Regulation of Gata1 gene expression levels during erythroid cell development

转录因子GATA-1是红系细胞分化的主要调控因子。在红细胞分化过程中，Gata1基因的表达水平是动态变化的，这种变化对正常的红细胞生成至关重要。为了研究Gata1基因在红系发育过程中表达变化的分子机制，我们利用含有Gata1基因的细菌人工染色体（Gata1- bac）建立了一种新的报告基因转基因小鼠系统。在Gata1- bac转基因小鼠中，报告基因的表达以拷贝数依赖但整合位置独立的方式忠实地再现了Gata1基因在时间和空间上的表达。由于Gata1基因造血增强子（G1HE）的核心区域（235 bp）位于红系特异性IE第一外显子上游3.9 kbp处，在人和小鼠之间高度保守，并且在我们的质粒转基因小鼠实验中似乎很重要，因此我们分析了含有G1HE-核心缺陷BAC报告基因的转基因小鼠系。结果表明，G1HE-Core在整个红细胞分化过程中对Gata1基因的上调至关重要。特别是，在G1HE-Core缺失的情况下，Gata1的表达在早期红系祖细胞（EEP）阶段（与BFU-E相对应）和红母细胞中被取消。相反，Gata1基因在晚红系祖细胞（LEP）阶段仍然存在g1he - core不依赖的表达，该阶段含有CFU-E和原红细胞。突变分析进一步表明，G1HE-Core中保守的GATA-box对该增强子活性至关重要。因此，对Gata1- bac报告基因转基因小鼠系的分析首次表明，G1HE-Core在体内指导Gata1基因的阶段特异性表达。

Transcription factor GATA-1 is the master regulator of erythroid cell differentiation. Expression level of the Gata1 gene changes dynamically during erythroid differentiation, and this change is essential for the proper erythropoiesis. To investigate the molecular mechanism how Gata1 gene expression changes during the erythroid lineage development, we established a novel reporter transgenic mouse system utilizing bacterial artificial chromosome containing the Gata1 gene (Gata1-BAC). In the Gata1-BAC transgenic mice, reporter gene expression faithfully recapitulates temporal and spatial expression of the Gata1 gene in a copy number-dependent but integration position-independent manner. Since the core region (235 bp) of Gata1 gene hematopoietic enhancer (G1HE), which localizes at 3.9-kbp upstream of erythroid specific IE first exon, is highly conserved between human and mouse and appeared to be important in our plasmid transgenic mouse experiments, we analyzed reporter transgenic mouse lines harboring the G1HE-Core-deficient BAC reporter transgene. Results demonstrated that G1HE-Core is essential for up-regulation of the Gata1 gene throughout the erythroid differentiation. Especially, in the absence of G1HE-Core, the expression of Gata1 is abrogated in early erythroid progenitor (EEP) stage, which corresponds to BFU-E, and in erythroblasts. On the contrary, there remains G1HE-Core-independent expression of the Gata1 gene in late erythroid progenitor (LEP) stage, which contains to CFU-E and proerythroblasts. Mutational analyses further revealed that the conserved GATA-box in G1HE-Core is essential for this enhancer activity. Thus, analyses with the Gata1-BAC reporter transgenic mouse lines demonstrate for the first time that G1HE-Core directs the stage-specific expression of the Gata1 gene in vivo.