REGULATION OF ESTROGEN RESPONSIVE GENES

我们研究的长期目标是通过 哪种雌激素指导基因的转录。这项建议是 被设计用来研究生物效应的分子基础 鸟类繁殖过程中的雌激素。蛋黄蛋白基因都是 对雌激素有反应的；然而，特别是个体差异 诱导的动力学叠加在这个坐标响应上 到雌激素。在给幼鸟注射雌激素后， 极低密度载脂蛋白II(apo-II)mRNA在4小时内出现， 卵黄蛋白原(VTG)II和III的mRNA在6小时内出现，并在 观察20小时后，VTG-I mRNA才能被检测到。分子 滞后的基础和观察到的差异的原因不是 明白了。我们的假设是，特定的蛋白质-DNA相互作用 它们发生在鸡的肝脏中，作为对雌激素的反应 染色质结构，从而以独特的方式标记每个单独的基因。 染色质的这些变化决定了转录的通路 影响基因的因素和个体基因的时间进程 已被激活。拟议中的实验旨在识别和 确定监管要素的功能，以确定时间 这些区域染色质结构的变化过程如下 雌激素的使用，以及与这些结构变化的关系 与伴随着激活的转录速率的改变有关 基因表达。这项建议的具体施舍是：1)确定 载脂蛋白II基因第一内含子内的调控元件，2) 鉴定VTG-I基因启动子中的调控元件，以及 3)确定发生的染色质变化的时间进程 跨越四个卵黄蛋白基因的调控元件 对雌激素的反应。这些研究将主要依靠DNA 足迹法、定点突变、瞬时转染法和 连续转录分析。 雌激素对生殖和发育以及健康都是至关重要的 和疾病。了解雌激素调节的机制 基因表达将为理解异常疾病提供基础 国家和协助治疗设计。

The long range goal of our research is to understand the mechanisms by which estrogen directs the transcription of genes. This proposal is designed to examine the molecular basis for the biological effects of estrogen during avian reproduction. The egg yolk protein genes are all responsive to estrogen; however, individual differences, in particular the kinetics of induction, are superimposed on this coordinate response to estrogen. Following the administration of estrogen to young birds, very low density apolipoprotein II (apo-II) mRNA appears within 4 hours, vitellogenin (VTG) II and III mRNA appear within 6 hours and a lag of 20 hours is observed before VTG-I mRNA can be measured. The molecular basis of the lag and the reasons for the observed differences is not understood. Our hypothesis is that the specific protein-DNA interactions which occur in chicken liver in response to estrogen perturb the chromatin structure thus marking each individual gene in a unique way. These changes in chromatin determine both the access of transcription factors to the gene and the time course with which the individual gene is activated. The proposed experiments are designed to identify and establish the function of the regulatory elements, to define the time course of changes in chromatin structure across these regions following the administration of estrogen, and to relate these structural changes to the altered rates of transcription that accompany the activation of gene expression. The specific alms of this proposal are to 1) identify the regulatory elements within the first intron of the apo-II gene, 2) identify the regulatory elements in the promoter of the VTG-I gene, and 3) determine the time course of the chromatin alterations that occur across the regulatory elements of the four yolk protein genes in response to estrogen. These studies will rely predominantly on DNA footprinting, site directed mutagenesis, transient transfection, and run-on transcription assays. Estrogen is crucial in reproduction and development as well as in health and disease. Understanding the mechanisms by which estrogen regulates gene expression will provide a basis for understanding abnormal disease states and assisting in therapeutic design.