Thyroid hormone control of a gene expression network

描述（申请者提供）：非洲爪蟾（Xenopus laevis）蛙的变态是了解发育过程中核受体信号传导的一个重要模型系统。甲状腺激素在蝌蚪幼虫转变为成年蛙的过程中引起了显著的功能和形态发生变化。在所有脊椎动物甲状腺激素受体功能的分子机制方面取得了很大进展。除了高度保守的甲状腺激素受体基因本身的特征外，已经确定了几种受体相互作用蛋白，分别介导它们在配体缺失或存在时抑制或激活转录的能力。这些共激活因子和共抑制因子，统称为共调节因子，募集复合物，修饰靶基因染色质或调节RNA聚合酶II进入激素应答启动子。共调节复合物的差异表达和/或活性被认为会影响细胞和启动子对特定激素的特异性反应，但这些实验大多依赖于瞬时转染和体外转录系统或基因敲除技术，这些技术会使动物在受精后失去这些基因。我们的中心假设是，在变态过程中，配体依赖性共激活和配体非依赖性共抑制的平衡对于甲状腺激素的适当的时间和空间反应至关重要。为了验证这一假设，我们将开发转基因蝌蚪，使我们能够确定活蝌蚪中甲状腺激素受体介导的转录的精确模式。然后，我们将把辅助激活因子和辅助抑制因子基因表达与发育能力的开始联系起来，以应对天然和合成的配体以及这些反应的组织特异性。在体内，辅抑制因子和辅激活因子与甲状腺激素受体相互作用的相对重要性将通过两种策略进行测试：首先，通过NH-3治疗，一种破坏辅激活因子和辅抑制因子与甲状腺激素受体相互作用的化合物；第二，通过组织特异性和诱导表达全长和显性负性辅抑制蛋白。最后，我们将分析由TH在脑和下颌软骨增生区域调节的基因表达网络。我们特别感兴趣的是确定NH-3对神经元与软骨增殖的选择性激动作用的分子基础。这种选择性激动/拮抗作用是雌激素受体调节剂（如他莫昔芬）的共同特征，但在甲状腺激素受体中尚未见报道。这些最新的研究将为两栖动物和哺乳动物（包括人类）两种依赖TH正常发育的组织中导致细胞增殖的基因表达级联提供新的信息。综上所述，非洲爪蟾变态是确定甲状腺激素受体及其共调节因子在脊椎动物发育过程中对基因表达网络的组织特异性控制中的作用的有力模型。

DESCRIPTION (provided by applicant): An important model system for understanding nuclear receptor signaling during development is metamorphosis of the frog Xenopus laevis. Thyroid hormone induces remarkable functional and morphogenetic changes as the larval tadpole transitions into the adult frog. A great deal of progress has been made on the molecular mechanisms of thyroid hormone receptor function in all vertebrates. Besides characterization of the highly conserved thyroid hormone receptor genes themselves, several receptor interacting proteins have been identified that mediate their ability to repress or activate transcription in the absence or presence of ligand, respectively. These coactivators and corepressors, collectively termed coregulators, recruit complexes that modify target gene chromatin or modulate RNA polymerase II access to hormone responsive promoters. Differential expression and/or activity of coregulator complexes have been suggested to influence cell and promoter specific responses to a given hormone, but these experiments have mostly relied on transient transfection and in vitro transcription systems or gene knockout technologies that deprive the animal of these genes from fertilization onward. Our central hypothesis is that the balance of ligand-dependent coactivation and ligand-independent corepression is critical for proper temporal and spatial responses to thyroid hormone during metamorphosis. To test this hypothesis, we will develop transgenic tadpoles that allow us to determine the precise pattern of thyroid hormone receptor mediated transcription in living tadpoles. We will then correlate coactivator and corepressor gene expression with the onset of developmental competence to respond to natural and synthetic Iigands and the tissue specificity of those responses. The relative importance of corepressor and coactivator interactions with the thyroid hormone receptor in vivo will then be tested by two strategies: first, by treatment with NH-3, a compound that disrupts both coactivator and corepressor interaction with the thyroid hormone receptor; and second, by tissue specific and inducible expression of full-length and dominant negative corepressor proteins. Finally, we will analyze gene expression networks regulated by TH in proliferating regions of the brain and jaw cartilage. We are particularly interested in determining the molecular basis for the selective agonism of NH-3 on neuronal versus cartilage proliferation. This selective agonism/antagonism is a common feature of estrogen receptor modulators such as tamoxifen but has not been previously described for thyroid hormone receptors. These last studies will provide new information on gene expression cascades leading to cellular proliferation in two tissues that are dependent on TH for proper development in both amphibians and mammals, including humans. In summary, Xenopus laevis metamorphosis is a powerful model for determining the role of thyroid hormone receptors and their coregulators in tissue specific control of gene expression networks during vertebrate development.