Brain specific kinases (BRSKs) are serine/threonine kinases, preferentially expressed in the brain after Embryonic Day 12. Although BRSKs are crucial neuronal development factors and regulation of their enzymatic activity has been widely explored, little is known of their transcriptional regulation. In this work, we show that Neuronal Growth Factor (NGF) increased the expression of Brsk1 in PC12 cells. Furthermore, during neuronal differentiation, Brsk1 mRNA increased through a MAPK-dependent Sp1 activation. To gain further insight into this regulation, we analyzed the transcriptional activity of the Brsk1 promoter in PC12 cells treated with NGF. Initially, we defined the minimal promoter region (−342 to +125 bp) responsive to NGF treatment. This region had multiple Sp1 binding sites, one of which was within a CpG island. In vitro binding assays showed that NGF-induced differentiation increased Sp1 binding to this site and that DNA methylation inhibited Sp1 binding. In vitro methylation of the Brsk1 promoter reduced its transcriptional activity and impaired the NGF effect. To evaluate the participation of DNA methyltransferases in Brsk1 gene regulation, the 5′Aza-dC inhibitor was used. 5′Aza-dC acted synergistically with NGF to promote Brsk1 promoter activity. Accordingly, DNMT3B overexpression abolished the response of the Brsk1 promoter to NGF. Surprisingly, we found Dnmt3b to be a direct target of NGF regulation, via the MAPK pathway. In conclusion, our results provide evidence of a novel mechanism of Brsk1 transcriptional regulation changing the promoter's methylation status, which was incited by the NGF-induced neuronal differentiation process.

脑特异性激酶（BRSK）是丝氨酸/苏氨酸激酶，在胚胎第12天后优先在大脑中表达。尽管BRSK是至关重要的神经元发育因子，并且对其酶活性的调节已广为探索，但对其转录调节知之甚少。在这项工作中，我们表明神经元生长因子（NGF）增加了PC12细胞中Brsk1的表达。此外，在神经元分化过程中，Brsk1 mRNA通过MAPK依赖性Sp1激活而增加。为了进一步了解该调节，我们分析了Brsk1的转录活性。NGF处理的PC12细胞中的启动子。最初，我们定义了对NGF处理有反应的最小启动子区域（-342至+125 bp）。该区域具有多个Sp1结合位点，其中一个位于CpG岛内。体外结合试验表明，NGF诱导的分化增加了Sp1与该位点的结合，而DNA甲基化抑制了Sp1的结合。Brsk1启动子的体外甲基化会降低其转录活性并损害NGF的作用。为了评估DNA甲基转移酶在Brsk1基因调控中的参与，使用了5'Aza-dC抑制剂。5′Aza-dC与NGF协同作用促进Brsk1启动子活性。因此，DNMT3B的过表达消除了Brsk1启动子对NGF的应答。出乎意料的是，我们发现Dnmt3b通过MAPK途径成为NGF调控的直接靶标。总之，我们的研究结果提供了一种新的Brsk1转录调控机制，可改变启动子的甲基化状态，这是由NGF诱导的神经元分化过程所激发的。