Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
J Biol Chem. 2010 Apr 2;285(14):10179-88. doi: 10.1074/jbc.M109.088740. Epub 2010 Feb 5.
GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knock-out mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative 3 (DN3) stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of prearranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively indicate that in addition to regulating IL-7Ralpha expression, GABP is critically required for TCR rearrangements and hence normal T cell development.
GABP 结合蛋白（GABP）由 GABPalpha 和 GABPbeta 亚基组成。GABPalpha 是 Ets 家族转录因子的成员，通过其保守的 Ets 结构域结合 DNA，而 GABPbeta 不结合 DNA，但具有转录激活活性。在 T 细胞中，已经证明 GABP 调节白细胞介素 7 受体 alpha 链（IL-7Ralpha）的基因表达，并被假设在 T 细胞发育中至关重要。为了直接研究其在早期胸腺细胞发育中的功能，我们使用了 GABPalpha 条件性敲除小鼠，其中编码 Ets DNA 结合域的外显子被 LoxP 位点包围。用 Lck-Cre 转基因敲除 GABPalpha 大大降低了胸腺细胞的细胞数量，阻断了双阴性 3（DN3）阶段的胸腺细胞发育，并导致 DN4 胸腺细胞中 T 细胞受体（TCR）beta 链的表达减少。通过染色质免疫沉淀，我们在 DN 胸腺细胞中证明，GABPalpha 与编码 TCR 重排关键分子的基因的转录起始位点相关联。在这些与 GABP 相关的基因中，通过 RNA 干扰敲低 GABPalpha 表达会降低 DNA 依赖性蛋白激酶复合物中 DNA 连接酶 IV、Artemis 和 Ku80 成分的表达。有趣的是，预先排列的 TCR 的强制表达而不是 IL-7Ralpha 的表达可以缓解 GABPalpha 靶向小鼠中的 DN3 阻断。我们的观察结果表明，除了调节 IL-7Ralpha 的表达外，GABP 对于 TCR 重排和正常 T 细胞发育也是至关重要的。
