In fetal testis, SOX9 acts on transcription and splicing of its targets genes through binding to genomic regions with conserved signatures [ChIP-seq]. In fetal testis, SOX9 acts on transcription and splicing of its targets genes through binding to genomic regions with conserved signatures [ChIP-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. Overall design: examination of the role of the transcription factor SOX9 in the fetal testis: ChIP-seq

在哺乳动物中，雄性发育命运由核心转录调控因子SOX9掌控：当存在SOX9时，胚胎性腺的体细胞前体细胞会分化为支持细胞（Sertoli cell）——睾丸分化的核心组织者。因此，解析该转录因子的靶基因，能够从基因组层面阐明细胞定向分化的分子机制。本研究通过对野生型小鼠与牛的睾丸开展染色质免疫共沉淀测序（ChIP-seq），并结合SOX9缺失小鼠睾丸的RNA测序（RNA-seq）数据，鉴定出哺乳动物胎儿性腺中SOX9的靶基因。小鼠和牛胎儿睾丸中的SOX9可结合哺乳动物间保守的大量基因，其中包含在睾丸与卵巢发育中功能已得到证实的基因。RNA-seq分析结果显示，睾丸与卵巢存在性别特异性的RNA剪接，且SOX9同时介导靶基因转录与差异剪接过程。SOX9结合的区域主要位于5'近端或基因内部，且具备特定的基因组特征，我们将其命名为"支持细胞特征序列"（Sertoli cell signatures, SCS）。SCS在哺乳动物中具有保守性，且包含支持细胞重编程因子SOX9、GATA4与DMRT1的多个结合基序；此外，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指引。例如，我们在核因子TRIM28的靶基因中检测到了SCS序列，并通过实验证实SOX9与TRIM28蛋白在胎儿睾丸中存在相互作用。研究整体设计：探究转录因子SOX9在胎儿睾丸中的功能作用，所用实验手段包含ChIP-seq。