ERα与Sp1相互作用激活LRP16启动子转录活性

根据已报道的LRP1 6启动子序列 (2 6kb) ,采用PCR反应获得 6个启动子 5′删除突变体 ,分别插入pGL3 Basic载体 ,构建 6种 5′缺失报告基因表达载体 (pS1 ～pS6) .分别与ERα真核表达载体共转染MCF 7细胞 .用双荧光素酶报告系统测定荧光素酶活性 ,以明确LRP1 6基因上游启动子区域中的雌激素反应序列 .结果显示 ,pS1 ～pS6均有雌二醇反应性 .进而对pS5的 3′端缺失分析发现LRP1 6基因翻译起始位点上游 - 2 1 4至 - 2 5 1位置的序列具有雌激素应答 ,序列分析发现该片段序列中包含了一个供转录因子Sp1结合的GC富含位点和一个ERα反应元件的半位点 (1 2ERE Sp1 ) ,进一步突变分析显示这两个元件均为雌激素反应性所必需 .以含这两个顺式元件的序列 (- 2 5 3bp至 - 2 2 4bp)作为探针 ,超级迁移凝胶电泳试验结果表明了ERα和Sp1蛋白均可以和探针结合 .研究发现了雌激素上调LRP1 6基因表达的一个增强子元件— 1 2ERE Sp1 ,ERα和Sp1蛋白需要与DNA结合形成复合体 ,通过其相互作用激活转录     

HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells

Estrogen receptor (ER) toxicity has hampered the development of vertebrate cell lines stably expressing substantial levels of recombinant wild-type ER. To isolate clonal lines of HeLa cells stably expressing epitope-tagged ER, we used a construction encoding a single bicistronic mRNA, in which FLAG-epitope-tagged human ER alpha (fER) was translated from a 5'-translation initiation site and fused to the neomycin resistance gene, which was translated from an internal ribosome entry site. One stable HeLa-ER-positive cell line (HeLa-ER1) produces 1,300,000 molecules of fER/cell (approximately 20-fold more ER than MCF-7 cells). The HeLa fER is biologically active in vivo, as judged by rapid death of the cells in the presence of either 17 beta-estradiol or trans-hydroxytamoxifen and the ability of the cell line to activate a transfected estrogen response element (ERE)-containing reporter gene. The FLAG-tagged ER was purified to near homogeneity in a single step by immunoaffinity chromatography with anti-FLAG monoclonal antibody. Purified fER exhibited a distribution constant (KD) for 17 beta-estradiol of 0.45 nM. Purified HeLa fER and HeLa fER in crude nuclear extracts exhibit similar KD values for the ERE (0.8 nM and 1 nM, respectively), which are approximately 10 times lower than the KD of 10 nM we determined for purified ER expressed using the baculovirus system. HMG-1 strongly stimulated binding of both crude and purified HeLa fER to the ERE (KD of 0.25 nM). In transfected HeLa cells, HMG-1 exhibited a dose-dependent stimulation of 17 beta-estradiol-dependent transactivation. At high levels of transfected HMG-1 expression plasmid, transactivation by ER became partially ligand-independent, and transactivation by trans-hydroxytamoxifen was increased by more than 25-fold. These data describe a system in which ER, stably expressed in HeLa cells and easily purified, exhibits extremely high affinity for the ERE, and suggest that intracellular levels of HMG-1 may be limiting for ER action.     

Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter

Progesterone receptor gene expression is induced by estrogen in MCF-7 human breast cancer cells. Although it is generally thought that estrogen responsiveness is mediated through estrogen response elements (EREs), the progesterone receptor gene lacks an identifiable ERE. The progesterone receptor A promoter does, however, contain a half-ERE/Sp1 binding site comprised of an ERE half-site upstream of two Sp1 binding sites. We have used in vivo deoxyribonuclease I (DNase I) footprinting to demonstrate that the half-ERE/Sp1 binding site is more protected when MCF-7 cells are treated with estrogen than when cells are not exposed to hormone, suggesting that this region is involved in estrogen-regulated gene expression. The ability of the half-ERE/Sp1 binding site to confer estrogen responsiveness to a simple heterologous promoter was confirmed in transient cotransfection assays. In vitro DNase I footprinting and gel mobility shift assays demonstrated that Sp1 present in MCF-7 nuclear extracts and purified Sp1 protein bound to the two Sp1 sites and that the estrogen receptor enhanced Sp1 binding. In addition to its effects on Sp1 binding, the estrogen receptor also bound directly to the ERE half-site. Taken together, these findings suggest that the estrogen receptor and Sp1 play a role in activation of the human progesterone receptor A promoter.     

