Regulation of T cell Differentiation

在我们对RORGamma-t转录调控的初步研究中，我们用不同长度的RORGamma-t启动子片段驱动的荧光素酶报告结构表明，RUNX1与启动子的结合对于最佳的RORGamma-t转录是必不可少的。此外，我们还发现，在胸腺早期T细胞分化过程中起重要作用的转录因子E蛋白也是重要的转录因子。 在进一步的研究中，我们通过转导表达E-蛋白的逆转录病毒或通过使用E-蛋白特异性siRNA的基因沉默来评估生理细胞中的E-蛋白，并发现这些蛋白也是重要的RORGamma-t转录因子。有趣的是，高浓度的E-蛋白抑制而不是增强RORGamma-t转录。然而，这种负面影响可能是ID2，一种阻止E蛋白与DNA结合的转录因子。ID2由IL-6诱导，IL-17是表达IL-17所必需的因子。 在条件性E蛋白KO小鼠的广泛研究中，证实了E蛋白对IL-17和RORGamma-t表达的影响。这些小鼠在他莫昔芬应答启动子的作用下，携带有腺泡化的E蛋白基因和T细胞特异性的Cre转基因。因此，当小鼠或来自小鼠的细胞暴露于他莫昔芬时，E蛋白没有表达。 在最后一组研究中，我们发现，IL-6和转化生长因子-β的组合可以极大地提高E蛋白水平，但这两种因素都不是单独作用的。这一发现很好地解释了为什么这些细胞因子对IL-17的分化至关重要。

In our initial studies of the regulation of RORgamma-t transcription we showed with luciferase reporter constructs driven by RORgamma-t promoter fragment of various lengths that Runx1 binding to the promoter is essential for optimal RORgamma-t transcription. In addition, we showed that E-proteins, transcription factors important during early T cell differentiation in the thymus, were also important transcription factors. In further studies in which we evaluated E-proteins in physiological cells by either transduction of retroviruses expressing these proteins or with gene silencing studies using E-protein-specific siRNA and found that these proteins also served as important RORgamma-t transcription factors. Of interest, high concentrations of E-protein suppressed rather than enhanced RORgamma-t transcription. However, this negative effect is probably ID2, a transcription factor that prevents E-protein binding to DNA. ID2 is induced by IL-6, a factor necessary for IL-17 expressison. The effect of E-proteins on IL-17 and RORgamma-t expression was verified in extensive studies of conditional E-protein KO mice. These mice had phloxed E-protein genes and a T cell-specific Cre transgene under a tamoxifen responsive promoter. Thus, when the mice or cells from the mice were exposed to tamoxifen the E-proteins were not expressed. In a final set of studies we found that E-protein levels were greatly enhanced by a combination of IL-6 and TGF-beta but neither of these alone. This finding offers a good explanation of why these cytokines are essential to IL-17 differentiation.