In fetal testis, SOX9 acts on transcription and splicing of its targets genes through binding to genomic regions with conserved signatures [RNA-seq]

In mammals, male fate is under the control of the master transcriptional regulator, SOX9: in its presence, somatic precursor cells of the embryonic gonads differentiate into Sertoli cells, the main organizers of testicular differentiation. Therefore, analyzing target genes of this transcription factor allows understanding mechanisms of cellular commitment at the genomic level. With the use of ChIP-seq in murine and bovine wild-type testes combined with RNAseq from mouse testes lacking SOX9, we identified SOX9 target genes in the mammalian fetal gonad. SOX9 in murine and bovine fetal testes binds to a large set of genes conserved among mammals, including those with well-established roles in testis and ovary development. RNAseq analysis shows that testis and ovary display sex specific RNA splicing and that SOX9 mediates both target gene transcription and differential splicing. Regions bound by SOX9 are predominantly 5' proximal or intra-genic, and display a specific genomic features that we call "Sertoli cell signatures" or SCS. The SCS is conserved among mammals and comprises multiple binding motifs for the Sertoli reprogramming factors SOX9, GATA4 and DMRT1; indeed, independent DMRT1 ChIP-seq confirms the enrichment of the SCS. Bioinformatic analysis of SCSs regions predicts novel regulatory mechanisms prompting functional validation. For example, we detected SCS in target genes of the nuclear factor TRIM28 and we show experimentally that SOX9 and TRIM28 proteins interact in fetal testis. examination of the role of the transcription factor SOX9 in the fetal testis在哺乳动物中，雄性性别发育命运由核心转录调控因子SOX9掌控：当存在SOX9时，胚胎性腺的体前体细胞会分化为塞尔托利细胞（Sertoli cells）——睾丸分化的主要组织者。因此，对该转录因子的靶基因进行分析，能够在基因组层面解析细胞定型的分子机制。本研究通过在野生型小鼠和牛睾丸中开展染色质免疫共沉淀测序（ChIP-seq），并结合SOX9敲除小鼠睾丸的RNA测序（RNA-seq）数据，成功鉴定出哺乳动物胚胎性腺中的SOX9靶基因。小鼠和牛胚胎睾丸中的SOX9可结合哺乳动物间保守的大量基因，其中包括那些在睾丸与卵巢发育中已被充分证实功能的基因。RNA-seq分析结果显示，睾丸与卵巢存在性别特异性RNA剪接，且SOX9同时介导靶基因转录与选择性剪接。SOX9结合的区域主要位于5'近端或基因内部，且具有特定的基因组特征，我们将其命名为"塞尔托利细胞特征序列"（Sertoli cell signatures, 简称SCS）。SCS在哺乳动物中保守存在，且包含塞尔托利细胞重编程因子SOX9、GATA4与DMRT1的多个结合基序；后续独立开展的DMRT1 ChIP-seq实验证实了SCS的富集现象。对SCS区域的生物信息学分析预测了全新的调控机制，为后续功能验证提供了方向。例如，本研究在核因子TRIM28的靶基因中检测到了SCS序列，并通过实验证实SOX9与TRIM28蛋白在胚胎睾丸中存在相互作用。本研究针对转录因子SOX9在胚胎睾丸中的功能展开了系统探究。