Analysis of Regulatory Mechanisms of Proplatetet Formation by Small Maf Proteins.

本研究的目的是通过分析小MAF蛋白在巨核细胞分化和成熟过程中的功能，以及确定小MAF下游调控的靶基因来阐明血小板形成的调控机制。(1)巨核细胞中小MAF的结构-功能分析二聚体转录因子通常由一个普遍存在的亚基与一个限制性较高的配对分子组成。MafG是MafG：P45异源二聚体中比较常见的一个亚基，它是巨核细胞中一种不可或缺的转录激活剂，介导了原血小板的形成。过量的MafG通过形成抑制性同源二聚体来拮抗MafG：P45异源二聚体的活性，从而阻止原血小板的形成；因此，MafG依赖于其相对丰度来激活和抑制转录。在此，我们报道了MafG在体内与SUMO-2/3结合，当在巨核细胞中表达时，SUMO化缺陷的MAFG正常参与反式…更多的成熟激活，但不是抑制。在异染色质和常染色质中都发现了MafG，而突变的MafG未能定位在异染色质中。我们的结论是，MafG介导的异染色质抑制和定位需要SUMO化，因此这种二聚体转录因子广泛表达的成分的SUMO化决定了其转录潜力的一个方面。(2)巨核细胞中一个新的调控小MAF下游的基因的鉴定。为了阐明在血小板形成过程中的分子机制，我们寻找了野生型和小MAF突变的巨核细胞之间的差异表达基因。在与细胞骨架相关的各种因子中，通过消减筛选得到了一个新的因子，称为克隆325。从脑cDNA文库中获得了全长cDNA，推导的氨基酸序列显示有一个亮氨酸拉链基序，但没有典型的功能基序。克隆325在从骨髓中纯化的原代巨核细胞中高表达，但在未成熟的造血细胞和红系细胞中相对较低。我们在小鼠体内破坏了克隆325的基因，发现纯合子动物是存活的，外周血细胞计数没有异常。但当观察突变动物骨髓中的巨核细胞时，我们发现很少有细胞适当地突出正常的前血小板。这一结果表明，克隆325对原血小板的形成是重要的。较少

The goal of this study was to clarify the regulatory mechanisms of proplatelet formation through analyang the function of small Maf proteins in megakaryocyte differentiation and maturation and identifying the target genes regulated downstream of small Maf.(1)Structure-function analysis of small Maf in megakaryocytesDimeric transcription factors often consist of one ubiquitous subunit complexed with a more highly restricted partner molecule. MafG is a more common subunit of the MafG:p45 heterodimer, which is an indispensable transcriptional activator mediating proplatelet formation in megakaryocytes. Excess MafG antagonizes the activity of the MafG:p45 heterodimer by forming an inhibitory homodimer, thereby blocking proplatelet formation ; thus MafG both activates and represses transcription depending on its relative abundance. Here we report that MafG is conjugated to SUMO-2/3 in vivo and that SUMOylation-defective MafG, when expressed in megakaryocytes, participates normally in transc … More riptional activation, but not in repression. While MafG was found in both heterochromatin and euchromatin, the mutant MafG failed to be localized in heterochromatin. We conclude that SUMOylation is required for MafG-mediated repression and localization to heterochromatin, and thus SUMOylation of the broadly expressed component of this dimeric transcription factor determines one aspect of its transcriptional potential.(2)Identification of a novel gene regulated downstream of small Maf in megakaryocytes.In order to clarify the molecular mechanisms operating in the process of proplatelet formation, we searched for the differentially expressed genes between the wild type and small maf mutant megakaryocytes. Among various factors relating to the cytoskeleton, one novel factor called clone 325 was isolated through the subtraction screening. The full length cDNA was obtained from the brain cDNA library, and the deduced amino acid sequence revealed one leucine zipper-like motif but no canonical functional motifs. The clone 325 was highly expressed in the primary megakaryocytes purified from the bone marrow, but its abundance was relatively low in immature hematopoietic cells and in erythroid cells. We disrupted the gene of the clone 325 in mouse and found that the homozygous animals are viable and displayed no abnormality in the cell counts of the peripheral blood. But when megakaryocytes from the bone marrow of the mutant animals were observed, we found that very few cells properly protruded normal proplatelets. This result suggests that the clone 325 is important for the proplatelet formation. Less