Supporting data for "Mechanistic study of TSPYL2 stabilization in TSPYL1 depleted cells"

The homozygous or double heterozygous pathogenic mutations of TSPYL1 cause SIDDT. However, how the loss of TSPYL1 causes SIDDT is largely unknown and the biological function of TSPYL1 is not clear. In our previous studies in vivo, Tspyl1 KO mice on the inbred C57BL/6N background grew much smaller than WT or heterozygous littermates and died before weaning time. The abnormal lung alveolarization in Tspyl1 KO mice was associated with perturbed TGFβ signaling. Moreover, TSPYL2 protein expression increased in multiple organs of Tspyl1 KO mice, and it has been shown that the activation of TGFβ signaling induces TSPYL2 upregulation. TSPYL1 works as an essential transcriptional regulator to regulate its target genes upon recruitment by transcription factors. Our previous work in A549 cells demonstrated that the increased TGFβ signaling stabilizes TSPYL2 upon TSPYL1 depletion and therefore causes EMT. Although the importance of TSPYL1 in regulating TGFβ signaling has already been highlighted, the response to TGFβ is well known to be context dependent. As we found that the protein level of TSPYL2 increased in different cell lines upon TSPYL1 KD, we hypothesized that there are multiple mechanisms involved. Molecular technologies such as qPCR, co-immunoprecipitation, and gene expression studies are applied in my studies to explore the functional relationship between TSPYL1 and TSPYL2. A375 melanoma cells that belong to neural crest-derived cells and can further undergo EMT were chosen as a cellular model . To answer this question, the first phase of my project consists of identifying whether TSPYL1 deficiency induces EMT morphological-like changes and cell death by recording morphological changes of cells and using a trypan blue exclusion assay to quantify cells upon TSPYL1 KD. Furthermore, TSPYL2 upregulation upon TSPYL1 KD caused EMT-like changes and p53 associated cell death. However, TSPYL1 KD and overexpression (OE) studies demonstrated that TSPYL1 activated instead of repressed TGFBR1 and increased TGFβ signaling. Furthermore, addition of TGFβ reduced TSPYL2 protein levels in A375 cells, which is opposite to the case in A549, HEK293FT and HK-2 cell lines. Together, the upregulation of TSPYL2 upon TSPYL1KD is not due to upregulated TGFβ signaling in A375 cells. To explore the alternative mechanism for TSPYL2 stabilization upon TSPYL1 KD in A375 cells, CUT&RUN-seq data was analyzed and TSPYL1 target genes were found to be enriched in the Ubiquitin-proteasome system (UPS). Moreover, TSPYL1 KD decreased the total ubiquitination level. NEDD4L was selected as the first candidate among TSPYL1 target genes for further validation after the literature survey. TSPYL1KD did not affect NEDD4L transcript level but the protein level decreased. The data suggested that TSPYL1 KD caused a disturbance in the UPS system with overall reduced ubiquitination of proteins. USP7 is one of the Deubiquitinating enzymes that are required in the reversal process of ubiquitin modifications and prevent protein substrates from degradation. Our group previously identified that USP7 interacted with TSPYL2. In this study, the treatment of USP7 inhibitor HBX 41108 increased the level of TSPYL2 and decreased the level of the ubiquitin ligase including NEDD4L and MDM2. This agrees with a previous report that TSPYL2 is a substrate of MDM2, and MDM2 is a substrate of USP7. By contrast, TSPYL2 upregulation was attenuated under the treatment of USP7 inhibitor in the situation of TSPYL1 KD. The results suggest that TSPYL2 stabilization upon TSPYL1 KD was at least partly due to increased deubiquitination of TSPYL2 in A375 cells. In summary, this study demonstrates that there are multiple mechanisms by which TSPYL1 KD stabilizes TSPYL2. In A375 cells, this may be related to reduced rather than increased TGFβ signaling. The UPS system, in particular USP7, is involved although the detail mechanism is unknown. To illustrate the potential regulatory mechanism between TSPYL1 and TSPYL2 in different cell models could provide mechanistic insights into the pathological process of SIDDT.

