenocarcinoma (19, 22, 24, 25).

It is reported that ICBP90 has the ability to bind the DNA and exhibits affinity for CpG islands. As well, it has been suggested that ICBP90 may play a role in chromatin remodeling through protein/protein interaction (21). Interestingly, pRb1/p105 gene contains several putative binding sites for ICBP90 and this latter regulates pRb1/p105 both at the protein and gene transcription levels (21). In addition, we have data indicating a functional interaction between ICBP90 and DNMT1 in Jurkat cells.5 Here, our hypothesis is that ICBP90 and pRb2/p130 may participate in a common mechanism of ER-{alpha} gene transcription regulation through chromatin remodeling.

ICBP90 protein expression was investigated in cycling MCF-7, MDA-MB-231, and MDA-MB-361 breast cancer cells. All these cell lines showed appreciable levels of ICBP90, with both MDA-MD-231 and MDA-MB-361 exhibiting higher ICBP90 protein levels than MCF-7 (Fig. 4A ).


Figure 4
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  		Figure 4. A, protein expression levels of ICBP90 in MCF-7, MDA-MB-231, and MDA-MB-361 breast cancer cell lines. The relative ICBP90 protein level was detected by Western blotting using 40 µg of whole lysate from MCF-7, MDA-MB-231, and MDA-MB-361 cells. The expression of ß-actin protein was assessed to normalize protein loading. B, interaction between pRb2/p130 and ICBP90 in nuclear and cytoplasmic fractions of MCF-7, MDA-MB-361, and MDA-MDA-231 cells. Immunoprecipitation experiments were done using pRb2/p130 as immunoprecipitating antibody. The presence of ICBP90 in both pRb2/p130 nuclear and cytoplasmic precipitates was assessed with anti-ICBP90 antibody by Western blotting. Controls were done by doing Western blotting of the immunoprecipitates where the antibody against pRb2/p130 was omitted (data not shown). C, efficient cytoplasmic and nuclear fractionation was confirmed by Western blotting analysis using anti-GAPDH antibody for cytoplasmic fraction and anti-Oct-1 antibody for nuclear fraction.

 
To assess whether pRb2/p130 interacts with ICBP90, we did immunoprecipitation experiments using both cytoplasmic and nuclear fractions from asynchronously growing MDA-MB-231, MCF-7, and MDA-MB-361 breast cancer cell lines and pRb2/p130 as immunoprecipitating antibody. As shown in Fig. 4B, we observed an interaction between ICBP90 and pRb2/p130 in the nuclear fractions of all the cell lines investigated. Surprisingly, we also observed a cytoplasmic interaction between pRb2/p130 and ICBP90 in MCF-7 cells. The results were confirmed by immunoprecipitations from the cytoplasmic and nuclear fractions of MDA-MB-231, MCF-7, and MDA-MB-361 cells and using anti-ICBP90 as immunoprecipitating antibody (data not shown). Negative controls were done by Western blotting analyses of the immunoprecipitates where the antibody was omitted. Efficient cytoplasmic and nuclear fractionation was confirmed by Western blotting analysis using anti-GAPDH antibody for cytoplasmic fraction and anti-Oct-1 antibody for nuclear fraction (Fig. 4C).

At this time, we have not sufficient information to explain the significance of the exclusive interaction between pRb2/p130 and ICBP90 in the cytoplasm of MCF-7 cells. However, it is reasonable to hypothesize that, in MCF-7 cells, pRb2/p130 may be involved in the nuclear transportation of ICBP90 and that, in MDA-MB-231 cells, this "mechanism" may be altered.

Furthermore, we did XChIP experiments to determine whether ICBP90 binds to regions A and B of the ER-{alpha} promoter in MCF-7 and MDA-MB-231 cells. As shown in Fig. 5 , we found that ICBP90 binds only the region A of ER-{alpha} promoter in both the cell lines investigated. Interestingly, previously, we showed that the region A of ER-{alpha} promoter is also bound by pRb2/p130 complexes as well as by pRb1/p105 and p107 (Fig. 3; see ref. 14).


Figure 5
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  		Figure 5. In vivo binding of ICBP90 to ER-{alpha} promoter. XChIPs were done in MCF-7 and MDA-MB-231 cells using ICBP90 as immunoprecipitating antibody. The presence of regions A and B of ER-{alpha} promoter in the immunoprecipitates was tested by PCR using specific primers spanning region A (ER1/ER2) and region B (ER3/ER4) of ER-{alpha} promoter, respectively (see also Fig. 4). Total chromatin (0.5%, inputs) was used as positive control in PCRs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The molecular mechanisms governing ER-{alpha} transcriptional activity and/or silencing by chromatin remodeling in ER-{alpha}–positive and ER-{alpha}–negative breast cells are still unknown. However, we previously provided the first in vivo evidence that a specific fragment of ER-{alpha} promoter is bound by pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1 complexes in ER-{alpha}–positive and ER-{alpha}–negative breast cancer cells, respectively, and provided a link between pRb2/p130 and chromatin-modifying enzymes in regulating ER-{alpha} transcription by chromatin remodeling (14). Here, we show that the presence of a specific pRb2/p130 complex on the ER-{alpha} promoter strongly correlates with its methylation status. In fact, at the time of maximal ER-{alpha} RNA expression (72 h) in 5-Aza-2dC–treated MDA-MB-231 cells, a specific pRb2/p130 multimolecular complex is recruited to ER-{alpha} promoter (Fig. 2). This complex is identical to the complex that we previously showed to be associated with the ER-{alpha} promoter in untreated MCF-7 ER-{alpha}–positive cells (14).

