c-Myc target gene specificity is determined by a post-DNA-binding mechanism

Kathryn E. Boyd, Julie Wells, Julie Gutman, Stephanie M. Bartley, Peggy J. Farnham

Research output: Contribution to journalArticle

243 Scopus citations

Abstract

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA- binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

Original languageEnglish (US)
Pages (from-to)13887-13892
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume95
Issue number23
DOIs
StatePublished - Nov 10 1998
Externally publishedYes

ASJC Scopus subject areas

  • Genetics
  • General

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