Analysis of transcriptional network in pancreatic β-cells

胰腺β细胞转录网络分析

• 批准号: 12470228
• 负责人: SEINO Yutaka
• 金额: $ 9.15万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470228/
• 关键词: pancreatic β-cell line; Tet expression system; SAGE; Transcription factor; Target gene; GLUT2; Proliferation and differentiation; 膵Β細胞株; 膵β細胞の発生と機能; Tet Expression System

Mutations in the hepatocyte nuclear factor (HNF)-1α gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1α via sequences downstream of the transcriptional start site by interaction with transcriptional coactivator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1α expression plasmid into a pancreatic β-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1α enhanced human GLUT2 promoter activity sixfold. Site-direct mutagenesis and footprint analyses showed that the HNF-1α binding site (+200 to +218) is critical in human GLUT2 gene expression. Furthermore, mammalian two hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1α (amino acids 391-540) to interact with both the NH_2-terminal region (amino acids 180-662) and the COOH-terminal region (amino acids 1,818-2,079) of p300. These findings demonstrated that HNF-1α binds to the 5'-untranslated region of GLUT2 and that p300 acts as a transcriptional coactivator for HNF-1α. In addition, these results provided new insight into the regulatory function of HNF-1α or by suggesting a molecular basis for human GLUT2 gene expression.

肝细胞核因子（HNF）-1α基因突变与青年人成熟型糖尿病（MODY）3亚型有关，MODY是一种以胰岛素分泌原发性缺陷为特征的疾病。在这里，我们表明，人GLUT 2基因密切调控HNF-1α通过转录起始位点下游序列与转录辅激活因子p300的相互作用。将人GLUT 2基因的启动子区亚克隆到荧光素酶表达质粒中，将荧光素酶表达质粒与HNF-1α表达质粒一起转染到胰腺β细胞系HIT-T15中，以评价转录活性。HNF-1α增强人GLUT 2启动子活性6倍。定点突变和足迹分析表明，HNF-1α结合位点（+200至+218）在人GLUT 2基因表达中至关重要。此外，哺乳动物双杂交和免疫沉淀研究表明，HNF-1α的反式激活结构域（氨基酸391-540）与p300的NH_2末端区域（氨基酸180-662）和COOH末端区域（氨基酸1，818 - 2，079）相互作用。这些发现表明HNF-1α与GLUT 2的5 '非翻译区结合，并且p300作为HNF-1α的转录共激活因子。此外，这些结果为HNF-1α的调节功能提供了新的见解，或为人类GLUT 2基因表达提供了分子基础。

项目成果

Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oki A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino M, Tashita A, Kobara Y, Tsubamoto Y, Jinnouchi T, Jomori T, Seino Y: "Inhibition of gastric inhibitory polyp

Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H, Fujimoto S, Oki A, Tsuda K, Toyokuni S, Hiai H, Mizunoya W, Fushiki T, Holst JJ, Makino M, Tashita A, Kobara Y

Hamamoto Y, Tsuura Y, Fujimoto S, Nagata M, Takeda T, Mukai E, Fujita J, Yamada Y, Seino Y: "Recovery of function and mass of endogenous beta-cells in streptozotocin-induced diabetic rats treated with islet transplantation"Biochem Biophys Res Commun. 287(

Hamamoto Y、Tsuura Y、Fujimoto S、Nagata M、Takeda T、Mukai E、Fujita J、Yamada Y、Seino Y：“用胰岛移植治疗链脲佐菌素诱导的糖尿病大鼠中内源性 β 细胞的功能和质量的恢复”Biochem

Pamir N, Lynn FC, Buchan AM, Ehses J, Hinke SA, Pospisilik JA, Miyawaki K, Yamada Y, Seino Y, McIntosh CH, Pederson RA: "Glucose-dependem Insulinotropic Polypeptide Receptor Null Mice (GIPR-/-) Exhibit Compensatory Changes in the Enteroinsular Axis"Am J P

Pamir N、Lynn FC、Buchan AM、Ehses J、Hinke SA、Pospisilik JA、Miyawaki K、Yamada Y、Seino Y、McIntosh CH、Pederson RA：“葡萄糖依赖性促胰岛素多肽受体无效小鼠 (GIPR-/-) 展示补偿

Ban N, Yamada Y, Soxneya Y, Miyawaki K, Ihara Y, Hosokawa M, Toyokuni S, Tkuda K, Seino Y: "Hepatocyte Nuclear Factor-lα Recruits the Transcriptional Co-activator p300 on the GLUT2 Gene Promoter"Diabetes. 51(5). 1409-1418 (2002)

Ban N、Yamada Y、Soxneya Y、Miyawaki K、Ihara Y、Hosokawa M、Toyokuni S、Tkuda K、Seino Y：“肝细胞核因子-lα 在 GLUT2 基因启动子上招募转录共激活因子 p300”糖尿病 51（ 5). 1409-1418 (2002)。

S.Fujimoto et al.: "Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose."Am J Physiol Endocrinol Metab. 279. E927-E940 (2000)

S.Fujimoto 等人：“通过预先暴露于高浓度的葡萄糖，增强大鼠胰岛的基础胰岛素释放。”Am J Physiol Endocrinol Metab。