Intra- and intermolecular cooperative binding of high-mobility-group protein I(Y) to the beta-interferon promoter.
The mammalian high-mobility-group protein I(Y) [HMG I(Y)], while not a typical transcriptional activator, is required for the expression of many eukaryotic genes. HMG I(Y) appears to recruit and stabilize complexes of transcriptional activators through protein-DNA and protein-protein interactions. The protein binds to the minor groove of DNA via three short basic repeats, preferring tracts of adenines and thymines arranged on the same face of the DNA helix. However, the mode by which these three basic repeats function together to recognize HMG I(Y) binding sites has remained unclear. Here, using deletion mutants of HMG I(Y), DNase I footprinting, methylation interference, and in vivo transcriptional assays, we have characterized the binding of HMG I(Y) to the model beta-interferon enhancer. We show that two molecules of HMG I(Y) bind to the enhancer in a highly cooperative fashion, each molecule using a distinct pair of basic repeats to recognize the tandem AT-rich regions of the binding sites. We have also characterized the function of each basic repeat, showing that only the central repeat accounts for specific DNA binding and that the presence of a second repeat bound to an adjacent AT-rich region results in intramolecular cooperativity in binding. Surprisingly, the carboxyl-terminal acidic tail of HMG I(Y) is also important for specific binding in the context of the full-length protein. Our results present a detailed examination of HMG I(Y) binding in an important biological context, which can be extended not only to HMG I(Y) binding in other systems but also to the binding mode of many other proteins containing homologous basic repeats, which have been conserved from bacteria to humans.
Yie
J
J
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
Liang
S
S
Merika
M
M
Thanos
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D004331:Drosophila melanogaster; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D025741:HMGA1a Protein; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D016899:Interferon-beta / Q000235:genetics*; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D006609:High Mobility Group Proteins; D046911:Macromolecular Substances; D025741:HMGA1a Protein; D016899:Interferon-beta