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Molecular and Cellular Biology, January 2002, p. 430-441, Vol. 22, No. 2
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.2.430-441.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of the ECB Binding Complex Responsible for the M/G1-Specific Transcription of CLN3 and SWI4

Bernard Mai,,{dagger} Shawna Miles, and Linda L. Breeden*

Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington 98109-1024

Received 15 March 2001/ Returned for modification 4 May 2001/ Accepted 24 September 2001

The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes. In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G1 phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB). Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein. Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences. These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element. Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes.

* Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, Division of Basic Sciences, 1100 Fairview Ave. N., Seattle, WA 98109-1024. Phone: (206) 667-4484. Fax: (206) 667-6526. E-mail: lbreeden{at}fhcrc.org.

{dagger} Present address: Aventis Pharma GmbH, Center for Functional Genomics, Fraunhoferstr. 13, D-82152 Martinsried, Germany.

Molecular and Cellular Biology, January 2002, p. 430-441, Vol. 22, No. 2
0022-538X/01/$04.00+0     DOI: 10.1128/MCB.22.2.430-441.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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