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The cytochromes P450 (CYPs) represent a large gene superfamily that plays an important role in the metabolism of both exogenous and endogenous compounds. We have reported that the testis-specific Y-encoded-like proteins (TSPYLs) are novel CYP gene transcriptional regulators. However, little is known of mechanism(s) by which TSPYLs regulate CYP expression or the functional consequences of that regulation. The TSPYL gene family includes six members, TSPYL1 to TSPYL6. However, TSPYL3 is a pseudogene, TSPYL5 is only known to regulates the expression of CYP19A1, and TSPYL6 is expressed exclusively in the testis. Therefore, TSPYL 1, 2 and 4 were included in the present study. To better understand how TSPYL1, 2, and 4 might influence CYP expression, we performed a series of pull-downs and mass spectrometric analyses. Panther pathway analysis of the 2272 pulled down proteins for all 3 TSPYL isoforms showed that the top five pathways were the Wnt signaling pathway, the Integrin signaling pathway, the Gonadotropin releasing hormone receptor pathway, the Angiogenesis pathway and Inflammation mediated by chemokines and cytokines. Specifically, we observed that 177 Wnt signaling pathway proteins were pulled down with the TSPYLs. Subsequent luciferase assays showed that TSPYL1 knockdown had a greater effect on the activation of Wnt signaling than did TSPYL2 or TSPYL4 knockdown. Therefore, in subsequent experiments, we focused our attention on TSPYL1. HepaRG cell qRT-PCR showed that TSPYL1 regulated the expression of CYPs involved in cholesterol-metabolism such as CYP1B1 and CYP7A1. Furthermore, TSPYL1 and β-catenin regulated CYP1B1 expression in opposite directions and TSPYL1 appeared to regulate CYP1B1 expression by blocking β-catenin binding to the TCF7L2 transcription factor on the CYP1B1 promoter. In β-catenin and TSPYL1 double knockdown cells, CYP1B1 expression and the generation of CYP1B1 downstream metabolites such as 20-HETE could be restored. Finally, we observed that TSPYL1 expression was associated with plasma cholesterol levels and BMI during previous clinical studies of obesity. In conclusion, this series of experiments has revealed a novel mechanism for regulation of the expression of cholesterol-metabolizing CYPs, particularly CYP1B1, by TSPYL1 via Wnt/β-catenin signaling, raising the possibility that TSPYL1 might represent a molecular target for influencing cholesterol homeostasis.