DOI: 10.3724/SP.J.1249.2015.02128
提出一种多学科交叉结合的策略,试图初步获得系统性调控γ-氨基丁酸(gamma-aminobutyric acid, GABA)受体基因的转录因子线索. 利用基于功能基因组学方法的DNA元件百科全书计划已发表的数据,系统性地获得GABA受体基因的开放染色质序列,并以此作为固相化探针,捕获与其特异性相互作用的蛋白质分子,并利用质谱分析鉴定蛋白. 结果发现,对不同脑区获得的核蛋白,探针都能捕获到同样的特异性条带,然而,作为对照的天门冬氨酸(N-methyl-D-aspartate, NMDA)受体基因相关的开放染色质探针,在不同脑区中并未检获上述特异信号,说明结合蛋白是特异的. 质谱分析表明,与GABA受体基因相关的开放染色质特异相互作用的蛋白是核膜血影重复蛋白-1(nuclear envelop spectrin repeatprotein-1, Nesprin-1),又称synaptic nuclear envelope-1(SYNE-1). 进一步的调控网络生物信息学分析表明,Nesprin-1可能与MAFA、IRX2、BCL6、CEBPA以及RP58等转录因子形成复合物,并与GABA受体基因GABRA5、GABRA6、GABBR1和GABBR2等共表达. 表明GABA受体基因在不同脑区是通过相同的转录调控机制进行表达的,Nesprin-1可能与MAFA、IRX2、BCL6、CEBPA以及RP58等转录因子形成复合物进而调控GABA受体基因表达,该特异的转录因子调控网络有望用于诱导多能干细胞或是前体细胞直接分化为GABA能神经元.
By using a multidisciplinary strategy, we try to systematically find out the transcription factors that regulate gamma-aminobutyric acid(GABA)receptor expression. Based on the published data in the encyclopedia of DNA elements(ENCODE), we obtain open chromatin sequences of GABA receptor genes. These sequences are used as solid phase probes to capture the specific proteins that could interact with the sequences directly. The captured protein is identified with mass spectrometry for further regulatory network analysis. The experimental results show that the GABA receptor gene probes can capture one specific band in different brain domains while the N-methyl-D-aspartate(NMDA)gene probes, which are designed as negative control, could not capture this specific band. Mass spectrometry results indicate that the protein, which could specifically bind with GABA receptor gene open chromatin sequence, is nuclear envelop spectrin repeatprotein-1(Nesprin-1)(synaptic nuclear envelope-1, SYNE-1). Further bioinformatics analysis results suggest that Nesprin-1 could form a complex with transcription factors such as MAFA, IRX2, BCL6, CEBPA and RP58 and could co-express with GABA receptor genes such as GABRA5, GABRA6, GABBR1 and GABBR2. In conclusion, GABA receptor genes are regulated through the same transcriptional regulation mechanism in different brain domains. Nesprin-1 could interact with transcription factors such as MAFA, IRX2, BCL6, CEBPA and RP58 to form a regulatory complex that regulates the expression of GABA receptor genes. This specific regulatory network can be used as a tool for further inductions of the differentiation of embryonic stem cells or other stem cells into GABA receptor expressing neurons.
