Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice

Background: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet Syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency, however putative causal non-coding promoter mutations have been identified. Methods: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We bred a transgenic mouse line with deletion of the extended evolutionarily-conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alteration in behavior. Results: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and some behavioral deficits. Conclusions: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence Characterization of differential gene expression changes with Scn1a heterozygous and homozygous knockout.研究背景：大脑中存在多个共激活启动子的基因较为常见，但目前针对这类潜在冗余的基因组调控元件的功能需求，仍缺乏深入认知。编码NaV1.1钠通道α亚基的SCN1A，便是这类拥有两个共激活启动子的基因之一。SCN1A的突变与癫痫相关，包括德雷夫综合征（Dravet Syndrome, DS）。多数DS患者携带可引发SCN1A单倍体剂量不足的编码区突变，同时也已发现疑似致病的非编码启动子突变。 研究方法：为明确上述潜在冗余的Scn1a启动子之一的功能作用，我们将研究聚焦于非编码区Scn1a 1b调控区域——该区域此前被定义为非经典可变转录起始位点。我们构建了携带经延伸的进化保守1b非编码区间缺失的转基因小鼠品系，对其基因与蛋白质表达变化进行表征，并评估了癫痫发作活动与行为改变情况。 研究结果：携带1b非编码区间缺失的小鼠，其全脑范围内Scn1a与NaV1.1的表达量出现了意料之外的严重下调。该表型伴随脑电图异常、热诱发性癫痫发作以及部分行为缺陷。 研究结论：本研究助力对癫痫核心风险基因SCN1A的调控网络进行功能解析。我们证实1b区域是与疾病密切相关的关键调控元件，并提供了Scn1a杂合与纯合敲除模型中差异基因表达变化的相关表征数据。