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Molecular and Cellular Biology, April 2000, p. 2400-2410, Vol. 20, No. 7
Department of Pharmacology and Molecular
Sciences1 and Oncology
Center,3 Johns Hopkins School of Medicine,
Baltimore, Maryland 21205, and Molecular Biology Institute,
University of California, Los Angeles, California
900952
Received 18 October 1999/Returned for modification 13 December
1999/Accepted 12 January 2000
Notch proteins are transmembrane receptors that mediate intercell
communication and direct individual cell fate decisions. The activated
intracellular form of Notch, NotchIC, translocates to the nucleus,
where it targets the DNA binding protein CBF1. CBF1 mediates
transcriptional repression through the recruitment of an SMRT-histone
deacetylase-containing corepressor complex. We have examined the
mechanism whereby NotchIC overcomes CBF1-mediated transcriptional
repression. We identified SKIP (Ski-interacting protein) as a CBF1
binding protein in a yeast two-hybrid screen. Both CBF1 and SKIP are
highly conserved evolutionarily, and the SKIP-CBF1 interaction is also
conserved in assays using the Caenorhabditis elegans and
Drosophila melanogaster SKIP homologs. Protein-protein interaction assays demonstrated interaction between SKIP and the corepressor SMRT. More surprisingly, SKIP also interacted with NotchIC.
The SMRT and NotchIC interactions were mutually exclusive. In
competition binding experiments SMRT displaced NotchIC from CBF1 and
from SKIP. Contact with SKIP is required for biological activity of
NotchIC. A mutation in the fourth ankyrin repeat that abolished Notch
signal transduction did not affect interaction with CBF1 but abolished
interaction with SKIP. Further, NotchIC was unable to block muscle cell
differentiation in myoblasts expressing antisense SKIP. The results
suggest a model in which NotchIC activates responsive promoters by
competing with the SMRT-corepressor complex for contacts on both CBF1
and SKIP.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
SKIP, a CBF1-Associated Protein, Interacts with the
Ankyrin Repeat Domain of NotchIC To Facilitate NotchIC
Function
*
Corresponding author. Mailing address: Department of
Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-2548. Fax:
(410) 955-8685. E-mail: dhayward{at}jhmi.edu.
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