DNA sequences impact estrogen and antiestrogen activity

DNA 序列影响雌激素和抗雌激素活性

• 批准号: 6845279
• 负责人: Carolyn M. Klinge
• 金额: $ 21.68万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845279
• 关键词: Baculoviridae; CHO cells; DNA directed RNA polymerase; MCF7 cell; acetylation; affinity chromatography; biological signal transduction; cathepsin D; chromatin; conformation; dimer; estrogen receptors; estrogens; fluorescence resonance energy transfer; gel mobility shift assay; genetic regulatory element; genetic transcription; histones; immunoprecipitation; nucleic acid sequence; protein protein interaction; receptor expression; tamoxifen; transfection /expression vector; vascular endothelial growth factors

DESCRIPTION (provided by applicant): Estrogen receptors (ER) alpha and beta are members of the nuclear receptor superfamily of proteins that regulate target gene transcription in response to hormones. Although co-expressed in certain tissues, the properties of ERalpha-ERbeta heterodimers are unknown. A number of studies indicate that ERbeta is a dominant negative inhibitor of ERalpha action, but these studies used an N-terminal truncated version of ERbeta and therefore further experiments are needed to address differences between the long and short forms of ERbeta. Tamoxifen (TAM) binds ER and is used to prevent recurrence of estrogen-dependent breast tumors and has mixed agonist-antagonist effects in other estrogen target tissues. The precise molecular mechanisms allowing estrogen response element (ERE) - containing genes to be selectively responsive to TAM versus estradiol are not well understood and may play a role in TAM resistant breast cancer. Several classes of nuclear accessory proteins are recruited to ERalpha and act as coactivator or corepressor complexes to activate or suppress transcription from EREs to which ERalpha is bound, usually in the promoter region. We reported significant differences in the ligand and ERE binding characteristics and transcriptional responsiveness of ERalpha and ERbeta, revealed by the use of natural and synthetic EREs that varied in sequence and spacing. Of note was our finding that the conformation of ERalpha changes when bound to different EREs, confirming our central hypothesis that DNA is an allosteric modulator of ER action. We hypothesize that these DNA-induced alterations in ER conformation will impact ER interaction with and activation by coactivator proteins. Comparatively little is known about ERbeta interaction with coactivators or corepressors. Using purified ERalpha and ERbeta for FRET and human normal and neoplastic mammary cells for transfection assays and ChIP, we will test the hypotheses that: ERalpha/ERbeta heterodimer differs in ERE and coactivator interaction from either ERalpha or ERbeta homodimers; alterations in ERalpha or ERbeta conformation induced by binding to different ERE sequences modulates ER interaction with coactivators SRC-1, SRC-2 (GRIP1), SRC-3 (AIB1), PRMT, and SRA; ERE regulated ER-coactivator interaction in turn modulates acetylation of histones H3 and H4, and the assembly of the coactivator/RNA polymerase II transcription initiation complex. Results will define the molecular mechanisms of ERalpha, ERbeta, and ERalpha/beta heterodimers as transcriptional regulators, with emphasis on the action of antiestrogens.

描述（由申请人提供）：雌激素受体（ER）α和β是核受体蛋白超家族的成员，其响应激素调节靶基因转录。尽管在某些组织中共表达，但 ERα-ERβ 异二聚体的特性尚不清楚。许多研究表明 ERbeta 是 ERalpha 作用的显性失活抑制剂，但这些研究使用了 ERbeta 的 N 端截短版本，因此需要进一步的实验来解决长形式和短形式 ERbeta 之间的差异。他莫昔芬 (TAM) 与 ER 结合，用于预防雌激素依赖性乳腺肿瘤的复发，并在其他雌激素靶组织中具有混合的激动剂-拮抗剂作用。含有雌激素反应元件（ERE）的基因选择性地对 TAM 和雌二醇做出反应的精确分子机制尚不清楚，并且可能在 TAM 耐药性乳腺癌中发挥作用。几类核辅助蛋白被招募到 ERα 上，并充当共激活子或辅阻遏物复合物，以激活或抑制 ERα 所结合的 ERE 的转录，通常位于启动子区域。我们报道了 ERα 和 ERβ 的配体和 ERE 结合特征以及转录反应性的显着差异，这是通过使用序列和间距不同的天然和合成 ERE 揭示的。值得注意的是，我们发现 ERalpha 的构象在与不同 ERE 结合时发生变化，证实了我们的中心假设，即 DNA 是 ER 作用的变构调节剂。我们假设这些 DNA 诱导的 ER 构象改变将影响 ER 与共激活蛋白的相互作用和激活。关于 ERbeta 与共激活剂或辅阻遏物的相互作用知之甚少。使用纯化的 ERα 和 ERβ 进行 FRET，使用人类正常和肿瘤性乳腺细胞进行转染测定和 ChIP，我们将测试以下假设： ERα/ERβ 异二聚体在 ERE 和共激活剂相互作用方面不同于 ERα 或 ERβ 同二聚体；与不同 ERE 序列结合诱导的 ERα 或 ERβ 构象改变可调节 ER 与共激活剂 SRC-1、SRC-2 (GRIP1)、SRC-3 (AIB1)、PRMT 和 SRA 的相互作用； ERE 调节 ER 与辅激活因子的相互作用，进而调节组蛋白 H3 和 H4 的乙酰化以及辅激活因子/RNA 聚合酶 II 转录起始复合物的组装。结果将定义 ERα、ERβ 和 ERα/β 异二聚体作为转录调节剂的分子机制，重点是抗雌激素的作用。