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.
摘要：
类维生素A在平衡增殖中至关重要,分化和凋亡，它们通过视黄酸受体（RAR）和类视黄醇X受体（RXR）发挥作用。RARβ是一种通过表观遗传机制沉默的抑癌基因，如乳腺DNA甲基化，宫颈癌和非小细胞肺癌。RARβ表达的增加与乳腺癌特异性生存率的提高有关。支架蛋白SIN3A的PAH2域与几种转录因子的特异性Sin3相互作用域（SID）相互作用，如MAD1，带来染色质修饰蛋白，如组蛋白脱乙酰酶，它针对染色质进行特定修饰。以前,我们已经确定，使用SID肽或小分子抑制剂（SMI）阻断PAH2介导的Sin3A与含SID蛋白的相互作用增加了RARβ表达并诱导了乳腺癌细胞中的视黄酸代谢，在体外和体内模型。这里,我们报告旨在了解RARβ诱导和功能的机理基础的研究。使用用MAD1SID转染或用MADSID肽处理的人乳腺癌细胞，我们在免疫共沉淀试验中观察到MAD1，RARα和RARβ从Sin3A中解离。通过荧光素酶测定，这与RARα和RARβ表达和功能增加有关，通过添加特定的RARα激动剂AM580而增强；EMSA显示MAD1与E-Box结合，类似于MYC,在RARβ启动子上，显示ChIP对Sin3A和HDAC1的富集减少，并且是MAD1/SID细胞中AM580增强的RARβ激活所必需的。这些数据表明Sin3A/HDAC1/2复合物在调节RARβ表达中与经典阻遏物合作。这些数据表明，SIN3A/MAD1充当第二个RARβ阻遏物，可能参与微调类维生素A敏感性。
