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Molecular and Cellular Biology, September 2001, p. 6222-6232, Vol. 21, No. 18
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.18.6222-6232.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Nuclear Localization of CBF1 Is Regulated by Interactions with the SMRT Corepressor Complex

Sifang Zhou1 and S. Diane Hayward1,2,*

Department of Pharmacology and Molecular Science1 and Oncology Center,2 Johns Hopkins University School of Medicine, Baltimore, Maryland 21231

Received 18 May 2001/Returned for modification 14 June 2001/Accepted 25 June 2001

The CSL family protein CBF1 is a nuclear mediator of Notch signaling and has been predicted to contain an N-terminal nuclear localization signal in exon 4. Surprisingly, we found that CBF1 carrying mutations at codon 233 or 249 within exon 7 was restricted to the cytoplasm. In mammalian and yeast two-hybrid assays, these mutations were also associated with a loss of CBF1-mediated transcriptional repression and a severely impaired interaction with the corepressors SMRT and CIR. Overexpression of SMRT rescued the ability of mutant CBF1 to target to the nucleus of transfected cells and similarly rescued nuclear targeting of enhanced green fluorescent protein (EGFP)-CBF1 exons 6 to 9 CBF1(6-9)carrying the codon 233 or 249 mutations. Carboxy-terminally truncated SMRT with amino acids (aa) 1291 to 1495 deleted was unable to rescue the nuclear targeting of mutant EGFP-CBF1(6-9). In yeast two-hybrid assays, the SMRT aa 1291 to 1495 domain interacted with SKIP and SMRT aa 1291 to 1495 colocalized with SKIP within the nuclei of cotransfected cells. Comparison of the intracellular localization of CBF1(6-9) with that of CBF1(5-9) further supported the suggestion that nuclear targeting of CBF1 is dependent on the formation of a CBF1-SMRT-SKIP corepressor complex. These observations suggest that nuclear targeting of CBF1 is itself a component of CBF1-mediated gene regulation and that in the absence of signaling, CBF1 enters the nucleus precommitted to a transcriptional repression function. The activators NotchIC (the intracellular domain of Notch) and Epstein-Barr virus EBNA2 also mediated nuclear targeting of mutant CBF1, consistent with the competition model for activator versus corepressor binding to CBF1.

* Corresponding author. Mailing address: Oncology Center, Johns Hopkins University School of Medicine, Bunting-Blaustein Building, CRB 308, 1650 Orleans St., Baltimore, MD 21231. Phone: (410) 614-0592. Fax: (410) 502-6802. E-mail: dhayward{at}jhmi.edu.

Molecular and Cellular Biology, September 2001, p. 6222-6232, Vol. 21, No. 18
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.18.6222-6232.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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