Investigation of Notch1 transcriptional regulation in the activated CD4+ T cells

Abstract

Notch1 signaling pathway is one of the most important signaling pathways of immune system. Notch1 is involved in TCR-mediated activation and proliferation of CD4+ T cells. Even though function of Notch1 in its downstream signaling is well known, mechanism of Notch1 transcriptional regulation is not so well known. To reveal it, Notch1 transcriptional regulation was investigated with several methods including luciferase assay, EMSA, qPCR and Flow cytometry. First, to identify Notch1 promoter region that is important for Notch1 transcription, Notch1 promoter was serially deleted from -1140 to -150 and subcloned into pGL3 basic vector. Each vector was used for luciferase assay and I could find that Notch1 promoter -300~-270 was important for Notch1 transcription during T cell activation. In silico analysis of -300~-270 region with MatInspector suggested NF-B proteins RelA and p50 could be the transcription factors that regulates Notch1 -300~-270 region. Each transcription factor was used for luciferase assay and I could find out that NF-B proteins increase Notch1 transcription by interacting with Notch1 -300~-270 region. Further, electromobility shift assay and supershift assay showed that NF-B proteins actually bind to Notch1 -300~-270 region. Also, qPCR and flow cytometry with NF-B-overexpressed cells verified that overexpression of RelA and p50 in Jurkat cells increase Notch1 mRNA and surface Notch1 expression. Finally, Notch1 reporter vectors were electroporated to mouse CD4+ cells and showed that Notch1 promoter region -300~-270 is crucial for Notch1 transcription in mouse primary cells. To summarize, NF-B could regulate Notch1 transcription by binding to Notch1 promoter region -300~-270