Prg1 is regulated by the basic helix-loop-helix transcription factor Math2

Math2 (NEX-1/NeuroD6) is a member of the bHLH transcription factor family and is involved in neuronal differentiation and maturation. In the present study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. Examining the promoter region of rat Prg1, we found four E-boxes designated -E1 to -E4 (CANNTG) which were recognized by the bHLH transcription factor. Using chromatin immunoprecipitation (ChIP) assays, we found that Math2 directly bound to the E-box(es) in the Prg1 promoter. The reporter assay of Prg1 showed that -E1 was critical for the regulation of the Prg1 expression by Math2. Then, the functional role of Math2 and Prg1 was investigated in PC12 cells. Seventy-two hours after transfection of Math2 or Prg1, neurite length and number was significantly induced in PC12 cells. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.Math2（NEX-1/NeuroD6）属于碱性螺旋-环-螺旋（basic Helix-Loop-Helix, bHLH）转录因子家族，参与神经元分化与成熟过程。本研究借助DNA微阵列（DNA microarrays）技术，并结合转染了Math2的大鼠皮层培养细胞，鉴定了Math2的靶向调控基因。在Math2调控的基因中，我们重点关注了可塑性相关基因1（plasticity-related gene 1, Prg1）。通过实时定量PCR（real-time quantitative PCR）分析，我们在大鼠皮层培养细胞与PC12细胞中验证了Math2诱导的Prg1表达。对大鼠Prg1的启动子区域进行分析后，我们发现了4个分别命名为-E1至-E4的E-box基序（CANNTG），此类基序可被bHLH转录因子识别。通过染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验，我们证实Math2可直接结合Prg1启动子区域的E-box位点。Prg1的报告基因实验结果显示，-E1位点对于Math2调控Prg1的表达至关重要。随后我们在PC12细胞中探究了Math2与Prg1的功能作用：在转染Math2或Prg1 72小时后，PC12细胞的神经突长度与数量均显著增加。共转染Prg1-siRNA可完全抑制Math2介导的细胞形态学变化。本研究结果表明，Math2可直接调控Prg1的表达，且Math2-Prg1信号级联在PC12细胞的神经突生长中发挥重要作用。