Regulation of Estrogen-Responsive Genes

雌激素反应基因的调控

• 批准号: 6723446
• 负责人: Ann Nardulli
• 金额: $ 34.02万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723446
• 关键词: chromatin immunoprecipitation; estrogen receptors; estrogens; gene expression; genetic regulation; genetic regulatory element; genetic transcription; hormone regulation /control mechanism; mammary gland; mass spectrometry; progesterone receptors; uterus

DESCRIPTION (provided by applicant): The steroid hormone estrogen plays a critical role in development and maintenance of female reproductive and mammary tissues, but is also involved in maintenance of cardiovascular, skeletal, and neural function, proliferation of breast cancer cells, and concentration of sperm in the male reproductive tract. Moreover, estrogens and selective estrogen receptor modulators (SERMs) are widely used in regulating fertility, alleviating postmenopausal symptoms, and preventing and treating breast cancer. Although it is widely accepted that the estrogen-occupied receptor mediates its effects by interacting with estrogen response elements (EREs) residing in target genes, a number of estrogen-responsive genes contain no identifiable ERE. To understand how estrogen-responsive genes containing AP-1 and Sp1 sites but lacking EREs respond to hormone treatment, we will study the regulation of the endogenous progesterone receptor (PR) gene. The PR plays a crucial role in reproductive and mammary cell development and fertility and has been used clinically as an indicator of estrogen-responsiveness in human breast cancer tumors. Recent studies from our laboratory indicate that the human PR gene, which lacks an ERE, is controlled by multiple cis elements including an AP-1 site and two Sp 1 sites adjacent to an ERE half site. Thus, the PR gene provides an excellent model to define mechanisms by which target genes lacking an ERE can be regulated by hormone. We will monitor the ordered recruitment of transcription factors and coregulatory proteins to these AP-1 and Sp 1 sites in the endogenous PR gene to delineate the temporal sequence of events required for maintaining basal PR expression and initiating and sustaining high levels of transcription using highly sensitive chromatin immunoprecipitation assays. The cis elements and coregutatory proteins involved in mediating the effects of estrogen and the SERMs 4-hydroxytamoxifen and raloxifene will be determined using in vivo footprinting and chromatin immunoprecipitation experiments. Both mammary and uterine cell lines will be examined to define mechanisms by which these clinically important SERMs mediate their tissue-specific effects. To define mechanisms by which estrogen-responsive genes containing AP-1 and Sp 1 sites are regulated, we will isolate and identify novel coregulatory proteins that associate with AP-1 and Sp 1 sites using agarose gel electrophoresis and mass spectrometry analysis and characterize the effects of these proteins on expression of genes containing AP-1 and Sp 1 sites. The insights gained in these combined studies will help to delineate how estrogens and SERMs regulate expression of genes containing AP-1 and Sp1 sites and enhance our understanding of cellular responsiveness of mammary tumor cells as well as normal tissues such as the uterus and breast to these clinically important pharmaceutical agents.

描述（由申请人提供）：类固醇激素雌激素在女性生殖和乳腺组织的发育和维持中起着关键作用，但也参与心血管、骨骼和神经功能的维持，乳腺癌细胞的增殖和男性生殖道精子的浓度。此外，雌激素和选择性雌激素受体调节剂（SERMs）在调节生育、缓解绝经后症状、预防和治疗乳腺癌方面被广泛应用。尽管人们普遍认为雌激素占据受体通过与靶基因中的雌激素反应元件（estrogen response elements, EREs）相互作用来调节其作用，但许多雌激素反应基因中不含可识别的雌激素反应元件。为了了解含有AP-1和Sp1位点但缺乏EREs的雌激素应答基因对激素治疗的反应，我们将研究内源性孕激素受体（PR）基因的调控。PR在生殖和乳腺细胞发育和生育中起着至关重要的作用，在临床上已被用作人类乳腺癌肿瘤中雌激素反应性的指标。我们实验室最近的研究表明，缺乏ERE的人类PR基因由多个顺式元件控制，包括一个AP-1位点和两个与ERE半位点相邻的Sp -1位点。因此，PR基因提供了一个很好的模型来定义缺乏ERE的靶基因可以被激素调节的机制。我们将监测内源性PR基因AP-1和Sp -1位点的转录因子和协同调节蛋白的有序募集，以描述维持PR基础表达和启动并维持高水平转录所需事件的时间序列，使用高度敏感的染色质免疫沉淀测定。参与介导雌激素和SERMs的顺式元件和协同调节蛋白将通过体内足迹和染色质免疫沉淀实验确定4-羟他莫昔芬和雷洛昔芬。乳腺和子宫细胞系将被检查，以确定这些临床重要的serm介导其组织特异性作用的机制。为了确定含有AP-1和Sp -1位点的雌激素应答基因的调控机制，我们将使用琼脂糖凝胶电泳和质谱分析分离和鉴定与AP-1和Sp -1位点相关的新型共调控蛋白，并表征这些蛋白对含有AP-1和Sp -1位点的基因表达的影响。在这些联合研究中获得的见解将有助于描述雌激素和serm如何调节含有AP-1和Sp1位点的基因表达，并增强我们对乳腺肿瘤细胞以及子宫和乳房等正常组织对这些临床重要药物的细胞反应性的理解。