雌激素通过LRP16基因5′侧翼GC富含区上调其转录的分子机制

LRP16是一个明确的雌激素(E2)反应性靶基因,已往研究在LRP16基因上游调控区鉴定了一个E2反应性1/2ERE/GC富含的ERα作用位点（-676 bp到-214 bp;命名为A区）.为进一步鉴定雌激素上调LRP16基因表达的最大化反应区域,对LRP16基因上游调控区进行缺失突变,通过相对荧光素酶活性分析观察到LRP16基因的一段5′近端侧翼序列（-213 bp到-24 bp;命名为B区）具有明显的E2反应性.通过与A区比较,认为B区最大化的呈递了E2对LRP16基因的转激活效应.序列分析表明,B区缺乏经典的ERE元件,而包含多个富含GC序列.针对Sp1的siRNA实验结果提示,Sp1参与了E2对该区域的转激活.针对GC富含区进一步的缺失突变,及荧光素酶活性分析,识别了一段30 bp（-213 bp到-184 bp）的序列在B区呈递E2反应活性中发挥核心作用.超级凝胶电泳实验结果表明,Sp1蛋白与这段30 bp的DNA序列在体外存在直接结合作用.染色质免疫共沉淀实验结果证实,ERα、Sp1与B区存在E2依赖性相互作用.本文在LRP16的5′侧翼区识别了一段最大化呈递E2活性的DNA片段.机制研究表明,在E2存在条件下,ERα通过Sp1与该区域的直接作用上调LRP16基因的表达.     

The aryl hydrocarbon receptor activates the retinoic acid receptoralpha through SMRT antagonism

Aryl hydrocarbon receptor (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo(a)pyrene interfere with hormonal regulatory pathways, leading to endocrine disruption. Notably, the activated AhR exerts complex effects on estrogens and retinoids at both levels of their metabolism and regulation of cognate genes. Our current investigation of these AhR effects revealed the TCDD-dependent activation of a subset of retinoid-dependent genes (tissue-transglutaminase, IGF binding protein-3, AhR) in MCF-7 breast cancer cells. A collection of in vitro hormone-dependent reporter gene models showed that AhR activation by TCDD stimulated transactivation by several class I heteromeric receptors (retinoic and thyroid hormone receptors) while it antagonized homodimeric nuclear receptors (estrogen and progesterone receptors, ER and PR). TCDD exerted a dose-dependent effect on a retinoic acid-dependent reporter gene expressed in MCF-7 cells. AhR was shown to be involved in a mutual antagonism with RARalpha corepressor SMRT (silencing mediator of retinoid and thyroid receptors). This, and the documented physical interaction between AhR and SMRT suggested that SMRT sequestration by AhR might activate RARalpha in the absence of ligand. Immunocytochemical studies of AhR and SMRT strongly suggested they colocalized in nuclear bodies during this sequestration. Concurring with this interpretation, we observed an interaction in vitro between AhR and the PML protein, the core component of nuclear bodies. This ability of AhR to elicit spurious activation of retinoid receptors expands the scope of AhR ligands influence beyond ER antagonism and specific Dioxin-responsive genes. Unknown AhR endogenous ligands may also elicit gene transactivation by class I receptors, while being inactive on classic xenobiotic-responsive genes.     

Androgen receptor transactivation assay using green fluorescent protein as a reporter

For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.     

Ligand dependence of estrogen receptor induced changes in chromatin structure.

To determine whether the human estrogen receptor requires ligand to bind to its cognate estrogen receptor element (ERE) in vivo, we have examined the structure of chromatin at a chromosomally integrated ERE-URA3 reporter gene in yeast, and the influence of ligand bound and ligand free estrogen receptors on that structure. Using indirect end-labelling to map DNaseI and micrococcal nuclease sensitive sites, we found that receptor induced alterations in chromatin structure were completely dependent upon the presence of estradiol. These same alterations in chromatin structure were induced by a truncated estrogen receptor with both TAF-1 and TAF-2 transactivation functions deleted, suggesting that DNA binding per se disrupts chromatin structure. These results support models in which the estrogen receptor requires ligand to bind to the ERE in vivo.