International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Padriciano, Trieste, Italy.
Department of Life Sciences, University of Trieste, Via A. Valerio 28, 34127, Trieste, Italy.
Sci Rep. 2021 May 17;11(1):10438. doi: 10.1038/s41598-021-89973-z.
The expression of TDP-43, the main component of neuronal intracellular inclusions across a broad spectrum of ALS and FTD disorders, is developmentally regulated and studies in vivo have shown that TDP-43 overexpression can be toxic, even before observation of pathological aggregates. Starting from these observations, the regulation of its expression at transcriptional level might represent a further key element for the pathogenesis of neurodegenerative diseases. Therefore, we have characterized the human TARDBP promoter, in order to study the transcriptional mechanisms of expression. Mapping of cis-acting elements by luciferase assays in different cell outlined that the activity of the promoter seems to be higher in SH-SY5Y, Neuro2A, and HeLa than in HEK293. In addition, we tested effects of two SNPs found in the promoter region of ALS patients and observed no significant effect on transcription levels in all tested cell lines. Lastly, while TDP-43 overexpression did not affect significantly the activity of its promoter (suggesting that TDP-43 does not influence its own transcription), the presence of the 5'UTR sequence and of intron-1 splicing seem to impact positively on TDP-43 expression without affecting transcript stability. In conclusion, we have identified the region spanning nucleotides 451-230 upstream from the transcription start site as the minimal region with a significant transcription activity. These results lay an important foundation for exploring the regulation of the TARDBP gene transcription by exogenous and endogenous stimuli and the implication of transcriptional mechanisms in the pathogenesis of TDP-43 proteinopathies.
TDP-43 是广泛存在于 ALS 和 FTD 疾病中神经元细胞内包涵体的主要成分，其表达受发育调控。体内研究表明，TDP-43 的过表达即使在观察到病理性聚集之前也可能具有毒性。鉴于这些观察结果，其在转录水平的表达调控可能代表神经退行性疾病发病机制的另一个关键因素。因此，我们对人类 TARDBP 启动子进行了特征分析，以研究其表达的转录机制。在不同细胞中的荧光素酶检测实验中对顺式作用元件进行作图，结果表明启动子的活性在 SH-SY5Y、Neuro2A 和 HeLa 中的似乎高于在 HEK293 中的。此外，我们测试了在 ALS 患者启动子区域发现的两个 SNP 的影响，并且在所有测试的细胞系中观察到对转录水平没有显著影响。最后，虽然 TDP-43 的过表达并没有显著影响其启动子的活性（表明 TDP-43 不会影响其自身的转录），但是 5'UTR 序列和内含子-1 剪接的存在似乎对 TDP-43 的表达产生积极影响，而不会影响转录物的稳定性。总之，我们已经确定了从转录起始位点上游的核苷酸 451-230 跨越的区域是具有显著转录活性的最小区域。这些结果为探索 TARDBP 基因转录的外源性和内源性刺激的调控以及转录机制在 TDP-43 蛋白病发病机制中的作用奠定了重要基础。
