Regulation of expression of the phospholipid hydroperoxide/sperm nucleus glutathione peroxidase gene -: Tissue-specific expression pattern and identification of functional cis- and trans-regulatory elements
Regulation of expression of the phospholipid hydroperoxide/sperm nucleus glutathione peroxidase gene -: Tissue-specific expression pattern and identification of functional cis- and trans-regulatory elements
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DOI：
10.1074/jbc.m209064200
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发表时间：
2003-01-24
影响因子：
4.8
通讯作者：
Kuhn, H
中科院分区：
文献类型：
--
作者：
Borchert, A;Savaskan, NE;Kuhn, H
最近在晚期精子细胞中发现了一种与磷脂氢谷胱甘肽过氧化物酶（phGPx）关系密切的精核谷胱甘肽过氧化物酶（snGPx）。这两种GPx亚型都起源于联合ph/snGPx基因，但它们的N-末端肽由交替的第一外显子编码。这两种酶的表达在不同的细胞中受到不同的调节，但对调节机制知之甚少。为了探索这两种同工酶表达的组织特异性调节，我们首先研究了它们的组织分布。phGPx在许多器官中表达水平较低，而snGPx仅在睾丸、肾脏和人胚肾细胞系HEK 293中检测到。亚细胞分级分离研究和免疫电镜显示胞质定位。为了探索差异表达模式的机制原因，我们首先测试了推定的phGPx和snGPx启动子的活性。联合ph/snGPx基因的5 '侧翼区表现出强启动子活性。相反，推定的snGPx启动子，其中包括334 bp的内含子序列，缺乏主要的启动子活性。然而，它强烈抑制ph/snGPx启动子的活性。这些数据表明，在第一内含子的ph/snGPx基因的负调控元件，和DNA酶保护试验揭示了几个蛋白结合位点的存在。鉴定了相应的反式调节蛋白（SP1、ERG 1、GATA 1、SREBP 1、USF 1和CREBP 1），并通过染色质免疫沉淀显示了EGR 1和SREBP 1的体内结合。这些数据表明，第一次体细胞表达的snGPx和内含子负顺式调控元件的存在提供证据的pH/snGPx基因。我们未能检测到替代snGPx启动子表明，ph/snGPx基因的转录可能是由一个联合的基本启动子。在给定细胞中表达哪种GPx同种型的决定似乎是由联合初级转录本的选择性剪接做出的。
A sperm nucleus glutathione peroxidase (snGPx), which is closely related to the phospholipid hydroperoxide glutathione peroxidase (phGPx), was recently discovered in late spermatids. Both GPx isoforms originate from a joint ph/snGPx gene, but their N-terminal peptides are encoded by alternative first exons. The expression of the two enzymes is differentially regulated in various cells, but little is known about the regulatory mechanisms. To explore the tissue-specific regulation of expression of the two isoenzymes, we first investigated their tissue distribution. Whereas phGPx is expressed at low levels in many organs, snGPx was only detected in testis, kidney, and in the human embryonic kidney cell line HEK293. Subcellular fractionation studies and immunoelectron microscopy revealed a cytosolic localization. To explore the mechanistic reasons for the differential expression pattern, we first tested the activity of the putative phGPx and snGPx promoters. The 5'-flanking region of the joint ph/snGPx gene exhibits strong promoter activity. In contrast, the putative snGPx promoter, which comprises 334 bp of intronic sequences, lacks major promoter activity. However, it strongly suppresses the activity of the ph/snGPx promoter. These data suggest negative regulatory elements in the first intron of the ph/snGPx gene, and DNase protection assays revealed the existence of several protein-binding sites. The corresponding trans-regulatory proteins (SP1, ERG1, GATA1, SREBP1, USF1, and CREBP1) were identified, and in vivo binding of EGR1 and SREBP1 was shown by chromatin immunoprecipitation. These data indicate for the first time somatic expression of the snGPx and provide evidence for the existence of intronic negative cis-regulatory elements in the ph/snGPx gene. Our failure to detect an alternative snGPx promoter suggests that transcription of the ph/snGPx gene may be regulated by a joint basic promoter. The decision, which GPx isoform is expressed in a given cell, appears to be made by alternative splicing of a joint primary transcript.