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

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.背景：大脑中携带多个共激活启动子的基因较为常见，但目前对于这类潜在冗余的基因组调控元件的功能需求仍知之甚少。编码NaV1.1型钠通道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的突变与包括德雷夫综合征（Dravet Syndrome, DS）在内的癫痫疾病密切相关。大多数德雷夫综合征患者携带可导致SCN1A单倍体剂量不足的编码区突变，不过目前也已发现疑似致病的非编码区启动子突变。 方法：为明确这类潜在冗余的Scn1a启动子之一的功能，我们聚焦于此前被定义为非经典可变转录起始位点的非编码Scn1a 1b调控区域。我们繁育了携带扩展进化保守1b非编码区间缺失的转基因小鼠品系，对其基因与蛋白表达变化进行表征，并评估其癫痫发作活动与行为改变。 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的表达量出现了出乎意料的严重降低。这一变化伴随脑电图异常与热诱发癫痫发作，同时部分小鼠出现行为缺陷。 结论：本研究为解析主要癫痫风险基因SCN1A的调控网络提供了功能层面的实验证据。我们确认1b区域为与疾病密切相关的关键调控元件，并针对Scn1a杂合及纯合敲除后的差异基因表达变化开展了表征研究。