Chiba-Falek O, Nussbaum R L
Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, 49 Convent Drive, MSC 4472, Bethesda, MD 20892-4472, USA.
Hum Mol Genet. 2001 Dec 15;10(26):3101-9. doi: 10.1093/hmg/10.26.3101.
Mutations in the alpha-synuclein gene (SNCA) have been implicated in familial Parkinson's disease (PD) while certain polymorphic alleles at a microsatellite repeat, NACP-Rep1, located approximately 10 kb upstream of the gene, have been associated with sporadic PD. In order to study the regulation of the human alpha-synuclein gene, we performed a deletion analysis of 10.7 kb upstream of the translational start site, using the luciferase reporter assay in 293T cells and the neuroblastoma cell line SH-SY5Y. The shortest fragment, 400 bp upstream of the transcriptional start site, was sufficient for transcription in both cell lines. The other constructs led to variable expression levels, with some showing maximum expression and others showing nearly complete extinction of expression. An 880 bp fragment located approximately 10 kb upstream of the gene and containing the NACP-Rep1 polymorphism, was shown to be necessary for normal expression. Additional analysis of the NACP-Rep1 locus and surrounding DNA suggested that two domains flanking the repeat interact to enhance expression while the repeat acts as a negative modulator. Next, we measured the activity of the entire 10.7 kb upstream region in the luciferase reporter assay when each of our different NACP-Rep1 alleles were present. The expression levels varied very significantly among the different alleles over a 3-fold range in the SH-SY5Y cells but showed little or no significant variation in the 293T cells. Given that even small changes in alpha-synuclein expression may, over many decades, predispose to PD, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of alpha-synuclein expression resulting from different NACP-Rep1 alleles.
α-突触核蛋白基因（SNCA）的突变与家族性帕金森病（PD）有关，而位于该基因上游约10 kb处的微卫星重复序列NACP-Rep1的某些多态性等位基因与散发性PD有关。为了研究人类α-突触核蛋白基因的调控，我们使用荧光素酶报告基因检测法，在293T细胞和神经母细胞瘤细胞系SH-SY5Y中，对翻译起始位点上游10.7 kb的区域进行了缺失分析。最短的片段，即转录起始位点上游400 bp，足以在两种细胞系中进行转录。其他构建体导致了不同的表达水平，有些显示出最大表达，而有些则显示出几乎完全的表达消失。位于该基因上游约10 kb处且包含NACP-Rep1多态性的一个880 bp片段，被证明是正常表达所必需的。对NACP-Rep1位点及周围DNA的进一步分析表明，重复序列两侧的两个结构域相互作用以增强表达，而重复序列则起负调节作用。接下来，当存在我们不同的NACP-Rep1等位基因时，我们在荧光素酶报告基因检测中测量了整个10.7 kb上游区域的活性。在SH-SY5Y细胞中，不同等位基因的表达水平在3倍范围内差异非常显著，但在293T细胞中几乎没有显著差异。鉴于即使α-突触核蛋白表达的微小变化在几十年中也可能使人易患PD，不同的NACP-Rep1等位基因与PD的关联可能是由于不同的NACP-Rep1等位基因导致α-突触核蛋白表达转录调控的多态性差异所致。
