Involvement of a cAMP-Responsive DNA Element in Mediating TRH Responsiveness of the Human Thyrotropin α-Subunit Gene

Kim, Dong S.,Ahn, Seung K.,Yoon, Jeong H.,Hong, Seung H.,Kim, Kyoon E.,Maurer, Richard A,Park, Sang D. 충남대학교 생물공학연구소 1996 생물공학연구지 Vol.4 No.-

TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH α-subunit gene, we have used transient transfection of GH_3 rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone α-subunit gene linked to luciferase. Analysis of 5'-deletions of the α-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinaseluciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a 5-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12-myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone. The calcium channel blocker, verapamil, and the calcium chelator, EGTA, completely blocked induction by TRH of the α-subunit reporter gene. The -151 to -100 region of the α-subunit gene contains two copies of a consensus CRE. Deletion of one copy of the CREs resulted in a loss of TRH and calcium responsivenss. Overall, these results indicate that the TRH responsive element of the α-subunit gene likely involves a previously characterized CRE. At least a portion of the mechanism of TRH effects on the α-subunit gene likely involves changes in intracellular calcium levels leading to activation of calcium-responsive protein kinases and phosphorylation of CRE-binding proteins. (Molecular Endocrinology 8:528-536, 1994)

Characterization of a Nontissue-Specific, 3', 5'-Cyclic Adenosine Monophosphate-Responsive Element in the Proximal Region of the Rat Prolactin Gene

Liang, Jie,Kim, Kyoon E.,Schoderbek, William E.,Maurer, Richard A. 충남대학교 생물공학연구소 1993 생물공학연구지 Vol.3 No.-

The nature of a DNA element located in the -100 to -85 region of the rat PRL gene has been characterized. Previous studies demonstrated that this region may contribute to basal and hormonally regulated expression of the PRL gene. As this region contains a sequence with similarity to a consensus cAMP-responsive element (CRE), a possible role for the cAMP response element binding protein (CREB) has been explored. A point mutation which made the PRL CRE-like sequence less like a consensus CRE had little effect on basal or cAMP-stimulated expression of a PRL-luciterase reporter gene. DNase footprint studies demonstrated that the proximal region of the PRL gene does not contain a high affinity CREB binding site. Mobility shift experiments demonstrated that the major GH_3 nuclear protein which interacts with the -100 to -85 region of the PRL gene in vitro is not CREB. Transfection of a dominant inhibitor of CREB action had little or no effect on expression of an indicator gene containing the PRL proximal region. Thus, the PRL proximal region does not contain a high affinity CREB binding site, and it is unlikely that CREB plays a major role in expression of the PRL gene. The functional capabilities of the -100 to -85 region of the PRL gene were then tested in a transfection assay. Synthetic multimers of this region were found to be sufficient to permit a transcriptional response to cAMP or TRH in GH_3 cells and cAMP in Rat-1 cells. These findings suggest that the proximal region of the rat PRL gene contains a functional, nontissue-specific CRE which likely contributes to the hormonal responsiveness of the PRL gene. (Molecular Endocrinology 6: 885-892,1992)

Analysis of DNA Sequences Required for Pituitary-Specific Expression of the Glycoprotein Hormone α-Subunit Gene

Schoderbek, William E.,Kim, Kyoon E.,Ridgway, E. Chester,Mellon, Pamela L.,Maurer, Richard A. 충남대학교 생물공학연구소 1993 생물공학연구지 Vol.3 No.-

