Analysis of biological function of regulatory network composed of bZip transcription factors

寻找bZip网络调控的靶基因。从mafG-/-::mafK+/-突变骨髓中获得的巨核细胞严重受损，而血小板形成被认为是血小板产生的主要途径。为了鉴定对血小板产生有重要作用的小maf蛋白的靶基因，我们从野生型巨核细胞中减去mag -/-::mafK+/-巨核细胞的CDNA库。获得了编码肌动蛋白和微管蛋白等细胞骨架蛋白的CDNA克隆。从减法中获得的一个克隆，#325，编码了新的未知蛋白质。#325在巨核细胞中表达量高，在红细胞中表达量低。这一表达谱表明#325在巨核细胞分化、成熟和血小板产生中起重要作用。神经细胞中bZip网络功能的分析。MafG-/-::mafK+/-复合小鼠在出生后两个月表现出严重的运动障碍。该征象类似于人的遗传性异常，是由甘氨酸受体功能不足引起的。当我们检测甘氨酸受体亚基基因的表达水平时，α和β亚基都降低了。这表明甘氨酸受体亚基基因直接或间接地通过MARE调控。Maf小蛋白对Bach蛋白亚细胞定位的影响。通过EGMSA检测了mafG-/-::mafK+/-小鼠脑核提取物与MARE的结合活性，我们发现Bach/小Maf异源二聚体的移位带强度显著降低。有趣的是，核定位表明Bach蛋白在mafG-/-::mafK中为imuuHcu；/ -大脑。结果表明，小Maf蛋白促进Bach的核定位，这取决于小Maf的bZip结构。

Searching for target genes regulated by bZip network.Proplatelet formation, which is regarded as a major pathway of platelet production, is severely impaired in megakaryocytes obtained from mafG-/-::mafK+/- mutant bone marrow. In order to identify the target genes of small Mafproteins which are important for platelet production, we subtracted CDNA pool of mafG-/-::mafK+/- megakaryocytes from that of wild type megakaryocytes. CDNA clones coding cytoskeletal proteins, such as actin and tubulin, were obtained. One clone obtained from the subtraction, #325, encoded novel unknown protein. Expression level of #325 was high in megakaryocytes but low in erythoid cells. This expression profile suggested that #325 plays an important role in megakaryocytic differentiation, maturation and platelet production.Analysis of bZip network function in neuronal cells.MafG-/-::mafK+/- compound mice display severe motor disorder which becomes apparent at two months after birth. This sign resembled hereditary hyperekplexia in human, which is caused by functional insufficiency of glycine receptor. When we examined the expression levels of glycine reseptor subunit genes, both alpha and beta subunits were reduced. This result indicated that glycine receptor subunit genes are regulated through MARE directly or indirectly.Effect of small Maf proteins to subcellular localization of Bach proteins.Binding activity to MARE in the brain nuclear extract from mafG-/-::mafK+/- mice was examined by EGMSA, and we found that a shifted band comnosed of Bach/small Maf heterodimers is remarkably reduced in intensity. Interestingly, nuclear localization that Bach proteins was imuuHcu in mafG-/-::mafK;/- brain. It was concluded that small Maf proteins promote the nuclear localization of Bach, which depends on bZip structure of small Maf.