Department of Neurology, University of Utah, Salt Lake City, UT 84132, USA.
Hum Mol Genet. 2012 Dec 1;21(23):5048-65. doi: 10.1093/hmg/dds349. Epub 2012 Aug 21.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 phenotype is characterized by cerebellar ataxia, neuropathy and slow saccades. SCA2 foreshortens life span and is currently without symptomatic or disease-modifying treatments. Identifying function-specific therapeutics for SCA2 is problematic due to the limited knowledge of ATXN2 function. As SCA2 is likely caused by a gain-of-toxic or gain-of-normal function like other polyglutamine disorders, targeting ATXN2 expression may represent a valid therapeutic approach. This study characterized aspects of ATXN2 expression control using an ATXN2 promoter-luciferase (luc) reporter construct. We verified the fidelity of construct expression by generating transgenic mice expressing the reporter construct. High reporter expression was seen in the cerebellum and olfactory bulb in vivo but there was relatively low expression in other tissues, similar to the expression of endogenous ataxin-2. We verified the second of two possible start codons as the functional start codon in ATXN2. By evaluating deletions in the ATXN2 promoter, we identified an E-twenty six (ETS)-binding site required for ATXN2 expression. We verified that endogenous ETS1 interacted with the ATXN2 promoter by an electromobility supershift assay and chromatin immunoprecipitation polymerase chain reaction. ETS1 overexpression increased ATXN2-luc (ATXN2-luciferase) as well as endogenous ATXN2 expression. Deletion of the putative ETS1-binding site abrogated the effects on the expression of ATXN2-luc. A dominant negative ETS1 and an ETS1 short-hairpin RNA both reduced ATXN2-luc expression. Our study broadens the understanding on the transcriptional control of ATXN2 and reveals specific regulatory features of the ATXN2 promoter that can be exploited therapeutically.
脊髓小脑性共济失调 2 型（SCA2）是一种常染色体显性疾病，由 ATXN2 基因中的 CAG 重复扩展引起。SCA2 的表型特征为小脑共济失调、周围神经病和缓慢扫视。SCA2 会缩短寿命，目前尚无症状或疾病修饰治疗方法。由于对 ATXN2 功能的了解有限，因此确定 SCA2 的功能特异性治疗方法存在问题。由于 SCA2 可能是由类似于其他多谷氨酰胺疾病的毒性或正常功能获得引起的，因此靶向 ATXN2 表达可能代表一种有效的治疗方法。本研究使用 ATXN2 启动子-荧光素酶（luc）报告基因构建体来描述 ATXN2 表达控制的各个方面。我们通过生成表达报告基因构建体的转基因小鼠来验证构建体表达的准确性。在体内，高报告基因表达见于小脑和嗅球，但在其他组织中的表达相对较低，类似于内源性 ataxin-2 的表达。我们验证了 ATXN2 中的两个可能起始密码子中的第二个作为功能起始密码子。通过评估 ATXN2 启动子中的缺失，我们确定了一个 E-twenty six（ETS）-结合位点，该位点是 ATXN2 表达所必需的。我们通过电泳迁移超转移测定和染色质免疫沉淀聚合酶链反应证实了内源性 ETS1 与 ATXN2 启动子的相互作用。ETS1 过表达增加了 ATXN2-luc（ATXN2-荧光素酶）以及内源性 ATXN2 的表达。缺失假定的 ETS1 结合位点消除了对 ATXN2-luc 表达的影响。显性负 ETS1 和 ETS1 短发夹 RNA 均降低了 ATXN2-luc 的表达。我们的研究拓宽了对 ATXN2 转录控制的理解，并揭示了 ATXN2 启动子的特定调节特征，这些特征可在治疗上得到利用。
