The use of transient chromatin immunoprecipitation assays to test models for E2F1-specific transcriptional activation

Jennifer L. Lavrrar, Peggy J. Farnham

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

The E2F family of transcription factors regulates the expression of genes involved in cell cycle progression, DNA synthesis, repair, and recombination, and a variety of other cellular processes. Although E2F proteins are often redundant in function, specificity of binding and activity can occur. For example, E2F1, but not other E2F family members, was shown previously to bind the murine carboxylesterase promoter in chromatin immunoprecipitation studies (Wells, J., Graveel, C. R., Bartley, S. M., Madore, S. J., and Farnham, P. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3890-3895). This promoter region lacks a consensus E2F binding site, suggesting that E2F1 may be recruited to the DNA in a unique fashion. To further investigate this E2F1-specific binding, we have employed a "transient chromatin immunoprecipitation" approach. Using various deletions and mutations of the promoter region, we localized the E2F1-specific binding site and demonstrated that it was required for E2F1-mediated transcription of the carboxylesterase promoter. The identified site was similar to the 8-bp consensus E2F site but differed from the consensus at a crucial position. To address whether E2F1 directly bound to this non-consensus site, we demonstrated that the DNA binding domain of E2F1 is necessary for E2F1-mediated activation of the carboxylesterase promoter. Interestingly, a "UP" mutation of the site, making it more similar to the consensus element, did not improve the ability of E2F1 to bind the promoter. Rather, E2F1 could no longer bind to the carboxylesterase promoter that contained the consensus E2F site. We propose a model in which E2F1-specific regulation of the carboxylesterase promoter requires both E2F1/DNA interactions and protein-protein interaction between E2F1 and a factor that binds adjacent to the non-consensus site.

Original languageEnglish (US)
Pages (from-to)46343-46349
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number44
DOIs
StatePublished - Oct 29 2004

ASJC Scopus subject areas

  • Biochemistry

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