Peptidyl arginine deiminase 2 (Padi2) is expressed in Sertoli cells in a specific manner and regulated by SOX9 during testicular development

Atsumi Tsuji-Hosokawa,Andrea Coschiera,Kenichi Kashimada,Shuji Takada,Tomoko Kato,David Schlessinger,Yuya Ogawa,Rowena Lavery,Vincent R Harley,Kei Takasawa,Tomohiro Morio,Risa Nomura

doi:10.1038/s41598-018-31376-8

Abstract

Peptidyl arginine deiminases (PADIs) are enzymes that change the charge of proteins through citrullination. We recently found Padi2 was expressed exclusively in fetal Sertoli cells. In this study, we analyzed the transcriptional regulation of Padi2 and the role of PADI2 in testicular development. We showed SOX9 positively regulated Padi2 transcription and FOXL2 antagonized it in TM3 cells, a model of Sertoli cells. The responsive region to SOX9 and FOXL2 was identified within the Padi2 sequence by reporter assay. In fetal testes from Sox9 knockout (AMH-Cre:Sox9flox/flox) mice, Padi2 expression was greatly reduced, indicating SOX9 regulates Padi2 in vivo. In vitro analysis using siRNA suggested PADI2 modified transcriptional regulation by SOX9. However, Padi2−/− XY mice were fertile and showed no apparent reproductive anomalies. Although, PADI2 is known as an epigenetic transcriptional regulator through H3 citrullination, no significant difference in H3 citrullination between wildtype and Padi2−/− XY gonads was observed. These results suggest Padi2 is a novel gene involved in testis development that is specifically expressed in Sertoli cells through the regulation by SOX9 and FOXL2 and PADI2 supports regulation of target genes by SOX9. Analysis of the Padi2−/− XY phenotype suggested a redundant factor compensated for PADI2 function in testicular development.

Highlights

SRY-box 9 (Sox9) is the major target of a male-determining gene on the Y chromosome, SRY, and its product SOX9 specifies the differentiation and function of Sertoli cells from somatic cell precursors, which orchestrate the development and maintenance of other testicular cell types
PADI2 function has been reported in other biological contexts, such as H3 citrullination during breast cancer oncogenesis[16,17,18] the estrus cycle in the uterus or in gonadotrope cells[11,19,20] peptidyl citrullination during colon cancer oncogenesis[21,22] and inflammation inducing multiple sclerosis and skin neoplasia[23,24,25]
The citrulline amino acid residues are only produced through post-translational modifications catalyzed by Peptidyl arginine deiminases (PADIs), which is encoded by the Padi gene[28]

Summary

Introduction

SRY-box 9 (Sox9) is the major target of a male-determining gene on the Y chromosome, SRY, and its product SOX9 specifies the differentiation and function of Sertoli cells from somatic cell precursors, which orchestrate the development and maintenance of other testicular cell types. Recent bioinformatics data analyses, including chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq), revealed that SOX9 recognizes numerous genes[7,8,9]. These data helped to produce an outline of the transcriptional network of SOX9, but the precise mechanisms of Sertoli cell differentiation and their maintenance remain mostly unclear. Padi[2] in fetal mice gonads obtained from the GEO Profiles database. (e) Padi[2] expression of gonad in WT and KitWv/Wv mice at 13.5dpc relative to ActB was quantified by qRT-PCR analysis. Statistical significance was determined using one-way ANOVA followed by a Tukey-Kramer post hoc test

Methods

Results

Conclusion