REGULATION OF ESTROGEN-RESPONSIVE GENES

我们研究的长期目标是了解雌激素指导基因转录的机制。本研究旨在从分子水平探讨雌激素在鸟类繁殖过程中的生物学效应。蛋黄蛋白基因都对雌激素有反应;然而，个体差异，特别是诱导的动力学，叠加在对雌激素的这种协调反应上。幼鸟雌激素给药后，4小时内出现极低密度载脂蛋白II（apo-II）mRNA，6小时内出现卵黄蛋白原（VTG）II和III mRNA，观察到20小时的滞后后才能测量VTG-I mRNA。滞后的分子基础和观察到的差异的原因尚不清楚。我们的假设是，鸡肝脏中发生的特异性蛋白质-DNA相互作用对雌激素的反应扰乱了染色质的结构，从而以独特的方式标记了每个基因。染色质的这些变化决定了转录因子对基因的接近和单个基因被激活的时间过程。所提出的实验被设计为鉴定和建立调控元件的功能，以定义在施用伴随基因表达的激活之后在这些区域中结构化的染色质的变化的时间过程。该建议的具体目的是：1）鉴定apo-II基因第一内含子内的调控元件，2）鉴定CTG-I基因启动子中的调控元件，3）确定响应雌激素的四种卵黄蛋白基因的调控元件发生染色质改变的时间过程。这些研究将主要依赖于DNA足迹法、定点诱变、瞬时转染和连续转录测定。雌激素在生殖和发育以及健康和疾病中至关重要。了解雌激素调节基因表达的机制将为了解异常疾病状态和辅助治疗设计提供基础。

The long range goal of our research is to understand the mechanisms by which estrogen directs the transcription of genes. This proposal designed to examine the molecular 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 structured 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 structured across these regions following the administration of accompany the activation of gene expression. The specific aims 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 CTG-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.