Abstract：
SET (SuVar3-9, Enhancer of Zeste, Trithorax) domain bifurcated histone lysine methyltransferase 1, setdb1, is the predominant histone lysine methyltransferase catalyzing H3K9me3. Prior studies have illustrated that setdb1 and H3K9me3 critically regulate sex differentiation and gametogenesis. However, the molecular details by which setdb1 is involved in these processes in fish have been poorly reported. Here, we cloned and characterized the setdb1 ORF (open reading frame) sequence from Chinese tongue sole (Cynoglossus semilaevis). The setdb1 ORF sequence was 3,669 bp, encoding a 1,222-amino-acid protein. Phylogenetic analysis showed that setdb1 was structurally conserved. qRT-PCR revealed that setdb1 had a high expression level in the testes at 12 mpf (months post fertilization). Single-cell RNA-seq data at 24 mpf indicated that setdb1 was generally expressed in spermatogenic cells at each stage except for sperm and was centrally expressed in oogonia. H3K9me3 modification was observed in gonads with the immunofluorescence technique. Furthermore, the overexpression experiment suggested that sox5 was a candidate target of setdb1. sox5 was abundantly expressed in male and pseudomale gonads at 24 mpf. Single-cell RNA-seq data showed that sox5 was mainly expressed in spermatogonia and its expression gradually declined with differentiation. Taken together, our findings imply that setdb1 regulates sox5 transcription in gonads, which provides molecular clues into histone modification-mediated orchestration of sex differentiation and gametogenesis.
摘要：
SET（SuVar3-9，Zeste的增强器，Trithorax）结构域分叉的组蛋白赖氨酸甲基转移酶1，setdb1，是催化H3K9me3的主要组蛋白赖氨酸甲基转移酶。先前的研究表明，setdb1和H3K9me3至关重要地调节性别分化和配子发生。然而,setdb1参与鱼类这些过程的分子细节报道很少。这里,我们克隆并表征了中国舌底（Cynoglossussemilaevis）的setdb1ORF（开放阅读框）序列。setdb1ORF序列为3,669bp，编码1,222个氨基酸的蛋白质。系统发育分析表明，setdb1在结构上是保守的。qRT-PCR显示，setdb1在12mpf（受精后几个月）时在睾丸中具有高表达水平。24mpf的单细胞RNA-seq数据表明，除精子外，setdb1通常在每个阶段的生精细胞中表达，并在卵原细胞中集中表达。用免疫荧光技术在性腺中观察到H3K9me3修饰。此外，过表达实验表明sox5是setdb1的候选靶标。sox5在24mpf时在雄性和假性性腺中大量表达。单细胞RNA-seq数据显示，sox5主要在精原细胞中表达，其表达随分化而逐渐下降。一起来看,我们的发现暗示setdb1调节性腺中的sox5转录，这为组蛋白修饰介导的性别分化和配子发生的编排提供了分子线索。
