title: Cooperative interaction between the DNA-binding domains of PU.1 and IRF4.
abstract: The two lymphoid-specific transcription factors PU.1 and IRF4 form a cooperative ternary complex at immunoglobulin enhancer elements such as the lambdaB and kappaE3' sites. We report here that the synergy of this interaction can be reconstituted in part with the DNA-binding domains of the two proteins. The minimal DNA binding-domain of IRF4 was mapped to residues 20 to 137, corresponding to the conserved DNA-binding region of other interferon regulatory factors (IRFs). This domain can bind weakly to a synthetic murine lambdaB element, while IRF4 constructs that contain residues 1 to 19 require the presence of PU.1 for DNA-binding at similar concentrations. Fluorescence polarization of fluorescein-labelled DNA was used to show that the presence of residues 1 to 19 decreases the binding affinity of IRF4 N-terminal constructs from two- to fivefold. However, all constructs bound better to the lambdaB element in the presence of the DNA-binding domain of PU.1. This cooperative interaction was not dependent on phosphorylation of the PEST domain of PU.1, but was dependent on the proper spacing of the binding sites for PU.1 and IRF4. These data suggest that at least part of the cooperative interaction between full-length PU.1 and IRF4 involves the DNA-binding domains of the two proteins. NMR spectroscopy of 15N-labelled PU.1 and IRF4 constructs indicates that the PEST domain of PU.1 and residues 1 to 19 of IRF4 may be unstructured in the isolated proteins.
authors.affiliation: Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5G 2M9, Canada.
authors.forename: A A
authors.initials: AA
authors.lastname: Yee
authors.forename: P
authors.initials: P
authors.lastname: Yin
authors.forename: D P
authors.initials: DP
authors.lastname: Siderovski
authors.forename: T W
authors.initials: TW
authors.lastname: Mak
authors.forename: D W
authors.initials: DW
authors.lastname: Litchfield
authors.forename: C H
authors.initials: CH
authors.lastname: Arrowsmith
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005454:Fluorescence Polarization; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry*; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1