转录因子Arx的细胞核质分布调控成肌细胞C2C12分化的分子机理研究

Nucleocytoplasmic localization of transcription factors is one of factors regulating physiological functions of eukaryotic cells. Arx, member of homeodomain-containing transcription factor family, regulates development of multiple tissues by repressing expression of genes in cells. Mutated Arx causes several genetic diseases. We observed in C2C12 cells that Arx relocates from the nucleus to the cytoplasm when cells start differentiation: Arx is mainly in the nucleus when cells do not differentiate but it is mainly in the cytoplasm in differentiated cells. Inhibitors of the Crm1-mediated export pathway block the export of Arx and stop the differentiation significantly, suggesting that the relocation of Arx from the nucleus to the cytoplasm is related to C2C12 differentiation. We also found that 530S (serine) of Arx could be the major phosphorylation site for PKA and its mutation from serine to alanine prevented both the interaction between Arx with Crm1 and the nuclear export of Arx.Based on these preliminary data, in this project, we will elucidate mechanisms of Arx export and undertand how nucleocytoplasmic localization of Arx regulated by PKA phosphorylation controls cell differentiation. We will identify genes which promote C2C12 differentiation but are repressed by Arx. These goals are part of our researches on functional analysis of nucleocytoplasmic localization of Arx.

转录因子在细胞核质间的分布是调控真核细胞生理功能的重要方式之一。Arx是homeodomain转录因子家族成员之一，它通过抑制其它基因的表达来调控多个组织的发育，突变的Arx导致多种遗传疾病。我们在C2C12细胞中发现，Arx的核质分布随细胞的分化而改变：分化前它在细胞核中，分化时它分布在细胞质。Crm1介导出核途径的特异抑制物能阻止Arx的细胞质分布，降低细胞的分化能力， 说明它的出核可能与C2C12分化相关；Arx的第530氨基酸-丝氨酸-可能是Arx主要的PKA磷酸化位点，它的突变(S to A)阻止Arx与Crm1相互作用，继而阻止Arx的出核。据此，我们将在本项目中确定Arx出核的分子机理；探索 PKA磷酸化调控Arx的细胞核质分布，继而调控C2C12分化的机理；分离和验证促进C2C12分化的Arx靶基因。本项目是我们系统研究Arx核质分布与其生物学功能关系的继续。

转录因子在细胞核质间的分布是调控真核细胞生理功能的重要方式之一。Arx是homeodomain转录因子家族成员之一，它通过抑制其它基因的表达来调控多个组织的发育，突变的Arx导致多种遗传疾病。Arx蛋白的细胞定位随C2C12细胞分化过程而改变：分化前定位在细胞核，分化时分布在细胞质,这一过程被Crm1核质转运受体的特异性抑制剂ratjadone抑制。我们构建了多个Arx蛋白的截短突变体，利用异核体细胞融合实验检测不同突变体的核质穿梭能力，发现其都保留了出核转运的能力。同时我们发现PKA激酶可以磷酸化修饰Arx蛋白的S266位点，但该位点的磷酸化修饰不影响其核质穿梭的能力。在C2C12细胞内过表达或敲低Arx蛋白表达都会影响C2C12细胞的分化能力，进一步研究发现SIX1是其作用靶基因，并参与调控C2C12细胞分化。过表达Arx蛋白突变体（ArxΔNLS/R382S：核定位信号突变体），可以促进C2C12细胞分化。