Correspondence Mastermind is a putative activator for Notch

During signaling by the Notch receptor, Notch’s intracellular domain is cleaved, moves to the nucleus and associates with a DNAbinding protein of the CSL class (CSL for CBF1, Suppressor of Hairless (Su(H)), LAG-1); as a result, target genes are transcriptionally activated (reviewed in [1,2]). In Caenorhabditis elegans, a glutamine-rich protein called LAG-3 forms a ternary complex with the Notch intracellular domain and LAG-1 and appears to serve as a transcriptional activator that is critical for signaling [3]. Although database searches failed to identify a LAG-3related protein, we surmised that Notch signaling in other organisms might involve an analogous activity. To search for a LAG-3-like activity in mice, we used a modified yeast two-hybrid screen similar to that used to identify LAG-3 [3]. Briefly, we used a complex bait to screen a library of mouse cDNAs fused to the Gal4 activation domain (Clontech). That bait included mouse CBF1 fused to the Gal4 DNA-binding domain (GD) as well as the intracellular domain of mouse Notch1. The bait proteins were co-expressed from a pBridge vector. Out of 6 million transformants, we recovered one positive with similarity to Drosophila Mastermind and human KIAA0200 (Figure 1a). We focused on this clone because Drosophila Mastermind is known to be critical for Notch signaling (reviewed in [2]) [4,5]. We call the murine ortholog of Mastermind mMam1, and the human one hMam1. The mMam1 fragment recovered in the two-hybrid screen consisted of 62 amino acids and included a conserved region present in both fly and human Mastermind proteins (Figure 1). To explore the idea that Mastermind might have a role similar to LAG-3 in Notch signaling, we conducted a series of two-hybrid assays (Figure 2). We first showed that mMam1 bound mCBF1–GD in the presence of either Notch1 or Notch3, but not in their absence (Figure 2a). We next asked whether Drosophila Mastermind might participate in a similar complex in flies. We made a fusion protein carrying the Gal4 activation domain and the amino-terminal 198 amino acids of fly Mastermind (dMam (1–198), Figure 1a; henceforth called dMam), which includes the conserved region of Mastermind that is critical for complex formation among mouse components. We found that dMam bound Su(H) strongly in the presence of the fly Notch intracellular domain, but not in its absence (Figure 2b). We next explored the interchangeability of proteins from different species. Remarkably, the fly protein, dMam, interacted with murine Notch1 or Notch3 and murine CBF1 (Figure 2c), and mMam1 interacted with fly Notch and Su(H) (Figure 2d). In contrast, C. elegans LAG-3 did not form a complex with either murine or fly components (Figure 2e), and mMam and dMam did not complex with worm components (Figure 2f). We conclude that both fly and murine Mastermind proteins form a ternary complex with either fly or murine receptors and CSL proteins. This interchangeability underscores the similarity between the fly and murine Notch pathways. Although murine Mastermind is not described, a full-length cDNA sequence for human Mastermind is available. Comparison of human and fly Mastermind sequences reveals only one short region of significant similarity that is limited to 60 amino acids at the amino terminus (Figure 1). Therefore, despite a low overall sequence similarity between mouse and Drosophila Mastermind proteins, the region crucial for complex formation is conserved. Finally, we examined the importance of the receptor’s ankyrin repeats for complex formation. In C. elegans, formation of the ternary complex is dependent on the ankyrin repeats of the Notch-related receptor GLP-1 [3]. To ask whether the same situation holds for the murine complex, we used two missense mutants, M1 and M2, each of which bears amino-acid substitutions in the Magazine R471