Transcriptional regulation of the mouse <i>EphA4</i>, <i>Ephrin-B2</i> and <i>Ephrin-A3</i> genes by the circadian clock machinery

Circadian rhythms originate from molecular feedback loops. In mammals, the transcription factors CLOCK and BMAL1 act on regulatory elements (i.e. E-boxes) to shape biological functions in a rhythmic manner. The EPHA4 receptor and its ligands Ephrins (EFN) are cell adhesion molecules regulating neurotransmission and neuronal morphology. Previous studies showed the presence of E-boxes in the genes of <i>EphA4</i> and specific Ephrins, and that <i>EphA4</i> knockout mice have an altered circadian rhythm of locomotor activity. We thus hypothesized that the core clock machinery regulates the gene expression of <i>EphA4</i>, <i>EfnB2</i> and <i>EfnA3</i>. CLOCK and BMAL1 (or NPAS2 and BMAL2) were found to have transcriptional activity on distal and proximal regions of <i>EphA4</i>, <i>EfnB2</i> and <i>EfnA3</i> putative promoters. A constitutively active form of glycogen synthase kinase 3β (GSK3β; a negative regulator of CLOCK and BMAL1) blocked the transcriptional induction. Mutating the E-boxes of <i>EphA4</i> distal promoter sequence reduced transcriptional induction. EPHA4 and EFNB2 protein levels did not show circadian variations in the mouse suprachiasmatic nucleus or prefrontal cortex. The findings uncover that core circadian transcription factors can regulate the gene expression of elements of the Eph/Ephrin system, which might contribute to circadian rhythmicity in biological processes in the brain or peripheral tissues.昼夜节律源自分子反馈环路。在哺乳动物体内，转录因子CLOCK与BMAL1可作用于调控元件（即E盒（E-box）），以节律性方式调控生物学功能。EPHA4受体（EPHA4）及其配体Ephrins（EFN）属于细胞黏附分子，可调控神经递质传递与神经元形态。既往研究已在EphA4基因与特定Ephrins的基因序列中发现E盒，且EphA4基因敲除小鼠的运动活动昼夜节律发生改变。据此我们提出假说：核心钟控机制可调控EphA4、EfnB2与EfnA3的基因表达。研究发现，CLOCK与BMAL1（或NPAS2与BMAL2）可在EphA4、EfnB2及EfnA3的推定启动子的远端与近端区域发挥转录活性。组成型激活形式的糖原合酶激酶3β（GSK3β，CLOCK与BMAL1的负调控因子）可阻断该转录诱导过程。对EphA4远端启动子序列中的E盒进行突变，可减弱转录诱导效应。在小鼠视交叉上核与前额叶皮层中，EPHA4与EFNB2的蛋白水平未呈现昼夜节律性变化。本研究结果揭示，核心昼夜节律转录因子可调控Eph/Ephrin系统相关元件的基因表达，这或可参与大脑或外周组织中生物学过程的昼夜节律调控。