Epigenetic and Transcriptional Functions of Nuclear Receptors and Chromatin Remodeling Proteins in Stem and Cancer Cells

干细胞和癌细胞中核受体和染色质重塑蛋白的表观遗传和转录功能

• 批准号: 8929755
• 负责人: TREVOR K ARCHER
• 金额: $ 365.65万
• 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/8929755
• 关键词: 26S proteasome; ATP phosphohydrolase; Acetylation; Animals; Architecture; Area; Attention; Basic Science; Binding; Breast Cancer Cell; Breast Cancer Treatment; Cancerous; Cell Cycle Regulation; Cell Lineage; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Cloning; Complex; Cyclin D1; DNA-Directed RNA Polymerase; Data; Development; Disease; Embryo; Embryonic Development; Endocrine Disruptors; Environment; Environmental Exposure; Epigenetic Process; Estrogens; Event; Exposure to; Flow Cytometry; Gene Expression Regulation; Gene Targeting; Genes; Genetic Programming; Genome; Glucocorticoid Receptor; Goals; Histones; Hormones; Human; Laboratory Scientists; Link; Maintenance; Mediating; Methylation; MicroRNAs; Modeling; Molecular; Molecular Machines; Mus; Nature; Nuclear Receptors; Pathway interactions; Play; Pluripotent Stem Cells; Population; Population Heterogeneity; Progesterone; Progesterone Receptors; Promoter Regions; Property; Proteins; Receptor Activation; Receptor Signaling; Regulation; Reproduction; Research; Response Elements; Role; SMARCA4 gene; Signal Transduction; Staging; Stem Cell Development; Stem cells; Steroid Receptors; Stimulus; System; Testing; Therapeutic Uses; Tissues; Transcriptional Activation; Transcriptional Regulation; Translational Repression; Tretinoin; Ubiquitin; Woman; Women' s Health; cancer cell; cancer diagnosis; cancer stem cell; chromatin remodeling; drug discovery; effective therapy; embryonic stem cell; genetic regulatory protein; human embryonic stem cell; human embryonic stem cell line; induced pluripotent stem cell; insight; interest; malignant breast neoplasm; mortality; multicatalytic endopeptidase complex; novel; pluripotency; promoter; protein degradation; receptor; receptor function; research study; response; self-renewal; sensor; stem; steroid hormone; steroid hormone receptor; transcription factor

Breast cancer will constitute greater than 30% of all new cancers diagnosed in women this year. Steroid hormones, such as estrogen and progesterone, play an important role in the development and treatment of breast cancer. These receptors represent critical sensors of environmental exposures on reproduction and development while being subject to epigenetic regulation of their activity. Despite intensive efforts, the mortality resulting from this disease has not decreased significantly over the last decade. In my laboratory, the scientists have undertaken detailed analysis of the mechanism of action of the steroid receptors and clinically important steroid receptor antagonists which are used to block their action. Our experiments are focused on the role of chromatin and epigenetics that are critical to understanding their function. It is hoped that these basic research studies will provide new insight into the role that steroid hormone receptors play in breast cancer and the possible development of novel and effective treatments. Our efforts are informed by the overwhelming evidence that a full understanding of transcriptional control requires an appreciation for roles played by the chromatin structure of target genes and the molecular machines that are required to unleash the regulatory potential of steroid receptors. To achieve this we have focused our attention on the mammalian BRG1 chromatin remodeling complex and the 26S proteasome, their interactions with and regulation by the glucocorticoid and progesterone receptors. The activity of these complexes has been evaluated in the context of the chromatin within human and mouse cells with particular attention to the activity of RNA polymerase and the acetylation/methylation of the core histones. Environmental exposure to low concentration hormones can have permanent epigenetic effects in animals and humans. Steroid hormone receptors initiate a genetic program tightly regulated by the chromatin environment of the responsive regions. Using the glucocorticoid receptor (GR) as a model factor for transcriptional initiation, we classified chromatin structure via diverse experimental approaches. We observed dynamic changes in architecture during GR activation specifically at regions of receptor interaction. We found a distribution of GR-responsive regions with diverse responses to activation and chromatin modulation. However, the majority GR-responsive regions shared a similar basal chromatin state, suggesting a common chromatin structure for GR recruitment. Brg-1 knockdown showed response element-specific effects of ATPase-dependent chromatin remodeling. Taken together, these data suggest classes of nuclear receptor response regions that react to activation through different chromatin regulatory events and identify a chromatin structure that classifies the majority of response elements tested. More recently my group has embarked on an exciting new area of research that examines the fundamental nature of human embryonic stem cells by characterizing the chromatin remodeling and chromatin modifying complexes that are present in these cells. The master regulatory proteins Oct4 and Sox2 are transcription factors required for pluripotency during early embryogenesis and maintenance of embryonic stem cell (ESC) identity. Functional mechanisms contributing to pluripotency are expected to be associated with genes transcriptionally activated by these factors. Here, we show that Oct4 and Sox2 bind to a conserved promoter region of miR-302, a cluster of eight miRNAs expressed specifically in ESCs and pluripotent cells. Expression of miR-302a is dependent on Oct4/Sox2 in human ESCs, and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis. Transcriptional activation of miR-302 and translational repression of its targets, such as Cyclin D1, may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells. The heterogeneous nature of stem cells is an important issue in both research and therapeutic use in terms of directing cell lineage differentiation pathways, as well as self-renewal properties. Using flow cytometry we have identified two distinct subpopulations by size, large and small, within cultures of human embryonic stem (hES) cell lines. These two cell populations respond differentially to retinoic acid (RA) differentiation and several endocrine disruptor compounds (EDC). Cloning studies indicate that both populations can revive the parental population. Furthermore, whole genome microarray identified approximately 400 genes with significantly different expression between the two populations (p<0.01). The findings that hES cells exist as heterogeneous populations with distinct responses to differentiation signals and environmental stimuli will be relevant for their use for drug discovery and disease therapy. The nature of many of our models, human and mouse, breast cancer cells, as well as embryonic stem cells, is indicative of our active interest in women's health and breast cancer specifically. Our research plan discovers and evaluates the contributions that chromatin remodeling proteins, 26S proteasome, nuclear receptors, pluripotency factors, mirRNAs and promoter chromatin architecture make to regulate the transcriptional response to endogenous and environmental signals in normal, human embryonic stem, induced pluripotent stem and cancerous cells.