同源或双杂合的TSPYL1致病突变可导致SIDDT。然而，TSPYL1缺失如何引发SIDDT尚不清楚，且TSPYL1的生物学功能尚不明确。在我之前的体内研究中，C57BL/6N近交系背景下的Tspyl1敲除小鼠相较于野生型或杂合子同胞鼠体型显著减小，且在断奶前死亡。Tspyl1敲除小鼠的肺部异常肺泡化与TGFβ信号通路失调有关。此外，Tspyl1敲除小鼠的多个器官中TSPYL2蛋白表达增加，已有研究表明，TGFβ信号通路的激活可诱导TSPYL2的上调。TSPYL1作为转录因子招募的必需转录调控因子，在其靶基因上发挥调节作用。我们之前在A549细胞中的研究表明，TSPYL1耗竭后TGFβ信号通路的增强稳定了TSPYL2，从而引发上皮间质转化（EMT）。尽管TSPYL1在调节TGFβ信号通路中的重要性已被强调，但TGFβ的响应众所周知是情境依赖的。我们发现，在TSPYL1敲除后，不同细胞系中TSPYL2的蛋白水平增加，据此我们假设存在多种机制。在我的研究中，分子技术如qPCR、共免疫沉淀和基因表达研究被应用于探索TSPYL1与TSPYL2之间的功能关系。选择A375黑色素瘤细胞作为细胞模型，因为这些细胞来源于神经嵴细胞，并可进一步发生EMT。为了回答这一问题，我的项目的第一阶段包括通过记录细胞形态变化和使用台盼蓝排除实验来量化TSPYL1敲除后的细胞，以确定TSPYL1缺失是否诱导EMT样形态变化和细胞死亡。此外，TSPYL1敲除后TSPYL2的上调引起了EMT样变化和p53相关的细胞死亡。然而，TSPYL1敲除和过表达（OE）研究显示，TSPYL1激活而非抑制TGFBR1并增加TGFβ信号通路。进一步地，添加TGFβ降低了A375细胞中的TSPYL2蛋白水平，这与A549、HEK293FT和HK-2细胞系中的情况相反。总的来说，TSPYL1敲除后TSPYL2的上调并非由于A375细胞中TGFβ信号通路的上调。为了探索A375细胞中TSPYL1敲除后TSPYL2稳定的替代机制，分析了CUT&RUN-seq数据，并发现TSPYL1靶基因富集于泛素-蛋白酶体系统（UPS）。此外，TSPYL1敲除降低了总泛素化水平。NEDD4L被选为TSPYL1靶基因中的第一个候选者，以进一步验证文献综述。TSPYL1敲除不影响NEDD4L转录水平，但蛋白质水平降低。数据表明，TSPYL1敲除导致UPS系统紊乱，蛋白质泛素化总体降低。USP7是参与泛素修饰逆转过程的去泛素化酶之一，防止蛋白质底物降解。我们小组之前发现USP7与TSPYL2相互作用。在本研究中，USP7抑制剂HBX 41108的处理增加了TSPYL2的水平，并降低了包括NEDD4L和MDM2在内的泛素连接酶的水平。这与之前的报道一致，即TSPYL2是MDM2的底物，而MDM2是USP7的底物。相比之下，在TSPYL1敲除的情况下，USP7抑制剂的处理下TSPYL2的上调被减弱。结果提示，TSPYL1敲除后TSPYL2的稳定至少部分是由于A375细胞中TSPYL2去泛素化增加。总之，本研究表明，TSPYL1敲除稳定TSPYL2存在多种机制。在A375细胞中，这可能与其TGFβ信号通路的降低而非增加有关。UPS系统，尤其是USP7，虽然其详细机制尚不明确，但参与了其中。阐明TSPYL1和TSPYL2在不同细胞模型间的潜在调控机制，可为SIDDT的病理过程提供机制性见解。