Jin Chunmei, Kang Hyae Rim, Kang Hyojin, Zhang Yinhua, Lee Yeunkum, Kim Yoonhee, Han Kihoon
Department of Neuroscience, College of Medicine, Korea University, Seoul, South Korea.
Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, South Korea.
Front Mol Neurosci. 2019 Sep 19;12:228. doi: 10.3389/fnmol.2019.00228. eCollection 2019.
Genetic variants of the SH3 and multiple ankyrin repeat domains 3 () gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of knock-out (KO) mice with deletions of different exons have previously been generated and characterized. The different KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of expression may contribute to this heterogeneity. However, whether such compensatory changes occur in KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in transcripts in two different mutant mouse lines ( and ) having partial deletions of exons. We validated an increase in transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1-12, but not the more C-terminal exons 19-22, was observed to increase in mice with deletion of exons 13-16. This suggests a selective compensatory activation of upstream promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased transcripts in KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of expression in the brain, which may also contribute to the phenotypic heterogeneity of different KO mouse lines.
编码兴奋性突触后核心支架的SH3和多个锚蛋白重复结构域3（SHANK3）基因的遗传变异会导致多种脑部疾病。此前已构建并鉴定了几种不同外显子缺失的SHANK3基因敲除（KO）小鼠品系。不同的SHANK3 KO小鼠品系具有共同和品系特异性的表型。SHANK3亚型多样性被认为是表型异质性的潜在机制，通过调节SHANK3表达产生的补偿性变化可能导致这种异质性。然而，尚未详细研究这种补偿性变化是否发生在SHANK3 KO小鼠品系中。利用先前报道的RNA测序分析，我们在两个不同的SHANK3外显子部分缺失的突变小鼠品系（Shank3ΔExon9-11和Shank3ΔExon13-16）中发现SHANK3转录本意外增加。我们使用qRT-PCR分析验证了SHANK3杂合子（HET）和KO小鼠海马体、皮质和纹状体中SHANK3转录本增加，但在小脑中未增加。特别是，在缺失外显子13-16的Shank3小鼠中，观察到N端外显子1-12的表达增加，而C端外显子19-22的表达未增加。这表明上游SHANK3启动子存在选择性补偿性激活。此外，使用结构域特异性的SHANK3抗体，我们证实了Shank3 KO小鼠中增加的SHANK3转录本产生了一种野生型小鼠中未检测到的小SHANK3亚型。综上所述，我们的结果揭示了大脑中SHANK3表达调控的另一层复杂性，这也可能导致不同SHANK3 KO小鼠品系的表型异质性。
