Functional study of testis-specific Y-encoded like protein 1 (TSPYL1)

Abstract

Testis-Specific Y-encoded Like Protein 1 (TSPYL1) and TSPYL4 belong to the nucleosome assembly protein superfamily. TSPYL1 and TSPYL4 are adjacent genes expressed in different tissues, including brain and testis. TSPYL1 mutation caused sudden infant death with dysgenesis of the testes syndrome in an Amish family. However, the biological functions of TSPYL1 and TSPYL4 remain elusive. Previously, our group has mutated TSPYL1 in the human neuroblastoma cell line BE2C. In the present study, these knockout (KO) cells were utilized to decipher the role of TSPYL1 in differentiation of neuroblastoma cells. In parallel, CRISPR/Cas9 system was utilized to KO Tspyl1 and Tspyl4 in mouse embryonic stem cells (ESCs). The ESCs were used for in vitro differentiation and blastocyst injection. A second approach of oocyte injection with sgRNAs and Cas9 protein was used to generate KO mice. Previously, we found that TSPYL1 KO BE2C cells had enhanced migration and invasion through gel matrix, which could be related to the upregulation of SNAI1, SNAI2, and SOX9. Here, my data show that the flattened, enlarged subpopulation in TSPYL1 KO BE2C clones were α-smooth muscle actin positive, and BE2C KO clones required a longer time to form tumours in nude mice. Mechanistically, the expression of KLF4, a gene for fibromuscular differentiation, was upregulated. Upregulation of SNAI1, SNAI2, and SOX9 could be directly due to TSPYL1 KO or a consequence of fibromuscular differentiation due to KLF4 expression. To address this, TSPYL1 was ectopically expressed in TSPYL1 KO cells. From RT-qPCR, expression of SNAI1 and SOX9 were significantly downregulated. Luciferase assays confirmed that TSPYL1 regulated the promoter activity of these genes. Lastly, bioinformatic analysis identified TSPYL1 binding motifs in the 1.2 kb promoter of KLF4, SNAI1, SNAI2, and SOX9. During neural differentiation of ESCs, the transcript levels of Tspyl1 and Tspyl4 increased. Tspyl1 and Tspyl4 KO ESC clones were generated by CRISPR/Cas9 to introduce small deletions. Karyotype analysis, proliferation rate by cell counting, differentiation through embryoid body formation and early neural differentiation in N2/B27 showed that Tspyl1 and Tspyl4 frameshift ESC clones behaved similar to parent ESC line L4. However, RT-qPCR analysis showed that induction of Klf4, Snai1 and Sox9 was impaired and the expression of Snai2 was upregulated in Tspyl1 KO during neural differentiation. Unlike the clone with 15 bp deletion in Tspyl4, the Tspyl1 and Tspyl4 frameshift ESC clones failed to give germline transmission. By injecting gRNAs and Cas9 proteins into F1 oocytes, two heterozygous Tspyl1 KO lines were generated. Around 10 % of Tspyl1+/- and Tspyl1-/- mice were weak and died at postnatal day 1-3 or 18-21. Significantly reduced body weights were recorded at postnatal day 35 and 130. In fertility assay, only 2 out of 20 young adult Tspyl1-/- males sired offspring once in 3 months and the others were sterile. Histologic analysis showed normal spermatogenesis in Tspyl1-/- testes. The cause of premature death and male infertility remained to be determined. In summary, TSPYL1 regulated KLF4, SNAI1 and SOX9 expression. TSPYL1 is important in male fertility and loss-of-function may cause reduced growth and premature death.