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节因子。GATA1基因的表达水平在红系分化过程中动态变化，这种变化对于正常的红系生成是必不可少的。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建立了一种新型的报告转基因小鼠系统(GATA1-BAC)。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无关的方式真实地概括了GATA1基因的时空表达。由于GATA1基因造血增强子(G1HE)的核心区(235bp)位于红系特异性IE第一外显子上游3.9kBP处，在人和小鼠之间高度保守，在我们的转基因小鼠实验中具有重要意义。结果表明，G1HE-Core对于GATA1基因在整个红系分化过程中的上调是必不可少的。尤其是在缺乏G1HE-Core的情况下，GATA1在早期红系祖细胞(EEP)和红细胞中的表达被抑制，与BFU-E相对应。相反，GATA1基因在红系祖细胞晚期仍有G1HE核心非依赖性表达，包括TO-CFU-E和前红系母细胞。突变分析进一步表明，G1HE-Core中保守的GATA-box是这种增强子活性所必需的。因此，对GATA1-BAC报告转基因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.