Characterization of a Functional ZBP-89 Binding Site That Mediates Gata1 Gene Expression during Hematopoietic Development*
Characterization of a Functional ZBP-89 Binding Site That Mediates Gata1 Gene Expression during Hematopoietic Development*
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DOI：
10.1074/jbc.m109.026948
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发表时间：
2009-09
期刊：
影响因子：
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通讯作者：
K. Ohneda;S. Ohmori;Y. Ishijima;Mayu A Nakano;Masayuki Yamamoto
中科院分区：
文献类型：
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作者：
K. Ohneda;S. Ohmori;Y. Ishijima;Mayu A Nakano;Masayuki Yamamoto
GATA-1 是一种谱系限制性转录因子，在造血发育中发挥重要作用。 Gata1基因造血增强子允许Gata1报告基因在转基因小鼠的红系细胞和巨核细胞中表达。 Gata1 造血增强子活性严格依赖于位于增强子 5' 区域的 GATA 位点。然而，也有人提出了与该 GATA 位点 3' 端相邻的 GC 丰富区域的重要性。在这项研究中，我们证明这个富含 GC 的区域包含五个连续的脱氧鸟苷残基（G5 串），它们与多个核蛋白结合。有趣的是，从 G5 串（G4 突变体）中删除一个脱氧鸟苷残基会特异性消除与 ZBP-89（一种 Krüppel 样转录因子）的结合，但不会消除与 Sp3 和其他结合因子的结合。我们证明 GATA-1 和 ZBP-89 占据 Gata1 增强子的染色质区域，并通过锌指结构域在体外物理关联。凝胶迁移率变化测定和 DNA 亲和沉淀测定表明，在没有 GATA-1 与 G1HE 结合的情况下，ZBP-89 与该区域的结合会减少。荧光素酶报告基因检测表明，ZBP-89 根据 G5 字符串序列激活 Gata1 增强子。最后，转基因小鼠研究表明，G4 突变显着降低了包含 Gata1 基因 8.5 kbp 区域的 Gata1 造血调节域的报告基因活性。这些数据提供了令人信服的证据，证明 G5 串对于体内 Gata1 基因表达是必需的，而 ZBP-89 是该顺式作用区域的功能性反式作用因子。
GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5′ region of the enhancer. However, the importance of the GC-rich region adjacent to the 3′-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G5 string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G5 string (G4 mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G5 string sequence. Finally, transgenic mouse studies reveal that the G4 mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G5 string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.