title: Quantitative hydroxyl radical footprinting reveals cooperative interactions between DNA-binding subdomains of PU.1 and IRF4.
abstract: Quantitative hydroxyl radical footprinting and fluorescence polarization measurements have been used to determine the dissociation constants (Kd) of complexes between the ets domain of the murine transcription factor PU.1 and three different DNA fragments. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Iglambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex. With the use of quantitative hydroxyl radical footprinting we obtained binding isotherms for individual protected nucleotides and contact sites on both strands of the DNA. Kd values of (1.53 +/- 0. 12) x 10(-)8 M were found for the lambdaB element, (3.60 +/- 0.65) x 10(-)8 M for the SV40 enhancer site, and (2.28 +/- 0.27) x 10(-)8 M for the sequence used in the crystal structure. In addition, the binding of a second protein, the DNA binding domain of IRF4, to the lambdaB site by itself and in the presence of PU.1 was analyzed. The IRF4 DBD shows three footprints on the TTCC strand and one footprint on the GGAA strand of the lambdaB element. The dissociation constant for the binary IRF4 DBD-lambdaB complex equals (5.59 +/- 0.60) x 10(-)7 M. The Kd value of the IRF4-lambdaB interaction is reduced by a factor of 5 in the presence of two different DNA-bound PU.1 protein constructs, PU.1 DBD and a PU.1 construct containing the PEST domain (PU.1-PEST). A similar decrease of the Kd value was observed for the binding of PU.1-PEST in the presence of DNA-bound IRF4 DBD demonstrating a cooperative interaction between the PU. 1-PEST and IRF4 DBD. On the basis of the hydroxyl radical footprints in the ternary PU.1/IRF4/lambdaB complex, a model for the interactions between the two proteins and the lambdaB site was developed. The DNA binding domains of both proteins bind the DNA in the major groove with potential protein-protein interactions near the intervening minor groove.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Ontario, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Gross
authors.forename: A A
authors.initials: AA
authors.lastname: Yee
authors.forename: C H
authors.initials: CH
authors.lastname: Arrowsmith
authors.forename: R B
authors.initials: RB
authors.lastname: Macgregor
mesh_terms: D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005454:Fluorescence Polarization; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism*; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D050835:Interferon Regulatory Factors; D007372:Interferons / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D000099232:Proto-Oncogene Protein Spi-1; D000099218:Interferon Regulatory Factor-4
chemical_list: D004247:DNA; D004268:DNA-Binding Proteins; D017665:Hydroxyl Radical; D007146:Immunoglobulin lambda-Chains; D050835:Interferon Regulatory Factors; D007372:Interferons; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; D000099218:Interferon Regulatory Factor-4