Abstract
Previous work from this laboratory demonstrated that 17-beta estradiol (E2) can directly stimulate the transcription rate of the rat luteinizing hormone beta (LH beta) gene and that an upstream portion of the LH beta gene between -2.0 and -0.6 kilobases could confer an E2-stimulated response to a reporter gene in transient expression assays. To localize the LH beta estrogen response element (ERE) by biological function, portions of the 5'-flanking region of the LH beta gene or synthetic oligonucleotides were inserted in expression vectors next to the herpes simplex virus thymidine kinase promoter fused to the chloramphenicol acetyltransferase gene. Constructs were transfected into GH3 cells, and transfected cells were treated for 48 h with E2. E2 stimulation of activity (2-4-fold) occurred with constructs containing the 15-base pair palindromic sequence (GGACACCATCTGTCC), found at bases -1173 to -1159 relative to the transcriptional start site in the LH beta gene. A construct containing a synthetic oligonucleotide of this putative LH beta ERE was stimulated 1.7-3-fold by E2, while a construct containing two copies of the sequence was stimulated to a slightly higher level (2.5-4.0-fold). An oligonucleotide in which the palindrome was mutated failed to confer E2 stimulation, and mutation of the palindromic region within the upstream region of the LH beta gene also eliminated the E2 response. The anti-estrogen tamoxifen could not elicit a response, nor could dehydrotestosterone or dexamethasone; however, thyroid hormone treatment resulted in a 2-2.5-fold stimulation. The 15-base pair LH beta gene palindrome was found to bind estrogen receptor (ER) complex directly by gel retardation experiments. Labeled LH beta ERE DNA formed three complexes with proteins from immature rat uterine extract. Two of these were associated with ER complexes, as determined by the comigration of [3H] estradiol bound to ER with these complexes, and by the ability of anti-ER antibody to associate with these complexes. The affinity of the LH beta ERE for ER was calculated by Scatchard analysis to be 2.2-5.0 nM, an approximately 5-10-fold lower affinity than for the ERE in the vitellogenin A2 gene region. The mutated ERE, which had no biological activity, could not compete effectively for binding to ER. ER which was heat-transformed at 30 degrees C had a similar affinity (2-5 nM) for the ERE as ER occupied with E2 (2-4 nM), while ER occupied by estrone had a lower affinity (9 nM).(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Scription rate of the rat luteinizing hormone j3 (LHB) There was no observed alteration in the pattern of gene and that an upstream portion of the LHB gene migration ofLHB ERE-estrogen receptor (ER) complexesformed with between -2.0 and -0.6 kilobases could confer an E, heat-transformed ER, or ER occupied by various estrostimulatedresponseto a reporter gene in transient gens
To localize the LHB estrogen re- through theER at a specific regulatory element in the sponse element (ERE) by biological function, portions upstream region of the ratLHB gene. This interaction of the 5”flanking regionof the LHB gene or synthetic results in Ea-stimulated transcriptionof the gene and oligonucleotides were inserted in expression vectors has important implications for thpehysiological regunext to the herpes simplex virus thymidine kinase lpartoio- n of the gene by sex steroids
Incubation of an oligonucleotide representingeither vitellogenin ( V I T ) or LH were added to cytosolic uterine protein extracts.Specific binding was determined by quantifying the amountof DNA inspecific ER-DNA complexes (designated senting the mutated ERE (LXA DNA) with identical preparations of GH3 cell nuclear proteinsinthe absence and by arrows in thetoppanel of Fig. 4)and performing Scatchard analysispresence of anti-ER did not result in the same pattern of to determine affinity of binding
Summary
T o localize the E*-responsive region in the rat LHP gene FIG. 2. 2. Ez-stimulated chloramphenicol acetyltransferase by biological activity, a series of deletions of the previously activity in tkCAT constructs containing either an intact or reported E2-responsive regions of the gene were made, and these gene fragments were inserted next to the thymidine kinase promoter in tkCAT constructs. E2stimulation to the thymidine kinase promoterw, hich was mutation of the LEA region within the context of not itself stimulated by EP (Fig. 1).Insertion of the putative theBamHI-EcoRIfragment of theLHP gene LHP ERE region LEA stimulated tkCAT activity 2-3- struct) eliminated the abiliotfythe gene region to confer fold, whereas insertion of a nearby gene region, LEB, with a stimulatory ESresponse. One copy of the LEA of the LHP ERfEused next to the thymidine kinase promoter, sequence (LEA1) could confear1.7-%fold response to tkCAT, were treated with EP as well as withseveral other steroids whereas two copies of the sequence (LEA,) gave aslightly (Fig. 3). LEA,tkCAT was stimulated 1.7-3.0-fold by Ezw, hereas treatment with estrone (E1), a weaker estrogen, resulted in
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