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Molecular and Cellular Biology, October 2004, p. 8790-8802, Vol. 24, No. 19
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.19.8790-8802.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Cyclin D3 as a Direct Target of E2A Using DamID

Siyuan Song,1 Jonathan Cooperman,1 Danielle L. Letting,2 Gerd A. Blobel,3 and John Kim Choi1,4*

Pathology,1 Division of Hematology, Children's Hospital of Philadelphia,3 Pathology and Laboratory Medicine,4 School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania2

Received 2 February 2004/ Returned for modification 9 March 2004/ Accepted 9 July 2004

The transcription factor E2A can promote precursor B cell expansion, promote G1 cell cycle progression, and induce the expressions of multiple G1-phase cyclins. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated the 1-kb promoter of cyclin D3, which contains two E boxes. However, deletion of the E boxes did not disrupt the transactivation by E2A, raising the possibility of indirect activation via another transcription factor or binding of E2A to non-E-box DNA elements. To distinguish between these two possibilities, promoter occupancy was examined using the DamID approach. A fusion construct composed of E2A and the Escherichia coli DNA adenosine methyltransferase (E47Dam) was subcloned in lentivirus vectors and used to transduce precursor B-cell and myeloid progenitor cell lines. In both cell types, specific adenosine methylation was identified at the cyclin D3 promoter. Chromatin immunoprecipitation analysis confirmed the DamID findings and localized the binding to within 1 kb of the two E boxes. The methylation by E47Dam was not disrupted by mutations in the E2A portion that block DNA binding. We conclude that E2A can be recruited to the cyclin D3 promoter independently of E boxes or E2A DNA binding activity.

* Corresponding author. Mailing address: Children's Hospital of Philadelphia, 802F ARC, 3516 Civic Center Blvd., Philadelphia, PA 19104. Phone: (215) 590-7194. Fax: (215) 590-0342. E-mail: jkchoi{at}mail.med.upenn.edu.

Molecular and Cellular Biology, October 2004, p. 8790-8802, Vol. 24, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.19.8790-8802.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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