The Farnham Laboratory

Transcriptional Regulation

Most transcription factors are members of multi-gene families and each family member can bind to same consensus sequence in vitro.  This low degree of in vitro specificity has made it difficult to clearly link one member of a family of transcription factors to a specific target promoter.  We have developed a chromatin immunoprecipitation assay that allows us to determine which members of a family of transcription factors binds to specific cellular promoters in living cells.  Surprisingly, we have found very little target gene specificity in DNA binding among members of the Myc or E2F families (Boyd et al., 98; Wells, et al., 2000).  The fact that different E2F and Myc family members can bind to a given target promoter raises very interesting questions concerning functional redundancy and the stochastic nature of transcriptional regulation; these issues are discussed below in the Cell Cycle Regulation section.  Our findings that DNA binding specificity is not the main regulator of target gene specificity indicates that transcriptional regulation is conferred by protein-protein interactions that occur after the family member has bound the DNA.  We have performed several types of experiments to address the mechanisms by which E2F and Myc family members activate transcription.  For example, we have shown that E2F family members can cooperate with some, but not all, transcription factors tested (van Ginkel et al., 97).  Furthermore we have demonstrated a strong correlation between activation of the dhfr promoter by E2F1 and binding of CBP (Fry et al., 99).  We have also used the chromatin immunoprecipitation assay to determine whether changes in histone acetylation regulate transcription of G1/S-phase promoters.  Our results suggest that recruitment of a Myc family member does not necessarily influence the degree of histone acetylation at that promoter (Eberhardy et al., 2 000).  Our current experiments in this area involve a further characterization of the mechanims by which E2F and Myc family members activate transcription. 

Relevant publications:

  • Weinmann, A.S., Bartley, S.M., Zhang, M.Q., Zhang, T., and Farnham, P.J. The use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol. Cell. Biol. 21: 6820-6832, 2001. [Abstract] [pdf]
  • Albert, T., Wells, J., Funk, J.-O., Pullner, A., Raschke, E.-E., Stelzer, G., Meisterernst, M., Farnham, P.J., and Eick, D. Chromatin remodeling and acetylation of the dual c-myc promoters P1/P2 is regulated by separate elements. J. Biol. Chem. 276: 20482-20490, 2001. [Abstract] [pdf]
  • Kel, A.E., Kel-Margoulis, O.V., Bartley, S. M., Farnham, P.J., Wingender, E.,and Zhang, M. Q.. Computer-assisted identification of cell cycle-related genes - new targets for E2F transcription factors. J. Mol. Biol. 309: 99-120, 2001.
  • Eberhardy, S.R. and Farnham, P.J. Direct examination of histone acetylation of c-Myc target genes using chromatin immunoprecipitation. J. Biol. Chem. 275: 33798-33805, 2000.[Abstract][pdf]
  • Wells, J., Boyd, K. E., Fry, C. J., Bartley, S. M., and Farnham, P. J. Target gene specificity of E2F and pocket protein family members in living cells. Mol. Cell Biol. 20: 5987-5807, 2000. [Abstract] [pdf]
  • Mac, S. M., D’Cunha, C., and Farnham, P.J. Direct recruitment of N-Myc to target gene promoters. Molecular Carcinogenesis 29:76-86, 2000.[Abstract] [pdf]
  • Boyd, K. E. and Farnham, P. J. Co-examination of site-specific transcription factor binding and promoter activity in living cells. Mol. Cel. Biol. 19: 8393-8399, 1999. [Abstract][pdf]
  • Fry, C. J., Bartley, S. M., Malinowski, E., Pearson, A., Greenblatt, J., and Farnham, P. J. E2F1-dependent transactivation of the dhfr promoter: A requirement for recruitment of CBP. J. Biol. Chem 274:15883-15891, 1999> [Abstract][pdf]
  • Fry, C.J. and Farnham, P. J. Context-dependent activation of transcription, J. Biol. Chem. 274: 29583-29586, 1999. [Full text] [pdf]