Molecular mechanism of thyroid hormone receptor function during metamorphosis

变态过程中甲状腺激素受体功能的分子机制

• 批准号: 7968611
• 负责人: Yun-Bo Shi
• 金额: $ 85.73万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/7968611
• 关键词: Adult; Affect; African; Amphibia; Animals; Binding; Biological Metamorphosis; Biological Models; Cell Culture Techniques; Cell Death; Cell Proliferation; Chemicals; Classification; Complex; Connective Tissue; Coupled; Development; Developmental Process; EP300 gene; Embryo; Endocrine; Endocrine Disruptors; Endocrine disruption; Endocrine system; Ensure; Epithelial Cells; Epithelium; Exposure to; Future; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Gene Transfer Techniques; Genes; Genetic Transcription; Goals; Growth; Growth and Development function; Health; Hormones; Human; Human Development; In Vitro; Intestines; Knowledge; Ligands; Limb structure; Mammalian Cell; Mediating; Metabolism; Methyltransferase; Microarray Analysis; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Morphogenesis; Nuclear Receptors; Oocytes; Organ; Organ Model; Organism; Pathway interactions; Plastics; Play; Population; Process; Property; RXR; Rana; Recruitment Activity; Regulation; Research; Response Elements; Role; Sex Differentiation; Signal Pathway; Signal Transduction; Staging; Stem Cell Development; Steroid Receptors; Structure; System; Tadpoles; Tail; Testing; Thyroid Hormone Receptor; Thyroid Hormones; Time; Tissues; Transcriptional Activation; Transgenic Organisms; Tubular formation; Uterus; Vertebrates; Water; Xenopus; Xenopus laevis; base; bisphenol A; cDNA Arrays; cofactor; estrogenic activity; genome wide association study; genome-wide; genome-wide analysis; hormone response element; in vivo; in vivo Model; model development; molecular marker; overexpression; prevent; programs; promoter; receptor; receptor binding; receptor expression; receptor function; response; success; tissue/cell culture; toad; tool

ROLES OF COFACTORS IN GENE REGULATION BY TR. TR Based on TR expression profiles and its molecular properties, we have previously proposed a dual function model for TR during frog development. That is, the heterodimers between TR and RXR (9-cis retinoic acid receptor) bind to target genes in vivo. In premetamorphic tadpoles, they repress gene expression in the absence of TH to prevent metamorphosis, thus ensuring a proper tadpole growth period. When TH is present either from endogenous synthesis during development or exogenous addition to the raring water of premetamorphic tadpoles, TR/RXR heterodimers activate TH-inducible genes to initiate metamorphosis. Our studies in the last several years have shown that TR is both necessary and sufficient for the metamorphic effects of TH. Thus metamorphosis provides the first example where TR is shown to mediate directly and sufficiently the developmental effects of TH. Furthermore, we have shown that TR regulates metamorphic timing by recruiting corepressor complexes to target genes in premetamorphic tadpoles to prevent precocious metamorphosis. During metamorphosis, TR needs to recruit coactivator complexes containing SRC3 (steroid receptor coactivator 3)/p300 to target genes for their activation and metamorphosis. These findings represent the first example whether specific cofactor complexes have been shown to play critical roles for developmental function of a nuclear receptor in vertebrates. The SRC/p300 complexes also contain the methyltransferase PRMT1, which has been implicated in TR function in mammalian cell culture studies. Thus, to further investigate the role and mechanisms of the SRC/p300 complexes in development, we have cloned and characterized Xenopus laevis PRMT1. By using intestinal remodeling during Xenopus laevis metamorphosis for in vivo molecular analysis, we showed that PRMT1 expression was upregulated during metamorphosis when both TR and TH were present. We then demonstrated a role