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Published on: June 21, 2016
The Sin3A/MAD1 Complex, through Its PAH2 Domain, Acts as a Second Repressor of Retinoic Acid Receptor Beta Expression
Nisha Rani Dahiya1, Boris A Leibovitch2, Rama Kadamb3
1The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Abstract:
Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARβ is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RARβ has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RARβ expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RARβ induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RARα and RARβ from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARβ expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RARα agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RARβ promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARβ activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARβ expression. These data suggest that SIN3A/MAD1 acts as a second RARβ repressor and may be involved in fine-tuning retinoid sensitivity.
Insights
Blocking the Sin3A-MAD1 interaction reactivates the tumor suppressor RARβ in breast cancer cells. This mechanism, involving Sin3A/HDAC1/2 complex, fine-tunes retinoid sensitivity and may improve cancer survival.
Area of Science:
- Molecular Biology
- Epigenetics
- Cancer Research
Background:
- Retinoids regulate cell proliferation, differentiation, and apoptosis via retinoic acid receptors (RARs) and retinoid X receptors (RXRs).
- RARβ, a tumor suppressor gene, is epigenetically silenced (e.g., DNA methylation) in various cancers, including breast cancer.
- Increased RARβ expression correlates with improved breast cancer survival.
Purpose of the Study:
- To elucidate the mechanistic basis of RARβ gene induction and function.
- To investigate the role of the Sin3A-MAD1 interaction in RARβ regulation.
Main Methods:
- Co-immunoprecipitation assays to assess protein complex dissociation.
- Luciferase assays to measure RARβ promoter activity.
- Electrophoretic Mobility Shift Assays (EMSA) to detect DNA binding.
- Chromatin Immunoprecipitation (ChIP) to quantify protein enrichment on the RARβ promoter.
Main Results:
- Blocking Sin3A interaction with MAD1 (using MAD1 SID or peptide) dissociated MAD1, RARα, and RARβ from Sin3A.
- This dissociation led to increased RARα and RARβ expression and function.
- MAD1 binds to the RARβ promoter E-Box, and Sin3A/HDAC1/2 complex is recruited to this site.
- Sin3A/HDAC1/2 complex is required for AM580-enhanced RARβ activation in MAD1/SID cells.
Conclusions:
- The Sin3A/HDAC1/2 complex acts as a repressor of RARβ expression, cooperating with other repressors.
- SIN3A/MAD1 functions as a novel RARβ repressor, potentially fine-tuning retinoid sensitivity in cancer cells.
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