Richard S, Zingg H H
Laboratory of Molecular Endocrinology, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1990 Apr 15;265(11):6098-103.
Gonadal steroids affect brain function primarily by altering the expression of specific genes, yet the specific mechanisms by which neuronal target genes undergo such regulation are unknown. Recent evidence suggests that the expression of the neuropeptide gene for oxytocin (OT) is modulated by estrogens. We therefore examined the possibility that this regulation occurred via a direct interaction of the estrogen-receptor complex with cis-acting elements flanking the OT gene. DNA-mediated gene transfer experiments were performed using Neuro-2a neuroblastoma cells and chimeric plasmids containing portions of the human OT gene 5'-glanking region linked to the chloramphenicol acetyltransferase gene. We identified a 19-base pair region located at -164 to -146 upstream of the transcription start site which is capable of conferring estrogen responsiveness to the homologous as well as to a heterologous promoter. The hormonal response is strictly dependent on the presence of intracellular estrogen receptors, since estrogen induced stimulation occurred only in Neuro-2a cells co-transfected with an expression vector for the human estrogen receptor. The identified region contains a novel imperfect palindrome (GGTGACCTTGACC) with sequence similarity to other estrogen response elements (EREs). To define cis-acting elements that function in synergism with the ERE, sequences 3' to the ERE were deleted, including the CCAAT box, two additional motifs corresponding to the right half of the ERE palindrome (TGACC), as well as a CTGCTAA heptamer similar to the "elegans box" found in Caenorhabditis elegans. Interestingly, optimal function of the identified ERE was fully independent of these elements and only required a short promoter region (-49 to +36). Our studies define a molecular mechanism by which estrogens can directly modulate OT gene expression. However, only a subset of OT neurons are capable of binding estrogens, therefore, direct action of estrogens on the OT gene may be restricted to a subpopulation of OT neurons.
性腺类固醇主要通过改变特定基因的表达来影响脑功能，然而神经元靶基因受此类调控的具体机制尚不清楚。最近的证据表明，雌激素可调节催产素（OT）神经肽基因的表达。因此，我们研究了这种调节是否通过雌激素受体复合物与OT基因侧翼的顺式作用元件直接相互作用而发生。使用Neuro-2a神经母细胞瘤细胞和含有与氯霉素乙酰转移酶基因相连的人OT基因5'侧翼区部分的嵌合质粒进行了DNA介导的基因转移实验。我们确定了一个位于转录起始位点上游-164至-146的19个碱基对区域，该区域能够赋予同源及异源启动子雌激素反应性。激素反应严格依赖于细胞内雌激素受体的存在，因为雌激素诱导的刺激仅发生在与人类雌激素受体表达载体共转染的Neuro-2a细胞中。所确定的区域包含一个新的不完全回文序列（GGTGACCTTGACC），其序列与其他雌激素反应元件（ERE）相似。为了确定与ERE协同作用的顺式作用元件，删除了ERE 3'端的序列，包括CCAAT框、与ERE回文序列右半部分相对应的另外两个基序（TGACC），以及一个类似于秀丽隐杆线虫中发现的“线虫框”的CTGCTAA七聚体。有趣的是，所确定的ERE的最佳功能完全独立于这些元件，只需要一个短的启动子区域（-49至+36）。我们的研究确定了雌激素可直接调节OT基因表达的分子机制。然而，只有一部分OT神经元能够结合雌激素，因此，雌激素对OT基因的直接作用可能仅限于OT神经元的一个亚群。
