Dynamic Changes in E-protein Activity are Essential for Treg cell Development

To gain a molecular view of E-proteins with respect to the development of Foxp3+ T cells, we perform microarray studies that would identify transcription factors that are up-regulated as E-proteins levels fall and and Foxp3 expression rises. We hypothesize that such transcription factors activate the synthesis of key proteins necessary for the development of Foxp3+ cells in the thymus (or in the periphery). Among the possible proteins (negatively regulated by E-protein are those involved in IL-2 signaling, since the latter has been shown to be critical for the development of Foxp3+ cells in the thymus. In the planned studies we will extract mRNA from sorted Foxp3+GFP+ thymocytes from wild type (WT) mice and similar cells from E-protein deficient thymocytes. However, since Foxp3+ thymocytes in WT mice already have reduced E-protein levels, differences between thymocytes from these mice and E-protein deficient mice might be minimal. We therefore will also extract mRNA from Foxp3-negative thymocytes from WT mice whose E-proteins levels have not fallen sufficient low to allow Foxp3 expression and Foxp3-negative thymocytes from E-protein deficient mice whose E-proteins are necessarily low but have not expressed Foxp3 for unrelated reasons. Isolate mRNA from sorted Foxp3+ and Foxp3- thymocytes from either wild type (WT) mice and from E-protein deficient (E2A and HEB knockout) thymocytes; 2 replicates of each of the 4 conditions