Freeman Dana M, Wang Zhibin
Laboratory of Environmental Epigenomes, Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States.
Front Genet. 2020 Jul 7;11:627. doi: 10.3389/fgene.2020.00627. eCollection 2020.
CCCTC-binding factor (CTCF) is a regulatory protein that binds DNA to control spatial organization and transcription. The sequence-specific binding of CTCF is variable and is impacted by nearby epigenetic patterns. It has been demonstrated that non-coding genetic variants cluster with CTCF sites in topological associating domains and thus can affect CTCF activity on gene expression. Therefore, environmental factors that alter epigenetic patterns at CTCF binding sites may dictate the interaction of non-coding genetic variants with regulatory proteins. To test this mechanism, we treated human cell line HEK293 with rotenone for 24 h and characterized its effect on global epigenetic patterns specifically at regulatory regions of Parkinson's disease (PD) risk loci. We used RNA sequencing to examine changes in global transcription and identified over 2000 differentially expressed genes (DEGs, >1.5-fold change, FDR < 0.05). Among these DEGs, 13 were identified as PD-associated genes according to Genome-wide association studies meta-data. We focused on eight genes that have non-coding risk variants and a prominent CTCF binding site. We analyzed methylation of a total of 165 CGs surrounding CTCF binding sites and detected differential methylation (|>1%|, < 0.05) in 45 CGs at 7 PD-associated genes. Of these 45 CGs, 47% were hypomethylated and 53% were hypermethylated. Interestingly, 5 out of the 7 genes had correlated gene upregulation with CG hypermethylation at CTCF and gene downregulation with CG hypomethylation at CTCF. We also investigated active H3K27ac surrounding the same CTCF binding sites within these seven genes. We observed a significant increase in H3K27ac in four genes (FDR < 0.05). Three genes (PARK2, GPRIN3, FER) showed increased CTCF binding in response to rotenone. Our data indicate that rotenone alters regulatory regions of PD-associated genes through changes in epigenetic patterns, and these changes impact high-order chromatin organization to increase the influence of non-coding variants on genome integrity and cellular survival.
CCCTC结合因子（CTCF）是一种调控蛋白，它通过与DNA结合来控制空间组织和转录。CTCF的序列特异性结合具有变异性，并受附近表观遗传模式的影响。已有研究表明，非编码基因变异在拓扑相关结构域中与CTCF位点聚集，因此可能影响CTCF对基因表达的活性。所以，改变CTCF结合位点表观遗传模式的环境因素可能决定非编码基因变异与调控蛋白的相互作用。为了验证这一机制，我们用鱼藤酮处理人细胞系HEK293 24小时，并具体研究其对帕金森病（PD）风险位点调控区域整体表观遗传模式的影响。我们使用RNA测序来检测整体转录变化，并鉴定出2000多个差异表达基因（DEG，变化倍数>1.5，错误发现率<0.05）。在这些差异表达基因中，根据全基因组关联研究元数据，有13个被鉴定为与PD相关的基因。我们重点关注了8个具有非编码风险变异和显著CTCF结合位点的基因。我们分析了CTCF结合位点周围总共165个CG的甲基化情况，在7个与PD相关的基因中检测到45个CG存在差异甲基化（|>1%|，<0.05）。在这45个CG中，47%发生了低甲基化，53%发生了高甲基化。有趣的是，7个基因中的5个基因在CTCF处CG高甲基化时基因上调，在CTCF处CG低甲基化时基因下调。我们还研究了这7个基因中相同CTCF结合位点周围的活性H3K27ac。我们观察到4个基因中的H3K27ac显著增加（错误发现率<0.05）。3个基因（PARK2、GPRIN3、FER）在鱼藤酮处理后CTCF结合增加。我们的数据表明，鱼藤酮通过表观遗传模式的变化改变了与PD相关基因的调控区域，这些变化影响高阶染色质组织，以增加非编码变异对基因组完整性和细胞存活的影响。
