Suzuki Mikiko, Moriguchi Takashi, Ohneda Kinuko, Yamamoto Masayuki
Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan.
Mol Cell Biol. 2009 Mar;29(5):1163-75. doi: 10.1128/MCB.01572-08. Epub 2008 Dec 22.
GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.
GATA1是红系细胞分化的关键调节因子。为了研究Gata1基因表达如何以阶段特异性方式受到调控，制备了在细菌人工染色体（G1BAC-GFP）中从Gata1基因座表达绿色荧光蛋白（GFP）报告基因的转基因小鼠品系。我们发现GFP报告基因的表达忠实地重现了Gata1基因的表达。利用GFP荧光与造血表面标志物相结合，我们建立了两种红系祖细胞组分的纯化方案，分别称为爆式红系集落形成单位相关红系祖细胞（BREP）和红系集落形成单位相关红系祖细胞（CREP）组分。我们研究了Gata1基因造血增强子（G1HE）及其增强子核心中高度保守的GATA框的功能。G1HE的缺失和GATA框的替代突变均导致BREP组分中GFP表达几乎完全丧失，但CREP阶段的表达仅部分受到抑制，这表明GATA框对Gata1在BREP阶段的表达起关键作用。一致地，从染色体Gata1基因座靶向缺失G1HE会导致BREP组分中Gata1基因表达受到抑制，进而导致BREP阶段造血祖细胞异常积累。这些结果证明了以分化阶段特异性方式动态调控Gata1基因表达的生理意义。
