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Molecular and Cellular Biology, March 2003, p. 1946-1960, Vol. 23, No. 6
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.6.1946-1960.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Activation of the Early B-Cell-Specific mb-1 (Ig-{alpha}) Gene by Pax-5 Is Dependent on an Unmethylated Ets Binding Site

Holly Maier, Jeff Colbert, Daniel Fitzsimmons, Dawn R. Clark, and James Hagman*

Integrated Department of Immunology, National Jewish Medical and Research Center, Denver, Colorado 80206, and University of Colorado Health Sciences Center, Denver, Colorado 80262

Received 2 October 2002/ Returned for modification 13 November 2002/ Accepted 30 December 2002

Methylation of cytosine in CpG dinucleotides promotes transcriptional repression in mammals by blocking transcription factor binding and recruiting methyl-binding proteins that initiate chromatin remodeling. Here, we use a novel cell-based system to show that retrovirally expressed Pax-5 protein activates endogenous early B-cell-specific mb-1 genes in plasmacytoma cells, but only when the promoter is hypomethylated. CpG methylation does not directly affect binding of the promoter by Pax-5. Instead, methylation of an adjacent CpG interferes with assembly of ternary complexes comprising Pax-5 and Ets proteins. In electrophoretic mobility shift assays, recruitment of Ets-1 is blocked by methylation of the Ets site (5'CCGGAG) on the antisense strand. In transfection assays, selective methylation of a single CpG within the Pax-5-dependent Ets site greatly reduces mb-1 promoter activity. Prior demethylation of the endogenous mb-1 promoter is required for its activation by Pax-5 in transduced cells. Although B-lineage cells have only unmethylated mb-1 genes and do not modulate methylation of the mb-1 promoter during development, other tissues feature high percentages of methylated alleles. Together, these studies demonstrate a novel DNA methylation-dependent mechanism for regulating transcriptional activity through the inhibition of DNA-dependent protein-protein interactions.

* Corresponding author. Mailing address: Integrated Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson St., K516B, Denver, CO 80206. Phone: (303) 398-1398. Fax: (303) 398-1396. E-mail: hagmanj{at}njc.org.

Molecular and Cellular Biology, March 2003, p. 1946-1960, Vol. 23, No. 6
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.6.1946-1960.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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