Function of the TAD/PEST Domain of the Mouse Notch1 and Notch4 Genes

Abstract

The Notch gene is highly conserved and found in most, if not all metazoans. The primary function of Notch proteins is to determine cellular fate during embryogenesis, but Notch plays a vital role in adult organisms as well. Improper Notch signaling has been linked to neoplasia, stroke and possibly schizophrenia in adult mammals. Figure 1 illustrates proper Notch signaling. Multiple Notch homologs exist in worms, zebrafish, chickens, mice and humans. The exact functions of the multiple Notch genes has yet to be determined. Mouse Notch proteins are the most similar to human Notch proteins, and for the purpose of this study, mouse Notch1 and Notch4 were used. It has been shown that the full length intracellular domain of mouse Notch4 (N4ICwt) exhibits very weak transcriptional activity in vitro. When the TAD/PEST domains are deleted (N4IC), however, Notch4 can upregulate downstream reporters. For Notch1, however, full length Notch1 (N1ICwt) shows greater activation than Notch1 without the TAD/PEST domains (N1IC, See Figure 2). Therefore, a construct was made with the intracellular domain of Notch1 with the Notch4 TAD/PEST domain (N1/N4). The activity was monitored using the 4XCSL, Hes1, and Hes5 promoters. 4XCSL is an artificial construct, while the Hes promoters have been linked to proper mammalian lung, retinal and pancreatic differentiation, neurogenesis, and T-cell development. Using these promoters and comparing the different Notch constructs, we are attempting to determine whether the Notch4 TAD/PEST domain is transferable. If so, the activitiy profile of N1/N4 should resemble that of N4ICwt versus N1ICwt in the different assays.