Functional analysis of transcription factor BACH1 on thrombopiesis and megakaryocyte diffrentiation

大约10%的唐氏综合征（DS）新生儿出生时伴有一种通常称为短暂性骨髓增生性疾病（TMD）的类白血病反应。部分退行性痴呆患者会在4岁前发展为恶性急性巨核母细胞白血病（AMKL）。人类BACH1基因定位于染色体21q22.1，位于潜在的ds相关基因区域内。我们在这个研究中有三个对象。首先，揭示转基因小鼠中转录因子BACH1过表达导致血小板减少的机制。第二，通过转录因子的过度表达寻找BACH1的靶基因。第三，探讨BACH1基因在DS患者TMD和AMKL发生中的作用。结果对BACH1转基因小鼠巨核细胞的电镜观察显示，巨核细胞的分界膜和α -颗粒形成明显受损，线粒体空泡化，细胞核内异染色质程度降低，含量增加。为了研究巨核祖细胞的增殖能力，我们进行了CFU-Meg集落形成实验。但是，在转基因小鼠和野生小鼠之间没有发现差异。这些结果表明，转基因小鼠巨核细胞的终末分化和成熟过程受到了损害。利用体外培养的巨核细胞，我们检测了血小板生成素存在下13只小鼠胎儿肝细胞的一些基因表达。结果显示β -微管蛋白和凝血素A合成酶（TXAS）基因下调。我们还通过染色质免疫沉淀法确认了BACH1蛋白与TXAS基因调控元件的结合。这些结果表明BACH1通过拮抗p45NF-E2活性调控巨核细胞的分化和成熟。目前，为了研究BACH1在白血病细胞中的功能，我们通过基因转染成功建立了BACH1过表达白血病细胞系。少

ObjectApproximately 10% of newborn babies with Down's syndrome (DS) are born with a leukemoid reaction commonly called transient myeloproliferative disorder (TMD). A portion of DS patients will develop malignant acute megakaryoblastic leukemia (AMKL) during their first 4 years of life. The human BACH1 gene is localized to chromosome 21q22.1 within the potential DS-associated gene region. We have three objects in this research. First, disclosing the mechanism of the thrombocytopenia caused by the over-expression of transcription factor BACH1 in transgenic mice. Second, finding the target genes of BACH1 by excessive expression of the transcription factor. Third, investigating the contribution of BACH1 gene in developing TMD and AMKL in DS patients.ResultsElectron microscopic analysis of megakaryocytes derived from BACH1 transgenic mice revealed remarkable impairment of demarcation membrane and alpha-granule formation, the vacuolation of mitochondria, and the lower degree of heterochromat … More in content in nuclei. To investigate the proliferative ability of the megakaryocytic progenitor cells, we performed CFU-Meg colony formation assay. But, no difference was detected between transgenic and wild mice. These results suggest that the terminal differentiation and maturation process were impaired in the transgenic mouse megakaryocytes.Using megakaryocytes developed in vitro culture of liver cells from 13 d. p. c. mouse fetuses in the presence of thrombopoietin, we examined some gene expression. The results of the assay showed the down-regulation of the beta1-tubulin and thromboxane A synthase (TXAS) gene. We also recognized the binding of BACH1 protein to the regulatory elements of TXAS gene by chromatin immunoprecipitation method. These results suggest that BACH1 regulates the megakaryocytic differentiation and maturation by antagonizing the p45NF-E2 activity.At present, to investigate the function of BACH1 in leukemic cells, we succeeded in establishment of BACH1 overexpressing leukemic cell line by the gene transfection. Less