SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor.
SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor.
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猪 GHR 内含子中的 SINE 插入可能通过充当阻遏物来降低其表达
DOI：
10.3390/ani11071871
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发表时间：
2021-06-23
期刊：
影响因子：
--
通讯作者：
Song C
中科院分区：
文献类型：
--
作者：
Chen C;Zheng Y;Wang M;Murani E;D'Alessandro E;Moawad AS;Wang X;Wimmers K;Song C
GH/IGF 轴基因在发育和生长过程中骨骼增生的调节中发挥核心作用，因此代表生长性状的候选基因。逆转录转座子插入多态性是结构变异的主要贡献者。由于它们携带功能元件，例如增强子、绝缘子或启动子，它们往往会产生大效应突变，导致靶基因活性和表型的变化。在本研究中，通过比较基因组学和 PCR 研究了四种 GH/IGF 轴基因（GH、GHR、IGF1 和 IGF1R）的 RIP。鉴定出 GHR 基因中的 4 个 RIP 和 IGF1 基因中的 1 个 RIP。进一步分析表明，GHR 第一个内含子中的一个 RIP 可能通过作为阻遏蛋白在 GHR 表达的调节中发挥作用。这些发现有助于了解 RIP 在猪 GH/IGF 轴基因遗传变异和表型变异中的作用。 GH/IGF轴基因的遗传多样性及其与基因表达和表型性状变异（主要以SNP为代表）的关联已被广泛报道。然而，逆转录转座子插入多态性（RIP）对 GH/IGF 轴基因活性的影响尚未见报道。本研究通过生物信息学预测和PCR验证来筛选4个GH/IGF轴基因（GH、GHR、IGF1和IGF1R）的RIP。通过PCR总共确认了5个RIP，包括IGF1内含子3中的1个SINE RIP，GHR 7内含子中的1个L1 RIP，以及GHR内含子1、内含子5和内含子9中的3个SINE RIP，显示了不同品种的多态性。双荧光素酶报告基因检测显示，GHR 内含子 1 中的 SINE 插入显着抑制 PK15、Hela、C2C12 和 3T3-L1 细胞中的 GHR 启动子活性。此外，qPCR 结果证实，这种 SINE 插入与腿部肌肉和背最长肌中 GHR 表达的降低相关，表明它可能作为参与 GHR 表达调节的阻遏蛋白。总之，我们的数据表明，RIP 有助于 GH/IGF 轴基因的遗传变异，其中 GHR 内含子 1 中的一个 SINE RIP 可能通过充当阻遏物来降低 GHR 的表达。
GH/IGF axis genes play a central role in the regulation of skeletal accretion during development and growth, and thus represent candidate genes for growth traits. Retrotransposon insertion polymorphisms are major contributors to structural variations. They tend to generate large effect mutations resulting in variations in target gene activity and phenotype due to the fact that they carry functional elements, such as enhancers, insulators, or promoters. In the present study, RIPs in four GH/IGF axis genes (GH, GHR, IGF1, and IGF1R) were investigated by comparative genomics and PCR. Four RIPs in the GHR gene and one RIP in the IGF1 gene were identified. Further analysis revealed that one RIP in the first intron of GHR might play a role in the regulation of GHR expression by acting as a repressor. These findings contribute to the understanding of the role of RIPs in the genetic variation of GH/IGF axis genes and phenotypic variation in pigs. The genetic diversity of the GH/IGF axis genes and their association with the variation of gene expression and phenotypic traits, principally represented by SNPs, have been extensively reported. Nevertheless, the impact of retrotransposon insertion polymorphisms (RIPs) on the GH/IGF axis gene activity has not been reported. In the present study, bioinformatic prediction and PCR verification were performed to screen RIPs in four GH/IGF axis genes (GH, GHR, IGF1 and IGF1R). In total, five RIPs, including one SINE RIP in intron 3 of IGF1, one L1 RIP in intron 7 of GHR, and three SINE RIPs in intron 1, intron 5 and intron 9 of GHR, were confirmed by PCR, displaying polymorphisms in diverse breeds. Dual luciferase reporter assay revealed that the SINE insertion in intron 1 of GHR significantly repressed the GHR promoter activity in PK15, Hela, C2C12 and 3T3-L1 cells. Furthermore, qPCR results confirmed that this SINE insertion was associated with a decreased expression of GHR in the leg muscle and longissimus dorsi, indicating that it may act as a repressor involved in the regulation of GHR expression. In summary, our data revealed that RIPs contribute to the genetic variation of GH/IGF axis genes, whereby one SINE RIP in the intron 1 of GHR may decrease the expression of GHR by acting as a repressor.
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