Transcriptional Functions of Nuclear Receptors in Cancer

癌症中核受体的转录功能

• 批准号: 6838568
• 负责人: TREVOR K ARCHER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6838568
• 关键词: DNA binding protein; DNA footprinting; breast neoplasms; cell cycle proteins; chromatin; corticosteroid receptors; cyclin dependent kinase; environmental toxicology; gene induction /repression; genetic transcription; histones; hormone inhibitor; hormone regulation /control mechanism; hormone related neoplasm /cancer; human tissue; immunoprecipitation; nuclear receptors; osteosarcoma; phosphorylation; progesterone receptors; receptor expression; transcription factor

During the last year we have made significant progress on this project both with respect to the understanding of the mechanisms by which steroid receptors function in vivo and the importance of chromatin to the regulation of transcription in cancer cells. The results from our work can be highlighted by exploring our progress in three specific areas indicated below. We developed a model breast cancer cell lines to examine potential mechanisms by which the ligand bound estrogen receptor (ERa) regulates glucocorticoid receptor (GRa)-mediated transcription. MCF-7 cells, which endogenously express ERa, were stably transfected with MMTV-luciferase reporter and GR expression constructs. Treatment with estrogen agonists [17b-estradiol (E2), diethylstilbestrol, genistein], but not antagonists [tamoxifen or raloxifene], inhibits GR-mediated MMTV transcription and chromatin remodeling. Furthermore, estrogen agonists inhibit glucocorticoid induction of p21 mRNA and protein levels, suggesting that the repressive effect applies to other GR regulated genes/proteins in MCF-7 cells. GR transcriptional activity is compromised because treatments with estrogen agonists down regulate GR protein levels. The protein synthesis inhibitor, cycloheximide and the proteasome inhibitor MG132 block E2-mediated decrease in GR protein levels, suggesting that estrogen agonists down regulate the GR via the proteasomal degradation pathway. In support of this, we demonstrate that E2-mediated GR degradation is coupled to an increase in both p53 and it?s key regulator protein Mdm2, an E3 ubiquitin ligase shown to target the GR for degradation. Using ChIP assay we demonstrate an E2-dependent recruitment of ERa to the Mdm2 promoter, suggesting a role of ER in the regulation of Mdm2 protein expression and hence the enhanced GR degradation in the presence of estrogen agonists. The molecular mechanisms by which nuclear hormone receptors recruit chromatin-remodeling activity are not fully elucidated. We showed that in the absence of its ligand-binding domain the glucocorticoid receptor (GR) is able to interact with both nuclear receptor co-activators and the BRG1 chromatin-remodeling complex in vivo. Individually, the GR makes direct interactions with BAF60a and BAF57, but not with BRG1, BAF155, or BAF170. Further, BAF60a possesses at least two interaction surfaces, one for GR and BRG1 and a second for BAF155, and BAF170. A GR mutant, GR (R488Q), that fails to interact with BAF60a in vitro has reduced chromatin-remodeling activity and reduced transcriptional activity from the promoter assembled as chromatin in vivo. Stable expression of a BAF60a truncation mutant, BAF60a4-140, caused chromatin-specific loss of GR functions in vivo. In the presence of the BAF60a mutant, the GR fails to interact with the BRG1 complex and consequently is also deficient in its ability to activate transcription from chromatin. Thus, in addition to previously identified BAF250, BAF60a may provide another critical and direct link between nuclear receptors and the BRG1 complex that is required for promoter recruitment and subsequent chromatin remodeling. To dissect the precise role of nuclear factor one (NF1) in chromatin remodeling and transcriptional activation, we used linker-scanning mutants of transcription factor binding sites on the MMTV promoter. We compared the NF1 mutant promoter in the context of non-chromatin templates (transient transfection) and templates organized as chromatin (stable transfection) to understand the effect of chromatin on factor binding and transcription. We showed that on a non-chromatin template, mutation in the NF1 binding site reduces both basal and hormone dependent transcription. This suggests that NF1 is required for transcription in the absence of organized chromatin. We also found that binding of NF1 on a non-chromatin template is independent of mutation in hormone response elements (HRE) or the octamer transcription factor (OTF) binding site. In contrast, the binding of OTF proteins to a non-chromatin template was found to be dependent on the binding of NF1, which may imply that NF1 has a stabilizing effect on OTF binding. On a chromatin template, mutation in the NF1 binding site does not affect the positioning of nucleosomes on the promoter. We also show that in the absence of NF1 binding, GR mediated chromatin remodeling of nucleosome B is reduced and hormone dependent activation of transcription is abolished. Further, we demonstrate that NF1 is required for the association of BRG1 complex on the promoter. These results suggest that NF1 may participate in chromatin remodeling activities in addition to directly enhancing transcription and that in the absence of its binding site GR is unable to effectively recruit the remodeling complex to the promoter.

