Rincón-Limas D E, Amaya-Manzanares F, Niño-Rosales M L, Yu Y, Yang T P, Patel P I
Department of Neurology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Cell Biol. 1995 Dec;15(12):6561-71. doi: 10.1128/MCB.15.12.6561.
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.
次黄嘌呤磷酸核糖转移酶（HPRT）基因在所有组织中均以低水平组成性表达，但在大脑中表达水平较高；这种差异表达的意义和机制尚不清楚。我们先前在人HPRT（hHPRT）基因的5'侧翼区域内鉴定出一个182bp的元件（hHPRT-NE），它不仅参与赋予神经元特异性，还参与抑制非神经元组织中的基因表达。在此我们报告，该元件与不同的核蛋白相互作用，其中一些核蛋白特异性存在于神经元细胞中（复合物I），而其他核蛋白则存在于显示该基因组成性表达的细胞中（复合物II）。此外，我们发现，视黄酸诱导人NT2/D1细胞发生神经元分化后，复合物I因子会表达。这一发现与神经元分化后HPRT基因转录增加相关。我们还将这两种复合物的结合位点定位到一个60bp的区域（Ff；位置-510至-451），在转染实验中分析时，该区域作为类似于全长hHPRT-NE序列的抑制元件发挥作用。甲基化干扰足迹法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核蛋白的结合位点。然而，对足迹区域进行的定点诱变表明，不同的核苷酸对于这两种复合物的结合至关重要。此外，紫外线交联实验表明，这两种复合物均由几种不同蛋白质的结合形成。综上所述，这些数据表明DNA结合因子与该调控元件的差异相互作用在该基因的脑优先表达中起关键作用，并且它们应能导致分离出对HPRT基因的神经元表达很重要的转录调节因子。
