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. 2002 Jun 1;16(11):1397–1411. doi: 10.1101/gad.991602

Figure 1.

Figure 1

MAM interacts with and is required for chromatin-dependent transcriptional activation by the Notch (CBF1:ICD) enhancer complex in vitro. (A) Primer-extension analysis of the transcriptional activity of purified recombinant Notch complexes on pNRE chromatin in vitro. Where indicated, the reactions either lacked enhancer factors (lanes 1,8), or contained CBF1 (120 nM, lanes 2,5–7,9–14), the wild-type Xenopus Notch ICD (120 nM, lanes 3,6,7,9), or different mutant ICD proteins (lanes 10–14), and human 1–301MM (120 nM, lanes 4,5,7,9–14). Arrows indicate transcripts originating from the pNRE or control (nonchromatin) α-globin (α-glo) templates. The Notch enhancer complex is assembled through binding of the ICD ankyrin repeat (ANK) domain to the central region of CBF1 (amino acids 179–361) and to an N-terminal domain of MAM, as indicated schematically at the bottom of the figure. (B) EMSA analysis of the ability of wild-type and mutant ICD proteins (lanes 3–18, as indicated above each lane) to support a stable ternary complex with CBF1 (lanes 2–18) and MAM (1–301MM; lanes 3,5,7,9,11,13,15) on DNA in vitro. Arrows indicate the various CBF1, ICD–CBF1, and MAM:ICD–CBF1 complexes on DNA. The different mutant ICD proteins tested are depicted to the right of the figure.