TMSG1/LASS2 inhibits cell proliferation, invasive ability through a novel transcriptional regulation way mediated by Homeodomain Running title: a novel transcription regulation role of TMSG1

Background: TMSG1/LASS2 is emerging as an effective tumor suppressor as a transmembrane protein since it has been first cloned. We for the first time found by accident in our previous study TMSG1/LASS2 could be located in the nucleus, and it contained Homeodomain and nuclear localization signal (NLS). We aimed to investigate whether TMSG1/LASS2 possessed the function of transcription factor and identify the target genes regulated by the transcriptional regulation function of TMSG1/LASS2. Methods: : We constructed and transfected three variants containing different functional domains of TMSG1/LASS2 to human prostate cancer cell PC-3M-1E8. Immunofluorescence was performed to detect the cellular localization of the three variants and identified its functional domain mediating it entering the nucleus. Chromatin immunoprecipitation (ChIP) associated with high-throughput sequencing was performed to select the target genes binding to TMSG1/LASS2 protein. Then we investigated the transcriptional regulation effect of TMSG1/LASS2 on one of the target genes Neural Proliferation, Differentiation and Control 1 (NPDC1) by dual-luciferase reporter system, and examined the expression of NPDC1 upon the overexpression or knockdown of TMSG1/LASS2, as well as the cell proliferation ability, apoptosis and cell cycle. Results: : Immunofluorescence result revealed the variants deleted Homeodomain or NLS could not enter the nucleus. Neural Proliferation, Differentiation and Control 1 (NPDC1) gene, whose promoter fragment was obtained from ChIP using TMSG1/LASS2 antibody for further study. Dual-Luciferase reporter system revealed TMSG1/LASS2, as well as the variant with the deletion of NLS could enhance the transcription of NPDC1 promoter rather than the variant deleted Homeodomain. RT-qPCR and Western blot results demonstrated the gene and protein levels of NPDC1 were significantly increased upon TMSG1/LASS2 overexpression other than the variants without Homeodomain or NLS. Moreover, the level of NPDC1 were markedly decreased upon TMSG/LASS2 knockdown. Furthermore, the full length TMSG1/LASS2 was more able to inhibit cell proliferation, invasive ability and promote the cell apoptosis than the variants without Homeodomain or NLS. Conclusions: : TMSG1/LASS2 could enter the nucleus to play transcription factor role mediated by its Homeodomain and NLS. TMSG1/LASS2 could promote the transcription and subsequent expression of NPDC1, one of its target genes. TMSG-1/LASS2 might inhibit cell proliferation, invasive ability, promote cell apoptosis and G0/G1 cell cycle arrest partially mediated by its Homeodomain and NLS. We for the first time propose a new transcriptional regulation function of TMSG1/LASS2 in addition to the known tumor suppressor function as a transmembrane protein.