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中的功能，确定小Maf下游调控的靶基因，阐明血小板形成的调控机制。(1)巨核细胞中小分子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和一个高度受限的伙伴分子组成。MafG是MafG:p45异源二聚体中更常见的亚基，是巨核细胞中介导血小板形成不可或缺的转录激活因子。过量的MafG通过形成抑制性同型二聚体来拮抗MafG:p45异源二聚体的活性，从而阻断血小板前体的形成；因此，MafG根据其相对丰度激活和抑制转录。在这里，我们报道了MafG在体内与SUMO-2/3结合，并且SUMO-2/3缺陷的MafG在巨核细胞中表达时，通常参与更多的交易激活，而不是抑制。虽然在异染色质和常染色质中都发现了MafG，但突变的MafG未能定位在异染色质中。我们得出结论，SUMOylation是mag介导的异染色质抑制和定位所必需的，因此这种二聚体转录因子的广泛表达成分的SUMOylation决定了其转录潜力的一个方面。(2)巨核细胞小Maf下游调控新基因的鉴定。为了阐明在血小板形成过程中起作用的分子机制，我们寻找野生型和maf小突变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，通过减法筛选分离出一个新的因子克隆325。从脑cDNA文库中获得全长cDNA，推导出的氨基酸序列显示一个亮氨酸拉链状基序，但没有典型的功能基序。克隆325在骨髓纯化的原代巨核细胞中高表达，但在未成熟造血细胞和红细胞中丰度相对较低。我们在小鼠体内破坏克隆325的基因，发现纯合子动物存活，外周血细胞计数无异常。但是，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能正常地突出正常的前血小板。这一结果表明克隆325对原血小板的形成起重要作用。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