在过去的一年中，我们在这个项目上取得了重大进展，这两个方面的类固醇受体在体内的功能和染色质的重要性，以调节癌细胞中的转录的机制的理解。通过探讨我们在以下三个具体领域取得的进展，可以突出我们工作的成果。 我们建立了一个乳腺癌细胞系模型，以研究配体结合的雌激素受体（ER α）调节糖皮质激素受体（GR α）介导的转录的潜在机制。用MMT-荧光素酶报告基因和GR表达构建体稳定转染内源性表达ER α的MCF-7细胞。用雌激素激动剂[17 b-雌二醇（E2），己烯雌酚，染料木黄酮]治疗，而不是拮抗剂[他莫昔芬或雷洛昔芬]，抑制GR介导的MMTV转录和染色质重塑。此外，雌激素激动剂抑制糖皮质激素对p21 mRNA和蛋白水平的诱导，表明抑制作用适用于MCF-7细胞中的其他GR调节基因/蛋白。GR转录活性受损，因为用雌激素激动剂治疗下调GR蛋白水平。蛋白质合成抑制剂放线菌酮和蛋白酶体抑制剂MG 132阻断E2介导的GR蛋白水平降低，表明雌激素激动剂通过蛋白酶体降解途径下调GR。在支持这一点，我们表明，E2介导的GR降解是耦合到增加p53和它？的关键调节蛋白Mdm 2，E3泛素连接酶的目标GR的降解。使用ChIP检测，我们证明了E2依赖性的招聘ERa的Mdm 2启动子，提示ER的作用，在Mdm 2蛋白表达的调节，因此增强GR降解的存在下，雌激素激动剂。 核激素受体募集染色质重塑活性的分子机制尚未完全阐明。我们发现，在缺乏其配体结合结构域的糖皮质激素受体（GR）是能够相互作用的核受体辅激活剂和BRG 1染色质重塑复合物在体内。单独地，GR与BAF 60 a和BAF 57直接相互作用，但不与BRG 1、BAF 155或BAF 170直接相互作用。此外，BAF 60 a具有至少两个相互作用表面，一个用于GR和BRG 1，第二个用于BAF 155和BAF 170。一个GR突变体，GR（R488 Q），在体外不能与BAF 60 a相互作用，具有降低的染色质重塑活性和降低的转录活性，从启动子组装成染色质在体内。BAF 60 a截短突变体BAF 60 a4 -140的稳定表达导致体内GR功能的染色质特异性丧失。在BAF 60 a突变体的存在下，GR不能与BRG 1复合物相互作用，因此也缺乏激活染色质转录的能力。因此，除了先前鉴定的BAF 250之外，BAF 60 a可能在核受体和BRG 1复合物之间提供另一种关键和直接的联系，这是启动子募集和随后的染色质重塑所需的。 为了剖析核因子1（NF 1）在染色质重塑和转录激活中的确切作用，我们使用MMTV启动子上转录因子结合位点的接头扫描突变体。我们在非染色质模板（瞬时转染）和染色质模板（稳定转染）的背景下比较了NF 1突变启动子，以了解染色质对因子结合和转录的影响。我们发现，在非染色质模板上，NF 1结合位点的突变降低了基础和激素依赖性转录。这表明，NF 1是转录所需的组织染色质的情况下。我们还发现NF 1在非染色质模板上的结合不依赖于激素反应元件（HRE）或八聚体转录因子（OTF）结合位点的突变。相反，OTF蛋白与非染色质模板的结合被发现依赖于NF 1的结合，这可能意味着NF 1对OTF结合具有稳定作用。在染色质模板上，NF 1结合位点的突变不影响核小体在启动子上的定位。我们还表明，在NF 1结合的情况下，GR介导的核小体B的染色质重塑减少，激素依赖的转录激活被废除。此外，我们证明，NF 1是所需的协会的BRG 1复合物的启动子。这些结果表明，NF 1可能参与染色质重塑活动，除了直接增强转录，并在其结合位点GR的情况下，是无法有效地招募的启动子的重塑复合物。