E2A turnover and Notch-controlled lymphocyte development

超出提供的空间。Notch信号通路在淋巴细胞发育过程中的几个关键决策中起着重要作用。基本的螺旋-环-螺旋E2a转录因子(E12/E47)也是B和T淋巴细胞发育所必需的。我们最近的发现表明，E2a转录因子可能是Notch信号的下游效应因子，对淋巴细胞的发育起作用。具体地说，我们已经证明，通过Notch的信号调节E2A蛋白的周转，这需要p42/p44 MAP激酶对这些蛋白的磷酸化。激活的N otchl的表达增强了E47与SCFSkp2 E3泛素连接酶及其泛素化的结合。Noch诱导的E2A蛋白降解发生在B细胞中，但没有发生在T细胞中。然而，这与这些细胞中MAP激酶的活性水平有关。因此，MAP激酶活性水平的差异可能是控制E2A周转的有效调控机制。在这项建议中，我们首先假设Notch信号加速E2A的降解可能通过阻止胸腺中B细胞的发育在Notch介导的T/B谱系选择中发挥重要作用。然后我们提出，一旦形成功能性的Pre-TCR和TCR，Notch信号可能负责降低T细胞中E2A的水平，而较低的E2A水平是T细胞在成熟过程中经过适当选择所必需的。这些假说将通过检测蛋白激活后B细胞和T细胞中E_(2A)的降解来检验。此外，突变将被引入到E2A基因组中，以编码抵抗Notch诱导的降解的E2A蛋白。转基因小鼠将被用来确定突变蛋白的表达是否允许胸腺中B细胞的形成，对Notch介导的抑制骨髓中B细胞发育的抵抗，以及对T细胞选择以及正负选择的影响。最后，我们打算了解Notch信号如何诱导E2A蛋白的泛素化和降解。也许，Notch信号促进了参与E2a蛋白泛素化的未知基因的表达，这将通过检测与E2a蛋白泛素化机制相关的蛋白质来鉴定。总之，本文概述的研究扩展了我们对Notch信号的一种新功能的研究，即诱导泛素介导的E2A降解，并评估了该功能在淋巴细胞发育背景下的生物学意义。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. Notch signaling pathways are important for several crucial decisions during lymphocyte development. T he basic helix-loop-helix E2A transcription factors (E12/E47) are also essential for the development for both B and T lymphocytes. Our recent findings suggest that E2A transcription factors may be downstream effectors of Notch signaling for lymphocyte development. Specifically, we have shown that signaling through Notch modulates the turnover of E2A proteins, which requires the phosphorylation of these proteins by p42/p44 MAP kinases. Expression of activated N otchl enhanced the association of E47 with the SCFSkp2 E3 ubiquitin ligase and its ubiquitination. Notch-induced degradation of E2A proteins occurred in B but not T cells studied. However, this correlated with the level of MAP kinase activity in these cells. Therefore, differences in the level of MAP kinase activity may be an effective regulatory mechanism to control E2A turnover. In this proposal, we first hypothesize that accelerated E2A degradation by Notch signals may play an important role in Notch-mediated T versus B lineage choice by blocking B cell development in the thymus. We then propose that Notch signals may be responsible for reducing E2A levels in T cells once functional pre-TCR and TCR are formed, and lower E2A levels are necessary for T cells to go through proper selections during maturation. These hypotheses will be tested by measuring E 2A degradation in prog enitor B cells and in T cells upon activati on of MAP kinases. Furthermore, mutations will be introduced into the E2A genome to encode E2A proteins resistant to Notch induced degradation. The knock-in mice will be used to determine if expression of the mutant protein allows B cell formation in the thymus, renders resistance to Notch-mediated inhibition of B cell development in the bone mar row, and impacts on T cell selection, as well as positive and negative selecti on. Finally, we propose to understand how Notch signals induce the ubiquitination and degradation of E2A proteins. Perhaps, Notch signals facilitate expression of unknown genes involved in the ubiquitination of E2A proteins, which will be identified by examination of proteins associated with the ubiquitination machinery of E2A proteins. In summary, studies outlined here expand our investigation on a novel function of Notch signaling, i.e., induction of ubiquitin-mediated E2A degradation, and evaluate the biological significance of this function i n the context of lymphocyte development. PERFORMANCE SITE ========================================Section End===========================================