of PRMT1 in TR-mediated transcription by showing that PRMT1 enhanced transcriptional activation by liganded TR in the frog oocyte transcription system and was recruited to the TH response element (TRE) of the target promoter in the oocyte as well as to endogenous TREs during frog metamorphosis. Surprisingly, we found that PRMT1 was only transiently recruited to the TREs in the target during metamorphosis and observed no PRMT1 recruitment to TREs at the climax of intestinal remodeling when both PRMT1 and TH were at peak levels. Mechanistically, we showed that overexpression of PRMT1 enhanced TR binding to TREs both in the frog oocyte model system and during metamorphosis. More importantly, transgenic overexpression of PRMT1 enhanced gene activation in vivo and accelerated both natural and TH-induced metamorphosis. These results thus indicate that PRMT1 functions transiently as a coactivator in TR-mediated transcription by enhancing TR-TRE binding and further suggest that PRMT1 has tissue specific roles to regulate the rate of metamorphosis. ANALYZING THE GENE EXPRESSION PROGRAMS UNDERLYING THE TEMPORAL AND TISSUE-DEPENDENT TRANSFORMATIONS DURING METAMORPHOSIS. The complexity of metamorphic changes in different organs argues for the presence of different gene regulation programs regulated by TR. Knowledge on this systematic gene regulation will help to identify not only molecular markers but also important cellular pathways or critical genes for future mechanistic studies. Thus, we have begun to use the recently developed Xenopus laevis cDNA array to analyze genome-wide gene expression changes associated with TH-induced intestinal remodeling. Our initial analysis of animals treated with TH for different number of days have provided a molecular description of the gene regulation pathways associated with different metamorphic processes in the intestine. The success of this study also prompted us to ask whether we could use metamorphosis coupled with cDNA array analysis as a model to study whether endocrine disrupting compounds (EDCs) can affect vertebrate development via the TH signaling pathway. EDCs are exogenous substances that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub) populations. As TH plays a central role in vertebrate development, growth, and metabolism, the effects of EDCs on TH signaling will undoubtedly pose a threat to human and wildlife health. However, the lack of a suitable in vivo model to study EDCs effects on TR function in vertebrate development impedes our understanding on whether and how persistent exposure to these bioaccumulative compounds affects human health. As a test case, we analyzed the effect of bisphenol A (BPA), on Xenopus metamorphosis. BPA, a chemical widely used to manufacture plastics, is estrogenic and capable of disrupting sex differentiation. However, recent in vitro studies have shown that BPA can also antagonize TH activation of TRs. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. We studied the effect of BPA on TH-dependent metamorphosis at both morphological and molecular levels. After 4 days of exposure, BPA inhibited TH-induced intestinal remodeling in premetamorphic Xenopus laevis tadpoles. Importantly, microarray analysis revealed that BPA antagonized the regulation of most TH-response genes, thereby explaining the inhibitory effect of BPA on metamorphosis. Surprisingly, most of the genes affected by BPA in the presence of TH were TH-response genes, suggesting that BPA predominantly affected TH-signaling pathways during metamorphosis. Our finding that this endocrine disruptor, well known for its estrogenic activity in vitro, functions to inhibit TH-pathways to affect vertebrate development in vivo thus not only provides a mechanism for the likely deleterious effects of BPA on human development but also demonstrates the importance of studying endocrine-disruption in a developmental context in vivo.

