An upstream region of the rat luteinizing hormone β gene binds estrogen receptor and confers estrogen responsiveness

• Published in: The Journal of biological chemistry 1989-01, Vol.264 (1), p.80-86
• Main Authors: Shupnik, M A, Weinmann, C M, Notides, A C, Chin, W W
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Biotechnology; Cattle; Cell Line; Cloning, Molecular; Estradiol - pharmacology; Estrogens - physiology; Female; Fundamental and applied biological sciences. Psychology; Gene expression; Genes - drug effects; Genes, Regulator; Genetic engineering; Genetic technics; Luteinizing Hormone - genetics; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Protein Binding; Receptors, Estrogen - isolation & purification; Receptors, Estrogen - metabolism; Transcription, Genetic; Transfection; Uterus - metabolism; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)31226-7

Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or by indirect effects of steroid on the hypothalamus. To study direct estrogen regulation of the rat luteinizing hormone β (LHβ) gene, we performed estrogen receptor-DNA binding studies and transient expression gene transfer experiments. Nitrocellulose filter binding studies were performed with purified estrogen receptor from calf uterus and labeled fragments of the LHβ gene. Dose-dependent specific binding to receptor occurred only with LHβ gene fragments containing a common 284-base region from −1388 to −1105 bases upstream from the transcriptional start site. This DNA region contained a 15-base imperfect palindromic region (GGACACCATCTGTCC) with sequence similarity to other estrogen-responsive elements. Biological function was tested by inserting portions of the 5′-flanking region of the gene next to the herpes simplex virus thymidine kinase promoter fused to the chloramphenicol acetyltransferase gene (LHβ-tkCAT) and performing gene transfer experiments with the pituitary GH3 cell line. Promoter activity in LHβ-tkCAT constructs containing LHβ gene sequences from bases −2013 to −613, or from bases −1388 to −613 in either orientation, exhibited stimulation with 17 β-estradiol (E2) treatment; in contrast, constructs containing bases −885 to −613 were not regulated by E2. Positive regulation by E2 exhibited dose- and time-dependent stimulation, with a maximum 2- to 6-fold effect achieved after 48 h of treatment with 10-8M E2. The estrogen receptor appeared to be required for this biological response. Stimulation of LH β-tkCAT constructs did not occur in L cells with undetectable levels of E2 receptor, but did occur after cotransfection of an LH β-tkCAT construct and an expression vector containing the human estrogen receptor cDNA. These studies demonstrate that a 5′-flanking region of the rat LHβ gene can bind to the estrogen receptor and that this region can confer hormonal responsiveness to a heterologous promoter. Thus, positive steroid regulation of luteinizing hormone may occur directly on the pituitary at the level of the LHβ gene.