乳腺癌将占今年女性确诊的所有新癌症的30%以上。类固醇激素，如雌激素和黄体酮，在乳腺癌的发生和治疗中发挥着重要作用。这些受体代表着环境暴露对生殖和发育的关键传感器，同时受制于其活动的表观遗传调节。尽管进行了密集的努力，但在过去十年中，这种疾病造成的死亡率并没有显著下降。在我的实验室里，科学家们对类固醇受体和临床上重要的类固醇受体拮抗剂的作用机制进行了详细的分析，这些拮抗剂被用来阻断它们的作用。我们的实验重点是染色质和表观遗传学的作用，这对理解它们的功能至关重要。人们希望，这些基础研究将为类固醇激素受体在乳腺癌中发挥的作用以及可能开发新的有效治疗方法提供新的见解。 我们的努力得到了压倒性证据的启发，即充分理解转录控制需要了解目标基因的染色质结构所起的作用，以及释放类固醇受体调节潜力所需的分子机器。为了实现这一点，我们将注意力集中在哺乳动物的BRG1染色质重塑复合体和26S蛋白酶体上，以及它们与糖皮质激素和孕激素受体的相互作用和调节。这些复合体的活性已经在人类和小鼠细胞内染色质的背景下进行了评估，特别关注了RNA聚合酶的活性和核心组蛋白的乙酰化/甲基化。 在环境中暴露于低浓度激素可能会对动物和人类产生永久性的表观遗传影响。类固醇激素受体启动了一个受反应区染色质环境严格调控的遗传程序。使用糖皮质激素受体(GR)作为转录启动的模型因子，我们通过不同的实验方法对染色质结构进行了分类。我们观察到GR激活过程中结构的动态变化，特别是在受体相互作用的区域。我们发现了对激活和染色质调节有不同反应的GR响应区的分布。然而，大多数GR响应区共享相似的基础染色质状态，这表明GR募集具有共同的染色质结构。BRG-1基因敲除显示了ATPase依赖的染色质重塑的反应元件特异性效应。综上所述，这些数据表明，核受体反应区的类别，通过不同的染色质调节事件对激活做出反应，并确定一种染色质结构，该结构对所测试的大多数反应元件进行分类。 最近，我的团队开始了一个令人兴奋的新研究领域，通过表征存在于人类胚胎干细胞中的染色质重塑和染色质修饰复合体来研究这些细胞的基本性质。主要调控蛋白Oct4和Sox2是早期胚胎发育和维持胚胎干细胞(ESC)特性所需的多能性转录因子。多能性的功能机制预计与这些因子转录激活的基因有关。在这里，我们发现Oct4和Sox2结合到miR-302的保守启动子区域，这是一个由8个miRNAs组成的簇，在ESCs和多能细胞中特异表达。MiR-302a在人胚胎干细胞中的表达依赖于Oct4/Sox2，在胚胎发育过程中miR-302a与Oct4在相同的发育阶段和相同的组织中表达。MiR-302的转录激活和其靶标的翻译抑制，如Cyclin D1，可能在Oct4/Sox2和多能细胞的细胞周期调控之间提供了联系。 干细胞的异质性在指导细胞谱系分化途径和自我更新特性方面是研究和治疗应用中的一个重要问题。使用流式细胞术，我们已经在人类胚胎干细胞(HES)细胞系的培养中根据大小鉴定了两个不同的亚群。这两个细胞群对维甲酸(RA)分化和几种内分泌干扰物(EDC)的反应不同。克隆研究表明，这两个种群都能恢复亲本种群。此外，全基因组微阵列鉴定了大约400个在两个群体之间有显著差异表达的基因(p&lt；0.01)。HES细胞作为异质群体存在，对分化信号和环境刺激有不同的反应，这一发现将与它们在药物发现和疾病治疗中的应用相关。 我们的许多模型的性质，人和小鼠，乳腺癌细胞，以及胚胎干细胞，表明我们对女性健康和乳腺癌特别是乳腺癌的积极兴趣。我们的研究计划发现并评估染色质重塑蛋白、26S蛋白酶体、核受体、多能性因子、MirRNAs和启动子染色质结构在调节正常细胞、人类胚胎干细胞、诱导的多能干细胞和癌细胞对内源性和环境信号的转录反应中所起的作用。