De Souza Rebecca A G, Kosior Natalia, Thomson Sarah B, Mathelier Anthony, Zhang Allen W, Bečanović Kristina, Wasserman Wyeth W, Leavitt Blair R
Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, BC Children's Hospital, University of British Columbia, Vancouver, BC, Canada.
Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden.
J Huntingtons Dis. 2018;7(3):223-237. doi: 10.3233/JHD-170272.
Huntington's disease is a late onset neurological disorder caused by a trinucleotide CAG repeat expansion mutation in the HTT gene encoding for the protein huntingtin. Despite considerable ongoing research, the wild-type function of huntingtin is not yet fully understood.
To improve knowledge of HTT gene regulation at the transcriptional level and inform future studies aimed at uncovering the HTT gene's normal function.
The HTT gene region was functionally characterized through an in silico analysis using publicly available data sets. ChIP-seq data sets and the online STRING database were used to identify putative transcription factor binding sites (TFBSs) and protein-protein interactions within the HTT promoter region. siRNA-mediated knockdown and ChIP-qPCR of STAT1, a TF identified from the in silico analysis, were used to validate the bioinformatics screen.
16 regions containing potential regulatory genomic markers were identified. TFBSs for 59 transcription factors (TFs) were detected in one or more of the 16 candidate regions. Using these TFs, 15 clusters of protein-protein interactions were identified using STRING. siRNA-mediated knockdown of STAT1 resulted in an increase in HTT expression, and ChIP-qPCR detected enrichment of STAT1 binding at one of the predicted regions. These assays confirmed the utility of the bioinformatic analysis.
Putative regulatory regions outside of the immediate HTT promoter region have been identified with specific protein-protein interactions. Future work will focus on in vitro and in vivo studies to examine the effect of modulating identified TFBSs and altering the levels of specific TFs of interest in regulating HTT gene expression.
亨廷顿舞蹈症是一种迟发性神经障碍疾病，由编码亨廷顿蛋白的HTT基因中的三核苷酸CAG重复扩增突变引起。尽管目前有大量研究，但亨廷顿蛋白的野生型功能尚未完全明确。
提高对HTT基因转录水平调控的认识，并为未来旨在揭示HTT基因正常功能的研究提供信息。
通过使用公开可用数据集进行计算机分析，对HTT基因区域进行功能表征。利用染色质免疫沉淀测序（ChIP-seq）数据集和在线STRING数据库来识别HTT启动子区域内的假定转录因子结合位点（TFBSs）和蛋白质-蛋白质相互作用。对从计算机分析中鉴定出的转录因子STAT1进行小干扰RNA（siRNA）介导的敲低和ChIP-qPCR，以验证生物信息学筛选结果。
鉴定出16个包含潜在调控基因组标记的区域。在16个候选区域中的一个或多个区域检测到59种转录因子（TFs）的TFBSs。利用这些TFs，通过STRING数据库鉴定出15个蛋白质-蛋白质相互作用簇。siRNA介导的STAT1敲低导致HTT表达增加，ChIP-qPCR检测到STAT1在其中一个预测区域的结合富集。这些实验证实了生物信息学分析的实用性。
已鉴定出HTT启动子区域之外的假定调控区域以及特定蛋白质-蛋白质相互作用。未来的工作将集中在体外和体内研究，以检查调节已鉴定的TFBSs以及改变特定目标转录因子水平对HTT基因表达调控的影响。
