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.