A Human Protein with Sequence Similarity to Drosophila Mastermind Coordinates the Nuclear Form of Notch and a CSL Protein To Build a Transcriptional Activator Complex on Target Promoters

Abstract

Mastermind (Mam) has been implicated as an important positive regulator of the Notch signaling pathway by genetic studies usingDrosophila melanogaster. Here we describe a biochemical mechanism of action of Mam within the Notch signaling pathway. Expression of a human sequence related to Drosophila Mam (hMam-1) in mammalian cells augments induction of Hairy Enhancer of split (HES) promoters by Notch signaling. hMam-1 stabilizes and participates in the DNA binding complex of the intracellular domain of human Notch1 and a CSL protein. Truncated versions of hMam-1 that can maintain an association with the complex behave in a dominant negative fashion and depress transactivation. Furthermore, Drosophila Mam forms a similar complex with the intracellular domain of Drosophila Notch andDrosophila CSL protein during activation of Enhancer of split, the Drosophila counterpart ofHES. These results indicate that Mam is an essential component of the transcriptional apparatus of Notch signaling.

ACKNOWLEDGMENTS

We thank T. Nagase for KIAA0200 cDNA; S. Artavanis-Tsakonas and R. Mann for Notch1 and Notch2 cDNA; T. Kitamura for pMx; R. Kageyama for pHES-1luc and pHES-5luc; T. Honjo for the cDNA and antibodies for RBP-J; M. Tomizawa and M. Higashi for computer software; K. Azuma for antibodies; T. Umemiya for immunocytochemistry; N. Yumoto for the expression vector for Notch1IC; M. Ito, K. Nakao-Sawai, and H. Okano for DNotchIC and S2 cells; L. Grimm for pMTNotch (ICN), pMTSu(H), andmγ Luc; M. Masada, N. Terada, and S. Masuda for support; T. Hiwasa for advice; and colleagues of the Department of Pathology, Chiba University School of Medicine, for discussions and encouragement. cDNA for RBP-J was supplied by from RIKEN DNA bank.

This work was supported by grants in aid from the Ministry of Education, Science, Sports, and Culture of Japan to M.K. (grant no. 11680671) and K.H. (grant no. 12215018); a grant from the Smoking Research Foundation to K.H.; grants from the Naito Foundation and the Sumitomo Foundation to K.M.; and a grant from the National Science Foundation (grant no. IBN 9904411) to B.Y.