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.

辅助因子在基因调节中的作用。 TR基于TR表达谱及其分子特性，我们先前已经提出了青蛙发育过程中TR的双功能模型。也就是说，TR和RXR之间的异二聚体（9-Cis视黄酸受体）与体内的靶基因结合。在未经TH的情况下，它们抑制基因表达，以防止变形，从而确保了适当的t骨生长期。当在发育过程中内源性合成或外源性添加到外源性the的稀有水的外源性中时，TR/RXR异二聚体会激活Th诱导基因以启动变质。在过去的几年中，我们的研究表明，对于Th的变质作用，TR既需要且足够。因此，变态提供了第一个例子，其中TR显示可直接介导Th的发育效果。此外，我们已经表明，TR通过募集核压形复合物为靶基t的靶基因来调节变质时机，以防止早产性变质。 在变形过程中，TR需要募集含有SRC3（类固醇受体共激活剂3）/P300的共激活因子络合物，以靶向基因激活和变形。这些发现是第一个例子，是否已证明特定的辅助因子对脊椎动物中核受体的发育功能起着关键作用。 SRC/P300复合物还包含甲基转移酶PRMT1，该甲基转移酶PRMT1与哺乳动物细胞培养研究有关。因此，为了进一步研究SRC/p300复合物在发育中的作用和机制，我们已经克隆并表征了Xenopus laevis prmt1。通过在爪蟾Laevis变形过程中使用肠重塑进行体内分子分析，我们表明当TR和Th都存在时，PRMT1表达在变质过程中上调。然后，我们通过证明PRMT1在Frog卵母细胞转录系统中增强了PRMT1在FROG卵母细胞转录系统中的转录激活，并被募集到卵母细胞中靶启动子的TH响应元件（TRE），以及在青蛙的内源性TRES中募集到靶启动子的TH响应元件（TRE），从而证明了PRMT1在TR介导的转录中的作用。令人惊讶的是，我们发现PRMT1仅在变态过程中仅暂时募集到靶标的TRE，并且在PRMT1和TH均处于峰值水平时，没有观察到在肠重塑的高潮时TRE的PRMT1募集。从机械上讲，我们表明PRMT1的过表达增强了TR与Frog卵母细胞模型系统和变态过程中的TR结合。更重要的是，PRMT1的转基因过表达在体内增强了基因激活，并加速了天然和TH诱导的变形。因此，这些结果表明，PRMT1通过增强TRE结合而在TR介导的转录中瞬​​时起作用，并进一步表明PRMT1具有组织特异性的作用以调节变形率。 分析变形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表明存在由TR调节的不同基因调节程序。有关此系统基因调控的知识将不仅有助于鉴定分子标记物，还有助于确定重要的细胞途径或关键基因，以实现未来的机械研究。因此，我们已经开始使用最近开发的爪蟾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。我们对用TH处理不同天的动物的初步分析提供了对与肠中不同变质过程相关的基因调节途径的分子描述。 这项研究的成功还促使我们询问我们是否可以使用变形，再加上cDNA阵列分析作为研究内分泌干扰化合物（EDC）是否可以通过TH信号途径影响脊椎动物的发育。 EDC是改变内分泌系统功能的外源物质，因此在完整的生物体或其后代或（亚）种群中会引起不良健康影响。由于TH在脊椎动物的发展，生长和代谢中起着核心作用，因此EDC对TH信号的影响无疑会对人类和野生动植物的健康构成威胁。然而，缺乏适合研究EDC对脊椎动物发育中TR功能的影响的体内模型阻碍了我们对这些生物蓄积化合物是否以及如何持续暴露对这些生物污染化合物的理解会影响人类健康。作为测试案例，我们分析了双苯酚A（BPA）对爪蟾变态的影响。 BPA是一种用于制造塑料的化学物质，具有雌激素，能够破坏性别分化。但是，最近的体外研究表明，BPA也可以拮抗TR的激活。研究子宫封闭的哺乳动物胚胎的困难阻碍了对脊椎动物发育过程中BPA的直接影响的分析。我们研究了BPA对形态学和分子水平上Th依赖性变态的影响。暴露4天后，BPA抑制了tH诱导的肠道重塑，在Xenopus laevis Tadpoles中。重要的是，微阵列分析表明，BPA拮抗了大多数TH响应基因的调节，从而解释了BPA对变态的抑制作用。出乎意料的是，在TH存在下受BPA影响的大多数基因都是Th响应基因，这表明BPA主要影响变质过程中的Th-Th-Signalail途径。我们的发现，这种内分泌破坏者以其体外的雌激素活性而闻名，其作用可抑制th轨道在体内影响脊椎动物发育，因此不仅为BPA对人类发育的可能有害影响提供了一种机制，而且还证明了在Vivo中研究内部分泌局部中断的重要性。