Genomic and functional analysis of transcriptome changes in Huntington's Disease

描述（由申请人提供）：亨廷顿舞蹈病基因产物亨廷顿蛋白外显子1的CAG重复扩增导致亨廷顿舞蹈病（HD），这是一种致命的神经退行性疾病，目前没有治愈或神经保护治疗方法。大量使用RNA阵列技术的工作表明，转录失调是HD发病机制的一个主要特征，并对特定的转录因子及其靶点进行了研究。最近的研究也表明了亨廷顿蛋白在RNA加工中的作用。先前对HD脑组织基因表达改变的研究使用了3'偏置基因表达阵列。在许多人类疾病，特别是神经退行性疾病中，异常前mrna剪接的发生越来越重要。然而，传统的基因表达技术并不适合于选择性剪接模式的定量分析，也不能很好地对罕见的转录物进行取样。我们初步工作的几条线索表明，HD中存在全局剪接异常。例如，我们之前报道过，在HD大脑中，microRNA miR-124显著降低。Maniatis及其同事的研究表明，miR-124通过下调重要的组织特异性剪接调节因子聚嘧啶束结合蛋白（PTBP1）来促进神经元特异性的选择性剪接事件。与miR-124的降低一致，我们有初步证据表明HD患者样本中PTBP1 mRNA水平显著升高。此外，初步数据表明，PTBP1调控的基因中的几个外显子在HD脑中显示出相应的外显子包含/排除变化。包含或排除非构成外显子会对转录物的稳定性和蛋白质活性产生巨大影响。因此，HD的转录组改变可能超出了上调和下调基因的范围，包括基因和蛋白质同种异构体的变化。在全球范围内评估HD的这些事件将有助于揭示疾病的病理生理学，并可能确定新的药物治疗靶点。在我们的工作中，包括三个目标，我们将从鉴定改变的HD转录组，到验证，再到体外和体内研究，以测试它们与HD表型的相关性。这些研究结合了Xing实验室的基因组学和生物信息学专业知识以及Ross和Davidson实验室的HD专业知识。这些变化的功能相关性将通过戴维森和罗斯实验室的功能增益和功能损失研究来阐明，这两个小组都有大量的HD模型经验。

DESCRIPTION (provided by applicant): A CAG repeat expansion in exon 1 of the HD gene product, huntingtin, causes Huntington's disease (HD), a fatal neurodegenerative disease for which there is no cure or neuroprotective treatment. Dysregulation of transcription is a major feature of HD pathogenesis, as indicated by a large body of work using RNA array techniques, and work on specific transcription factors and their targets. More recent studies have also suggested a role for huntingtin in RNA processing. Prior work on gene expression alterations in HD brain tissues used 3' biased gene expression arrays. Of increasing importance in many human diseases, particularly neurodegenerative diseases, is the occurrence of aberrant alternative pre-mRNA splicing. However, conventional gene expression techniques are not well suited to quantitative analysis of alternative splicing patterns, and do not sample rare transcript well. Several lines of evidence from our preliminary work suggest global splicing abnormalities in HD. For example, we reported earlier that microRNA miR-124 was significantly reduced in HD brains. Work by Maniatis and colleagues showed that miR-124 promotes neuronal-specific alternative splicing events by down- regulating an important tissue-specific splicing regulator, polypyrimidine tract-binding protein (PTBP1). Consistent with the decrease in miR-124, we have preliminary evidence for significantly increased PTBP1 mRNA levels in HD patient samples. Moreover, preliminary data suggest that several exons in genes regulated by PTBP1 show corresponding changes in exon inclusion/exclusion in HD brain. Inclusion or exclusion of non-constitutive exons can have dramatic effects on transcript stability and protein activity. Thus transcriptome alterations in HD may extend beyond up- and down-regulated genes to include changes in gene and protein isoforms. Assessing these events on a global scale for HD will aid efforts to unravel disease pathophysiology, and may identify new drug targets for therapy. In our work, which encompasses 3 aims, we will move from identification of the altered HD transcriptome, to validation, to in vitro and in vivo studies to test their relevance on HD phenotypes. These studies combine the genomics and bioinformatics expertise of the Xing lab and the HD expertise of the Ross and Davidson labs. The functional relevance of those changes will be elucidated using gain and loss of function studies in the Davidson and Ross labs, where both groups have substantial experience with HD models.