Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India.
Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India; Academy of Scientific and Innovative Research (AcSIR), New Delhi 110025, India.
J Mol Biol. 2019 Mar 15;431(6):1127-1147. doi: 10.1016/j.jmb.2019.01.042. Epub 2019 Feb 7.
Monoamine oxidase B (MAO-B), a flavoenzyme located in the outer mitochondrial membrane, is involved in the catabolism of monoamines. Altered levels of MAO-B are associated with cardiovascular/neuronal diseases. However, molecular mechanisms of MAO-B gene regulation are partially understood. We undertook a systematic analysis of the MAO-B gene to identify the key transcriptional/post-transcriptional regulatory molecules. Expression of MAO-B promoter-reporter constructs in cultured cells identified the -144/+25-bp domain as the core promoter region. Stringent in silico analysis of this core promoter predicted binding sites for several transcription factors. Over-expression/down-regulation of transcription factors Sp1/Egr1/CREB increased/decreased the MAO-B promoter-reporter activity and endogenous MAO-B protein level. Electrophoretic mobility shift assays and ChIP assays provided evidence for interactions of Sp1/Egr1/CREB with the MAO-B promoter. MAOB transcript level also positively correlated with the transcript level of Sp1/Egr1/CREB in various human tissue samples. Computational predictions using multiple algorithms coupled with systematic functional analysis revealed direct interactions of the microRNAs miR-1224 and miR-300 with MAO-B 3'-UTR. Dopamine dose-dependently enhanced MAO-B transcript and protein levels via increased binding of CREB to MAO-B promoter and reduced miR-1224/miR-300 levels. 8-Bromo-cAMP and forskolin augmented MAO-B expression, whereas inhibition of PKA diminished the gene expression suggesting involvement of cAMP-PKA axis. Interestingly, Sp1/Egr1/CREB/miR-1224 levels correlate with MAO-B expression in rodent models of hypertension/MPTP-induced neurodegeneration, indicating their roles in governing MAO-B gene expression in these disease states. Taken together, this study elucidates the previously unknown roles of the transcription factors Sp1/Egr1/CREB and microRNAs miR-1224/miR-300 in regulating MAO-B gene expression under basal/disease states involving dysregulated catecholamine levels.
单胺氧化酶 B（MAO-B）是一种位于线粒体外膜的黄素酶，参与单胺的分解代谢。MAO-B 水平的改变与心血管/神经元疾病有关。然而，MAO-B 基因调控的分子机制部分了解。我们对 MAO-B 基因进行了系统分析，以确定关键的转录/转录后调节分子。在培养细胞中表达 MAO-B 启动子报告构建体，确定了-144/+25-bp 区域作为核心启动子区域。对该核心启动子的严格计算机分析预测了几个转录因子的结合位点。转录因子 Sp1/Egr1/CREB 的过表达/下调增加/减少了 MAO-B 启动子-报告基因的活性和内源性 MAO-B 蛋白水平。电泳迁移率变动分析和 ChIP 分析为 Sp1/Egr1/CREB 与 MAO-B 启动子相互作用提供了证据。MAOB 转录本水平也与各种人组织样本中 Sp1/Egr1/CREB 的转录本水平呈正相关。使用多种算法进行的计算预测以及系统的功能分析表明，miR-1224 和 miR-300 与 MAO-B 3'-UTR 直接相互作用。多巴胺通过增加 CREB 与 MAO-B 启动子的结合和降低 miR-1224/miR-300 水平，剂量依赖性地增强 MAO-B 转录本和蛋白水平。8-溴-cAMP 和 forskolin 增强 MAO-B 表达，而 PKA 抑制减少基因表达，表明 cAMP-PKA 轴的参与。有趣的是，Sp1/Egr1/CREB/miR-1224 水平与高血压/MPTP 诱导的神经退行性变啮齿动物模型中 MAO-B 表达相关，表明它们在调节这些疾病状态下的 MAO-B 基因表达中起作用。总之，这项研究阐明了转录因子 Sp1/Egr1/CREB 和 microRNAs miR-1224/miR-300 在调节基础/疾病状态下 MAO-B 基因表达中的以前未知的作用，涉及调节儿茶酚胺水平。
