Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.
Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.
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普遍存在的神经元 DNA 结合蛋白与人次黄嘌呤磷酸核糖转移酶基因的负调控元件相互作用。
DOI：
10.1128/mcb.15.12.6561
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发表时间：
1995
影响因子：
5.3
通讯作者：
Patel,PI
中科院分区：
文献类型：
--
作者：
Rincón-Limas,DE;Amaya-Manzanares,F;Niño-Rosales,ML;Yu,Y;Yang,TP;Patel,PI
次黄嘌呤磷酸核糖基转移酶（HPRT）基因在所有组织中以低水平组成性表达，但在大脑中以较高水平表达;这种差异表达的意义和机制尚不清楚。我们以前在人HPRT基因5 ′侧翼区发现了一个182 bp的元件（hHPRT-NE），它不仅参与赋予神经元特异性，而且抑制非神经元组织中的基因表达。在这里，我们报告说，这个元素与不同的核蛋白相互作用，其中一些是专门存在于神经元细胞（复合物I）和其他人是存在于细胞中显示组成型表达的基因（复合物II）。此外，我们发现，复合物I因子在人NT 2/D1细胞中表达，诱导神经元分化的视黄酸。这一发现与神经元分化后HPRT基因转录的增加相关。我们还将两种复合物的结合位点定位到一个60 bp的区域（Ff;位置-510到-451），当在转染试验中分析时，该区域作为类似于全长hHPRT-NE序列的阻遏元件发挥作用。甲基化干扰足迹揭示了一个最小的独特DNA基序，5 ′-GGAAGCC-3 ′，作为神经元和非神经元来源的核蛋白的结合位点。然而，定点突变的足迹区域表明，不同的核苷酸是必不可少的协会，这两个复合物。此外，紫外交联实验表明，这两种复合物是由几种不同的蛋白质的协会形成的。两者合计，这些数据表明，差异相互作用的DNA结合因子与此调控元件在大脑优先表达的基因中起着至关重要的作用，他们应该导致隔离的转录调节重要的神经元表达的HPRT基因。
The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutively expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) within the 5′-flanking region of the human HPRT (hHPRT) gene, which is involved not only in conferring neuronal specificity but also in repressing gene expression in nonneuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation. We also mapped the binding sites for both complexes to a 60-bp region (Ff; positions −510 to −451) which, when analyzed in transfection assays, functioned as a repressor element analogous to the full-length hHPRT-NE sequence. Methylation interference footprintings revealed a minimal unique DNA motif, 5′-GGAAGCC-3′, as the binding site for nuclear proteins from both neuronal and nonneuronal sources. However, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the associations of these two complexes. Moreover, UV cross-linking experiments showed that both complexes are formed by the association of several different proteins. Taken together, these data suggest that differential interaction of DNA-binding factors with this regulatory element plays a crucial role in the brain-preferential expression of the gene, and they should lead to the isolation of transcriptional regulators important in neuronal expression of the HPRT gene.