Thyroid hormone control of a gene expression network

描述(由申请人提供)：理解发育过程中核受体信号的一个重要模型系统是非洲爪蛙的变态。当幼虫蝌蚪过渡到成蛙时，甲状腺激素会诱导显著的功能和形态发生变化。关于所有脊椎动物甲状腺激素受体功能的分子机制已经取得了很大的进展。除了高度保守的甲状腺激素受体基因本身的特征外，已经发现了几种受体相互作用蛋白，它们分别在没有或存在配体的情况下介导它们抑制或激活转录的能力。这些辅助激活因子和辅助抑制因子，统称为辅助调节因子，招募修饰靶基因染色质或调节RNA聚合酶II获得激素反应启动子的复合体。协同调节复合体的差异表达和/或活性被认为可以影响细胞和启动子对特定激素的特异性反应，但这些实验主要依赖于瞬时转基因和体外转录系统或基因敲除技术，这些技术剥夺了动物未来受精的这些基因。我们的中心假设是，在变态过程中，配体依赖的共激活和配体非依赖的协同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，使我们能够确定活着的蝌蚪中甲状腺激素受体介导的转录的准确模式。然后，我们将把辅活化子和辅抑制子基因的表达与对自然和合成配体的反应的发育能力的开始以及这些反应的组织特异性联系起来。体内辅抑制子和辅抑制子与甲状腺激素受体的相互作用的相对重要性将通过两种策略来检验：第一，通过NH-3处理，一种化合物可以破坏辅抑制子和辅抑制子与甲状腺激素受体的相互作用；第二，通过组织特异性和可诱导的全长和显性负辅助抑制蛋白的表达。最后，我们将分析TH在大脑和颌骨软骨增殖区调控的基因表达网络。我们特别感兴趣的是确定NH-3对神经元和软骨增殖的选择性兴奋的分子基础。这种选择性的激动剂/拮抗剂是雌激素受体调节剂如他莫昔芬的共同特征，但以前没有被描述为甲状腺激素受体。这些最新的研究将提供关于两种组织中导致细胞增殖的基因表达级联的新信息，这两种组织依赖于TH在两栖动物和哺乳动物(包括人类)中的正常发育。总之，非洲爪哇变态是确定甲状腺激素受体及其辅助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作用的有效模型。

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.