Characterization of the mouse islet-specific glucose-6-phosphatase catalytic subunit-related protein gene promoter by in situ footprinting -: Correlation with fusion gene expression in the islet-derived βTC-3 and hamster insulinoma tumor cell lines
Characterization of the mouse islet-specific glucose-6-phosphatase catalytic subunit-related protein gene promoter by in situ footprinting -: Correlation with fusion gene expression in the islet-derived βTC-3 and hamster insulinoma tumor cell lines
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DOI：
10.2337/diabetes.50.3.502
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发表时间：
2001-03-01
期刊：
影响因子：
7.7
通讯作者：
O'Brien, RM
中科院分区：
文献类型：
--
作者：
Bischof, LJ;Martin, CC;O'Brien, RM
6-磷酸葡萄糖酶 (G6Pase) 是一种位于内质网的多组分系统，包含催化亚基和 6-磷酸葡萄糖、无机磷酸盐和葡萄糖的转运蛋白。我们最近克隆了一种编码胰岛特异性 G6Pase 催化亚基相关蛋白（IGRP）的新基因（Ebert 等人，Diabetes 48：543-551，1999）。为了开始研究 IGRP 基因胰岛特异性表达的分子基础，一系列截短的 IGRP-氯霉素乙酰转移酶 (CAT) 融合基因被瞬时转染到胰岛来源的小鼠 β TC-3 和仓鼠胰岛素瘤肿瘤细胞系中。在这两种细胞系中，在-306和-66之间的IGRP启动子序列逐渐缺失后，基础融合基因表达降低，表明最大IGRP-CAT表达需要多个启动子区域。然后使用连接介导的聚合酶链反应足迹技术比较表达内源性 IGRP 基因的 β TC-3 细胞和不表达内源性 IGRP 基因的肾上腺皮质 Y1 细胞中反式作用因子与 IGRP 启动子的原位结合。在 β TC-3 细胞中选择性地鉴定了多个反式作用因子结合位点，这些位点与被确定为基础 IGRP-CAT 融合基因表达所需的 IGRP 启动子区域相关。数据表明，肝细胞核因子 3 可能对于基础 IGRP 基因表达很重要，就像它对于胰高血糖素、GLUT2 和 Pdx-1 基因表达一样。此外，还鉴定了以前与胰岛基因表达不相关的几种反式作用因子的结合位点，以及潜在的新蛋白质的结合位点。
Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-related protein (IGRP) (Ebert et al., Diabetes 48:543-551, 1999). To begin to investigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into the islet-derived mouse beta TC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fusion gene expression decreased upon progressive deletion of the IGRP promoter sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymerase chain reaction footprinting technique was then used to compare trans-acting factor binding to the IGRP promoter in situ in beta TC-3 cells, which express the endogenous IGRP gene, and adrenocortical Y1 cells, which do not. Multiple trans-acting factor binding sites were selectively identified in beta TC-3 cells that correlate with regions of the IGRP promoter identified as being required for basal IGRP-CAT fusion gene expression. The data suggest that hepatocyte nuclear factor 3 may be important for basal IGRP gene expression, as it is for glucagon, GLUT2, and Pdx-1 gene expression. In addition, binding sites for several trans-acting factors not previously associated with islet gene expression, as well as binding sites for potentially novel proteins, were identified.