Moreover, we suggest that the retinoblastoma family proteins pRb1/p105, p107, and pRb2/p130 may synergistically cooperate in modulating ER-{alpha} gene expression by inducing modifications of local chromatin structure through interaction with chromatin-modifying enzymes (Fig. 3).

Our hypothesis is that the sequence of epigenetic events for establishing and maintaining the silenced state of ER-{alpha} gene can be locus or pathway specific and that the remodeling of local chromatin structure of ER-{alpha} gene by pRb2/p130 complexes, and perhaps pRb1/p105 and p107, may influence its susceptibility to specific DNA methylation.

Several studies have suggested that DNA methylation can mark genes for inactivation. We strongly believe that the diversity of methylation patterns is too complex to be explained just by the activities of general nonsite-selective DNMTs and demethylases and then other factors must exist in determining a specific methylation pattern. Our observations suggest that the methylation pattern of ER-{alpha} gene could be determined by specific protein-protein and protein-DNA interactions around specific sites. The relationship between DNA methylation and chromatin modification could be viewed as a complex feedback loop. In this context, inactive chromatin states could lead to increased DNA methylation, suggesting that factors that alter chromatin structure could also alter DNA methylation patterns. Therefore, alterations in the protein complexes that act to regulate the transcription level of specific genes could result not only in altered transcription but also in change in the DNA methylation pattern itself.

In Fig. 6 , we pictured a hypothetical model of how pRb family proteins may regulate ER-{alpha} transcription.


Figure 6
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  		Figure 6. Hypothetical model of how pRb2/p130 complexes may regulate ER-{alpha} transcription (see further explanation in the text). A, pRb2/p130 corepressor complex represses the ER-{alpha} transcription by maintaining a close chromatin conformation in MDA-MB-231 cells. The interaction between the pRb2/p130 complex and ICBP90 leads to the recruitment of DNMT1 and concomitant release of p300. Histone deacetylation and methylation, perhaps ubiquitination of H3, and DNA methylation could set up a heritable mark to establish a heterochromatin state of long-term silencing. B, pRb2/p130 coactivator complex regulates the ER-{alpha} transcription by maintaining an open chromatin conformation in MCF-7 cells. The balance between HDAC1 and p300 activity permits high level of histone H3 and H4 acetylation. TAFs, activator transcription factors; TFIIs, transcription factors.

 
Our hypothesis is that in MDA-MB-231 ER-{alpha}–negative cells the high density of methylated CpG sites on ER-{alpha} promoter may permit a functional interaction between ICBP90 (which showed in vitro to have high affinity for methylated DNA) and the pRb2/p130 coactivator complex. In fact, this interaction could dictate the reorganization of the pRb2/p130 coactivator complex into a pRb2/p130 corepressor complex through the recruitment of DNMT1 by ICBP90 and/or pRb2/p130 itself and the concomitant release of p300. Therefore, the presence of the pRb2/p130 corepressor complex could induce a local chromatin remodeling by histone deacetylation (HDAC1) and methylation (SUV39H1), perhaps ubiquitination of H3 (ICBP90) and DNA methylation (DNMT1). This latter event of chromatin remodeling could be propagated over other nucleosomes (perhaps by pRb1/p105 and p107), leading to higher-order chromatin structure that denies access to basal transcriptional factors, and could set up a heritable mark to establish a heterochromatin state of long-term ER-{alpha} silencing. Thus, in addition to DNA methylation, inducing the formation of inactive chromatin states, inactive chromatin states could lead to increased DNA methylation in a feedback loop (Fig. 6A).

On the other hand, in MCF-7 ER-{alpha}–positive cells, the presence of pRb2/p130 coactivator complex on ER-{alpha} promoter could be dictated by low density of methylated CpG sites maybe due to de novo methylation, which does not permit any functional interaction between ICBP90 and pRb2/p130. Then, the absence of DNMT1 in the pRb2/p130 coactivator complex could facilitate p300 recruitment, leading to a biochemical balance between HDAC1 and p300 activities. This balance could be required to permit high level of histone H3 and H4 acetylation and maintain an open chromatin conformation on the ER-{alpha} promoter, leading to the transcriptional activation of this gene in MCF-7 cells (Fig. 6B). Moreover, it is reasonable to hypothesize that, in MDA-MB-231 cells, SUV39H1 can methylate histone H3, whereas in MCF-7 cells its histone methyltransferase activity is prevented by p300 activity, which can acetylate histone H3. Finally, in this complex scenario, we cannot exclude the possibility that ICBP90 may be involved in mechanisms of ER-{alpha} gene activation in MCF-7 cells because it has been reported that ICBP90 is able to interact with TIP60, a histone acetylase (23, 26).

Our model could provide a basis for understanding how the complex pattern of ER-{alpha} methylation and transcriptional silencing are generated and for understanding the relationship between this pattern and its function.


   Acknowledgments
 
Grant support: NIH grant CA060999, U.S. Army Medical Research (06062003), and Sbarro Health Research Organization (http://www.shro.org; A. Giordano) and Fondazione Italiana per la Ricerca sul Cancro fellowship (M. Macaluso).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: M. Macaluso and M. Montanari contributed equally to this work.

5 C. Bronner, unpublished data. Back

Received 4/23/07. Revised 5/18/07. Accepted 6/ 5/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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