Leroy C, Manen D, Rizzoli R, Lombès M, Silve C
Inserm U 426 et Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France.
J Mol Endocrinol. 2004 Feb;32(1):99-113. doi: 10.1677/jme.0.0320099.
The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.
本研究的目的是利用在人类序列中 -40 位鉴定出的 G/A 突变，分析位于 bp -45 至 -39 bp 处的假定近端 Myc 相关锌指蛋白（MAZ）位点对甲状旁腺激素（PTH）/PTH 相关肽（PTHrP）受体（PTHR1）基因 P2 启动子活性的功能重要性。将野生型“全长”（1285P2）和截短型（760P2）启动子序列插入到荧光素酶基础（pLucB）和增强子（pLucE）报告基因表达载体的上游。在成骨样 SaOS-2 细胞和肾细胞（RC.SV3A2）中的瞬时转染表明，-40 G/A 突变显著损害了野生型 1285P2-pLucB 和 760P2-pLucE 启动子构建体的转录活性。P2 序列的进一步截短表明，-109/-37 bp 序列对启动子活性至关重要。与 MAZ 表达载体共转染未改变野生型 1285P2-pLucB 构建体的报告基因活性，但显著提高了突变构建体活性 2 倍（P<0.05）。使用 SaOS-2 核提取物和包含 -45 至 -39 假定 MAZ 位点的双链 DNA 片段（ds-MAZ-oligo）进行的电泳迁移率变动分析揭示了两种特异性 DNA-蛋白质复合物。复合物 II（快速迁移）对突变的 MAZ 基序的亲和力低于对野生型 MAZ 基序的亲和力，而复合物 I（慢速迁移）对野生型或突变的 MAZ 序列具有相同的亲和力。用 Sp1 共有寡核苷酸（ds-Sp1-oligo）进行的竞争研究显著降低了复合物 I 的强度，同时复合物 II 的强度增加。最后，核糖核酸酶保护分析表明，与对照（P<0.001）和 ds-Sp1-oligo（P<0.05）相比，用 ds-MAZ-oligo 转染的 SaOS-2 细胞中 P2 特异性 PTHR1 mRNA 转录本表达显著降低。综上所述，我们的研究表明，假定的 -45 至 -39 MAZ结合位点调节人 PTHR1 P2 启动子的组成活性。
