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

寻找bZip网络调控的靶基因：mafG-/-：：mafK+/-突变骨髓巨核细胞的前血小板形成（Proplatelet formation，proplatelet）被认为是血小板生成的主要途径，但其功能严重受损。为了鉴定对血小板产生重要的小Maf蛋白的靶基因，我们从野生型巨核细胞的cDNA库中减去mafG-/-：：mafK+/-巨核细胞的cDNA库。获得了编码细胞骨架蛋白如肌动蛋白和微管蛋白的cDNA克隆。从减法获得的一个克隆，#325，编码新的未知蛋白。#325在巨核细胞中表达水平高，而在类巨核细胞中表达水平低。MafG-/-：：mafK+/-复合物小鼠表现出严重的运动障碍，在出生后两个月变得明显。这种现象类似于人类遗传性兴奋过度，这是由于甘氨酸受体功能缺陷所致。当我们检测甘氨酸受体亚单位基因的表达水平时，α和β亚单位都减少了。小Maf蛋白对Bach蛋白亚细胞定位的影响用EGMSA检测了mafG-/-：：mafK+/-小鼠脑核提取物与MARE的结合活性，发现由Bach/小Maf异二聚体组成的移动带强度明显降低。有趣的是，Bach蛋白质在mafG-/-：：mafK;/-脑中的核定位是imuuHcu。结果表明，小分子Maf蛋白促进Bach的核定位，这依赖于小分子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.