Studying the effects of the TEN1-ICD on transcriptional regulation

Teneurins are large type II transmembrane proteins that are necessary for the normal development of the central nervous system (CNS). While many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus where it can influence gene transcription. Target genes as well as mechanisms have yet to be elucidated, and thus we are investigating the transcriptional activity of the human teneurin-1 ICD in this study. For the whole transcriptome analysis of TEN1-ICD overexpression, we used a modified and improved tet-system. Two separate vectors are required to make the cell line stable. One contains the tet-activating domain fused to a glucocorticoid binding domain (GBD). The other contains the tetO operator sequences directly upstream of a CMV promoter and the gene to be overexpressed. BS149 cells were first transfected with pirtetR-GBD and made stable by Puromycin selection, and then after further transfection with either ptetO-eGFP-His (negative control) or ptetO-TEN1-ICD-eGFP-His by Hygromycin selection. The stable BS149 cell lines were split into three 10 cm Petri dishes each. The triplicate cell lines were cultured once before induction with Dexamethasone and Doxycycline. The overexpressing cells were then FACS-sorted directly into RLT lysis buffer (Qiagen) at a 3:1 volume ratio of lysis buffer to cells in PBS, 24 h post-induction.

腱蛋白（teneurins）是一类大型II型跨膜蛋白，对中枢神经系统（CNS）的正常发育至关重要。尽管已有诸多研究阐明了腱蛋白的生物学意义，尤其是在发育过程中的作用，但目前对其功能的分子机制仍知之甚少。既往研究表明，腱蛋白的N端胞内结构域（ICD）可在细胞膜处被剪切，随后转运至细胞核内，进而调控基因转录。其调控的靶基因及具体作用机制仍有待阐明，因此本研究旨在探究人源teneurin-1 ICD的转录活性。为开展TEN1-ICD过表达的全转录组分析，本研究采用了经过优化改良的四环素系统（tet-system）。构建稳定细胞系需要两种独立的载体：其中一种载体携带与糖皮质激素结合结构域（GBD）融合的tet激活结构域；另一种载体则在巨细胞病毒启动子（CMV promoter）的上游直接连接了tetO操纵子序列以及待过表达的目的基因。首先将pirtetR-GBD转染至BS149细胞中，通过嘌呤霉素筛选获得稳定株；随后分别转染ptetO-eGFP-His（阴性对照）或ptetO-TEN1-ICD-eGFP-His，并通过潮霉素筛选得到稳定细胞系。将各稳定BS149细胞系分别接种至3个直径10cm的培养皿中，即每个细胞系设置三个生物学重复；三份平行样本在经地塞米松与多西环素诱导前先进行一次传代培养。诱导处理24小时后，将过表达细胞按照裂解缓冲液与PBS重悬细胞3:1的体积比，直接通过荧光激活细胞分选（FACS）收集至RLT裂解缓冲液（凯杰Qiagen）中。