Jones Mary E, Kondo Motonari, Zhuang Yuan
Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.
BMC Dev Biol. 2009 Oct 12;9:51. doi: 10.1186/1471-213X-9-51.
E-proteins are transcription factors important for the development of a variety of cell types, including neural, muscle and lymphocytes of the immune system. E2A, the best characterized E-protein family member in mammals, has been shown to have stage specific roles in cell differentiation, lineage commitment, proliferation, and survival. However, due to the complexity of E2A function, it is often difficult to separate these roles using conventional genetic approaches. Here, we have developed a new genetic model for reversible control of E2A protein activity at physiological levels. This system was created by inserting a tamoxifen-responsive region of the estrogen receptor (ER) at the carboxyl end of the tcfe2a gene to generate E2AER fusion proteins. We have characterized and analyzed the efficiency and kinetics of this inducible E2AER system in the context of B cell development.
B cell development has been shown previously to be blocked at an early stage in E2A deficient animals. Our E2AER/ER mice demonstrated this predicted block in B cell development, and E2AER DNA binding activity was not detected in the absence of ligand. In vitro studies verified rapid induction of E2AER DNA binding activity upon tamoxifen treatment. While tamoxifen treatment of E2AER/ER mice showed inefficient rescue of B cell development in live animals, direct exposure of bone marrow cells to tamoxifen in an ex vivo culture was sufficient to rescue and support early B cell development from the pre-proB cell stage.
The E2AER system provides inducible and reversible regulation of E2A function at the protein level. Many previous studies have utilized over-expression systems to induce E2A function, which are complicated by the toxicity often resulting from high levels of E2A. The E2AER model instead restores E2A activity at an endogenous level and in addition, allows for tight regulation of the timing of induction. These features make our E2AER ex vivo culture system attractive to study both immediate and gradual downstream E2A-mediated events.
E蛋白是对多种细胞类型（包括免疫系统的神经细胞、肌肉细胞和淋巴细胞）发育至关重要的转录因子。E2A是哺乳动物中特征最明确的E蛋白家族成员，已被证明在细胞分化、谱系定向、增殖和存活中具有阶段特异性作用。然而，由于E2A功能的复杂性，使用传统的遗传方法往往难以区分这些作用。在此，我们开发了一种新的遗传模型，用于在生理水平上对E2A蛋白活性进行可逆控制。该系统是通过在tcfe2a基因的羧基末端插入雌激素受体（ER）的他莫昔芬反应区域来产生E2AER融合蛋白而创建的。我们在B细胞发育的背景下对这种可诱导的E2AER系统的效率和动力学进行了表征和分析。
先前已表明，在E2A缺陷动物中，B细胞发育在早期阶段受阻。我们的E2AER/ER小鼠在B细胞发育中表现出这种预测的阻滞，并且在没有配体的情况下未检测到E2AER DNA结合活性。体外研究证实，他莫昔芬处理后E2AER DNA结合活性迅速诱导。虽然用他莫昔芬处理E2AER/ER小鼠在活体动物中显示出对B细胞发育的低效挽救，但在体外培养中将骨髓细胞直接暴露于他莫昔芬足以挽救并支持从前proB细胞阶段开始的早期B细胞发育。
E2AER系统在蛋白质水平上提供了对E2A功能的可诱导和可逆调节。许多先前的研究使用过表达系统来诱导E2A功能，这因E2A高水平通常导致的毒性而变得复杂。相反，E2AER模型在内源水平上恢复E2A活性，此外，还允许对诱导时间进行严格调节。这些特性使我们的E2AER体外培养系统对于研究直接和渐进的下游E2A介导的事件具有吸引力。
