| Project/Area Number |
09671044
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
内分泌・代謝学
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| Research Institution | Nagoya University |
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Principal Investigator |
MURATA Yoshiharu Nagoya Univ., Res.Inst.Environ.Med., Professor, 環境医学研究所, 教授 (80174308)
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| Co-Investigator(Kenkyū-buntansha) |
NAGAYA Takashi Nagoya Univ., Res.Inst.Environ.Med., Research Associate, 環境医学研究所, 助手 (80262913)
KAMBE Fukushi Nagoya Univ., Res.Inst.Environ.Med., Associate Professor, 環境医学研究所, 助教授 (00211871)
SEO Hisao Nagoya Univ., Res.Inst.Environ.Med., Professor, 環境医学研究所, 教授 (40135380)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | thyroid hormone / transcription / human / transcription factor / cycloheximide / 転写因子 / 遺伝子発現 |
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| Research Abstract |
ZAKI-4 is a thyroid hormone-responsive gene that we have recently identified in human skin fibroblasts. Addition of 3, 3', 5-triiodothyronine (T_3), an active form of thyroid hormone, enhances the transcription of the ZAKII-4 gene in skin fibroblasts. However, transcriptional activation was completely blocked by the pretreatment with cycloheximide, a translation inhibitor of protein synthesis. The result indicates that de novo protein synthesis is required to activate the transcription of the ZAKI-4 gene by T_3. It appears that T_3 induces some factor(s) which enhances ZAKI-4 gene transcription. Since it has generally been accepted that T_3 exerts its effect through binding to its nuclear receptor and regulating expression of target genes, the identification of the T_3 induced factor(s) that enhance the transcription of the ZAKI-4 gene could uncover a novel mechanism of thyroid hormone action. Therefore, we designed the current research project in order to elucidate the mechanism of T_
… More
3 dependent transcriptional activation of ZAKI-4 gene by using a 5'-flanking region of the ZAKI-4 gene. We identified the transcription start site by the primer extension method. Using PromoterFinderTM, we cloned a DNA fragment (about 2.8 kb, designated as alphaE2-5) in the 5' flanking region of ZAKI-4. We constructed a chimeric luciferase reporter gene with alphaE2-5 and transfected this construct to a ZAKI-4 expressing human glioma cell line, U-251-MG.With alphaE2-5, a 5-fold increase in luciferase activity was observed, indicating that alphaE2-5 contains a region which mediates the promoter activity of ZAKI-4. We have also precisely analyzed the promoter region and found that a region from -28 to -4 mediated the promoter activity as well as the T_3 dependent transcriptional regulation. By electrophoretic mobility shift assay (EMSA) using the nuclear extract from U-251MG, we confirmed that the extract contained a protein which binds to TFZ-1. We further analyzed the protein by South Western blotting with TFZ-1 as a probe and demonstrated that the apparent molecular mass of the protein is 45 kDa and expression increased by the addition of 10^<-7>M T_3. These results suggest that the protein designated as TMZ45 is the factor which mediates transcriptional regulation of ZAKI-4 by T_3. Currently we are trying to clone the cDNA for TMZ45 using the yeast one hybrid system. Less
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