Leukemogenesis caused by incapacitated GATA-1 function

GATA-1 is essential for the development of erythroid and megakaryocytic lineages. We found that GATA-1 gene knockdown female (GATA-1.05/X) mice frequently develop a hematopoietic disorder resembling myelodysplastic syndrome that is characterized by the accumulation of progenitors expressing low levels of GATA-1. In this study, we demonstrate that GATA-1.05/X mice suffer from two distinct types of acute leukemia, an early-onset c-Kit-positive nonlymphoid leukemia and a late-onset B-lymphocytic leukemia. Since GATA-1 is an X chromosome gene, two types of hematopoietic cells reside within heterozygous GATA-1 knockdown mice, bearing either an active wild-type GATA-1 allele or an active mutant GATA-1.05 allele. In the hematopoietic progenitors with the latter allele, low-level GATA-1 expression is sufficient to support survival and proliferation but not differentiation, leading to the accumulation of progenitors that are easily targeted by oncogenic stimuli. Since such leukemia has not been observed in GATA-1-null/X mutant mice, we conclude that the residual GATA-1 activity in the knockdown mice contributes to the development of the malignancy. This de novo model recapitulates the acute crisis found in preleukemic conditions in humans.     

GATA-1 bends DNA in a site-independent fashion

The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.     

非洲爪蟾GATA-1b中与GATA-1a的差异位点突变后的功能

非洲爪蟾GATA 1因子分为GATA 1a和GATA 1b两种亚型。与其他种类的GATA 1因子相类似 ,GATA 1是红细胞分化成熟所必需的因子之一。二种亚型在造血方面具有同样的作用 ,但只有xGATA 1b具有抑制神经发生的功能。比较二者的结构 ,发现二者的核酸和蛋白质结构虽然都非常相似 ,但并不完全一致。为探讨这些差异结构与功能之间的关系 ,我们对xGATA 1b中与xGATA 1a差异的Ser1 6 8和His1 6 9位点进行缺失突变 ,并定点突变Thr30 4和Thr359位点。之后将体外制备好的野生型或突变型xGATA 1bmRNA单独或与DN BRmRNA共同导入非洲爪蟾 2细胞胚胎内 ,利用动物帽系统分析突变体在神经发生和造血方面的功能。结果发现 ,Ser1 6 8和His1 6 9以及Thr359位点突变后 ,xGATA 1b再不能抑制DN BR诱导的神经发生 ;但所有突变都未影响xGATA 1b在造血方面的功能。由此 ,我们首次证实了Ser1 6 8和His1 6 9以及Thr359位点可能是导致xGATA 1b和xGATA 1a之间功能分离的结构基础之一。