Transient transfection studies have been used to determine the DNA sequences of the glycoprotein hormone α-subunit gene that are required for tissuespecific expression. In the initial phase of these studies, a variant mouse α gene was identified which contains a fully palindromic cAMP response element (CRE). The corresponding region of a previously cloned and sequenced mouse α gene contains a single point mutation that disrupts the symmetrical nature of this element. DNase footprint studies demonstrate that the fully palindromic CRE binds the CRE-binding protein with much higher affinity than the imperfect palindrome. Thansfection experiments using both mouse α gene variants demonstrate differences in basal and cAMP-induced expression. Studies of the cAMP response of the human α gene indicated that this gene contains sequences other than the known CRE that are sufficient to permit a transcriptional response to cAMP in both placental and pituitary cells. Expression of human and mouse α-subunit genes has been examined in cells of the gonadotrope, thyrotrop, and trophoblast lineages to identify DNA sequences that mediate selective transcription of the α gene in these cells. The results demonstrate that sequences between about -500 and -200 are important for expression in the pituitary, but not the placenta. Clustered point mutations were used to further characterize sequences required for expression in the pituitary. Two regions, one at positions -445 to -438 and one at positions -337 to -330, were required for expression in cells of the gonadotrope lineage. One of these regions, at -337 to -330, is also important for expression in thyrotropes. When linked to a minimal promoter, multiple copies of the -344 to -300 region had transcriptional enhancer activity in gonadotropes and thyrotropes, but not in several other cell types. These results are consistent with a model involving different combinations of regulatory elements that determine cell-specific α expression in gonadotropes and thyrotropes. (Molecular Endocrinology 6:893-903, 1992)

Decreased Catalytic Subunit mRNA Levels and Altered Catalytic Subunit mRNA Structure in a cAMP-resistant Chinese Hamster Ovary Cell line

Howard, Paul,Day, Kathleen H.,Kim, Kyoon E.,Richardson, Jeanne,Thomas, James,Abraham,Irene,Fleischmann, Robert D.,Gottesman, Michael M.,Maurer, Richard A. 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

The mechansims responsible for decreased levels of cAMP-dependent protein kinase activity in a mutant Chinese hamster ovary cell line have been examined. The cAMP-resistant Chinese hamster ovary 10260 cell line was found to possess only 20% of the cAMP-dependent protein kinase activity found in wild-type cells. The pressence of decreased concentrations of the catalytic subunit in these cells was confirmed through binding studies using a radiolabeled, heat-stable inhibitor of the kinase. Cloned Chinese hamster ovary catalytic subunit cDNAs were isolated, characterized, and used as hybridization probes to examine the relative concentrations of catalytic subunit mRNAs in the wild-type and 10260 cell lines. A 40-50% decrease in the concentration of the mRNA for the Cα isozyme of the catalytic subunit was observed in 10260 cells, as compared with wild-type. This decrease in catalytic subunit mRNA concentration probably accounts for a portion of the decreased kinase activity in the mutant cell. Further analysis of Cα mRNA by polymerase chain reaction confirmed the decreased expression of Cα mRNA in 10260 cells and further demonstrated the presence of two different species of Cα cDNAs was indistinguishable from the wild-type cDNA, but the other species was shorter. Nucleotide sequence analysis of the amplified cDNAs led to the identification of a 191-base pair deletion in the shorter cDNA. Gene transfer studies using wild-type and 10260 Cα cDNAs demonstrated wild-type activity, but the shorter cDNA was inactive. These studies suggest that at least two alternations in gene expression are responsible for decreased cAMP-dependent protein kinase activity in the 10260 cell line. One alteration results in an approximately 2-fold decrease in the concentrations of Cα mRNA in the cells.

Nucleotide Sequence of the Bovine Gene for Follicle-Stimulating Hormone β-Subunit

KIM, KYOON E.,DAVID F. GORDON,RICHARD A. MAURER 충남대학교부설 생명공학연구소 1991 생물공학연구지 Vol.1 No.-

The gene for the β-subunit of follicle-stimulating hormone (FSH-β) was isolated from a library of bovine DNA fragments cloned in bacteriophage λ and the complete nucleotide sequence of the gene was determined. The bovine FSH-β gene contains approximately 4000 nucleotides and consists of three exons separated by two intervening sequences. The transcription initiation site of the gene was mapped by nuclease protection experiments. Analysis of RNA species present in pituitary mRNA demonstrated the presence of a 4.0-kb RNA containing FSH-β sequences, which is the appropriate size for the primary transcript of the gene. Comparison of nucleotide sequence of the 5′-flanking sequence of the FSH-β gene to the 5′-flanking regions of other pituitary glycoprotein hormone genes reveals little sequence similarity.