TR 辅助因子在基因调控中的作用。 TR 基于TR表达谱及其分子特性，我们之前提出了青蛙发育过程中TR的双功能模型。也就是说，TR和RXR（9-顺式视黄酸受体）之间的异二聚体在体内与靶基因结合。在变态前的蝌蚪中，它们在没有 TH 的情况下抑制基因表达以防止变态，从而确保蝌蚪有适当的生长期。当 TH 存在于发育过程中的内源合成或外源添加到变态前蝌蚪的稀有水中时，TR/RXR 异二聚体会激活 TH 诱导基因以启动变态。我们过去几年的研究表明，TR 对于 TH 的变质作用来说既是必要的也是充分的。因此，变态提供了第一个例子，其中 TR 被证明可以直接且充分地介导 TH 的发育效应。此外，我们还发现TR通过招募辅阻遏物复合物到变态前蝌蚪的靶基因来调节变态时间，以防止变态早熟。 在变态过程中，TR 需要招募含有 SRC3（类固醇受体共激活剂 3）/p300 的共激活剂复合物来靶向基因，以实现其激活和变态。这些发现代表了第一个例子，证明特定辅因子复合物是否已被证明对脊椎动物核受体的发育功能发挥关键作用。 SRC/p300 复合物还含有甲基转移酶 PRMT1，该酶与哺乳动物细胞培养研究中的 TR 功能有关。因此，为了进一步研究 SRC/p300 复合物在发育中的作用和机制，我们克隆并表征了非洲爪蟾 PRMT1。通过利用非洲爪蟾变态过程中的肠道重塑进行体内分子分析，我们发现当 TR 和 TH 都存在时，PRMT1 表达在变态过程中上调。然后，我们证明了 PRMT1 在 TR 介导的转录中的作用，表明 PRMT1 通过配体 TR 在青蛙卵母细胞转录系统中增强转录激活，并被招募到卵母细胞中靶启动子的 TH 反应元件（TRE）以及青蛙变态过程中的内源性 TRE。令人惊讶的是，我们发现PRMT1仅在变态过程中短暂地被招募到靶标的TRE上，并且在肠道重塑的高潮时，当PRMT1和TH都处于峰值水平时，没有观察到PRMT1被招募到TRE上。从机制上讲，我们发现在青蛙卵母细胞模型系统和变态过程中，PRMT1 的过度表达增强了 TR 与 TRE 的结合。更重要的是，PRMT1 的转基因过表达增强了体内基因激活并加速了自然和 TH 诱导的变态。因此，这些结果表明PRMT1通过增强TR-TRE结合而在TR介导的转录中短暂地发挥共激活剂的作用，并进一步表明PRMT1具有调节变态速率的组织特异性作用。 分析变态过程中时间和组织依赖性转化的基因表达程序。不同器官变质变化的复杂性证明了TR调节的不同基因调控程序的存在。关于这种系统基因调控的知识不仅有助于识别分子标记，还有助于识别未来机制研究的重要细胞途径或关键基因。因此，我们开始使用最近开发的非洲爪蟾 cDNA 阵列来分析与 TH 诱导的肠道重塑相关的全基因组基因表达变化。我们对接受 TH 治疗不同天数的动物进行了初步分析，提供了与肠道不同变质过程相关的基因调控途径的分子描述。 这项研究的成功也促使我们思考是否可以使用变态与 cDNA 阵列分析相结合作为模型来研究内分泌干扰化合物 (EDC) 是否可以通过 TH 信号通路影响脊椎动物的发育。 EDC 是一种外源性物质，会改变内分泌系统的功能，从而对完整的生物体或其后代或（亚）群体造成不利的健康影响。由于TH在脊椎动物的发育、生长和代谢中发挥着核心作用，EDCs对TH信号传导的影响无疑会对人类和野生动物的健康构成威胁。然而，由于缺乏合适的体内模型来研究 EDC 对脊椎动物发育中 TR 功能的影响，这阻碍了我们对持续接触这些生物累积性化合物是否以及如何影响人类健康的理解。作为测试案例，我们分析了双酚 A (BPA) 对非洲爪蟾变态的影响。 BPA 是一种广泛用于制造塑料的化学物质，具有雌激素作用，能够破坏性别分化。然而，最近的体外研究表明，BPA还可以拮抗TRs的TH激活。研究子宫内哺乳动物胚胎的困难阻碍了对 BPA 在脊椎动物发育过程中的直接影响的分析。我们在形态学和分子水平上研究了 BPA 对 TH 依赖性变态的影响。暴露 4 天后，BPA 抑制 TH 诱导的变态非洲爪蟾蝌蚪肠道重塑。重要的是，微阵列分析表明，BPA 拮抗大多数 TH 反应基因的调节，从而解释了 BPA 对变态的抑制作用。令人惊讶的是，在 TH 存在的情况下，大多数受 BPA 影响的基因都是 TH 反应基因，这表明 BPA 在变态过程中主要影响 TH 信号通路。我们的发现是，这种内分泌干扰物以其体外雌激素活性而闻名，其功能是抑制 TH 通路，从而影响体内脊椎动物的发育，因此，这不仅为 BPA 对人类发育可能产生的有害影响提供了一种机制，而且还证明了研究体内发育背景下的内分泌干扰。