Functional interaction between the DNA binding subunit trimerization domain of NF-Y and the high mobility group protein HMG-I(Y).
The mammalian transcription factor, NF-Y(CBF), contains three known subunit components, NF-YA (CBF-B), NF-YB(CBF-A), and NF-YC(CBF-C), which are all required to reconstitute specific CCAAT box DNA binding activity. In this study, the high mobility group chromosomal protein, HMG-I(Y), has been shown to activate NF-Y in transient transfections in vivo using the natural murine alpha2(I) collagen promoter and a multimerized version of the proximal NF-Y(CBF) CCAAT box element. In vitro analysis of the alpha2(I) collagen promoter region inclusive of the NF-Y(CBF) binding site (-106 to -65 base pairs) failed to identify any high affinity HMG-I(Y) DNA-binding sites. However, the heterotrimeric NF-Y complex, as well as the NF-YA subunit alone, was shown to stably interact in vitro with both HMG-I(Y) and phosphorylated HMG-I, as modified by casein kinase II, using far Western and protein-protein interaction solution assays in the absence of CCAAT box DNA. Furthermore, the interaction between HMG-I(Y) and NF-Y was mapped to the highly conserved DNA binding-subunit interaction domain (DBD) of the NF-YA subunit and to a single AT-hook motif in HMG-I(Y). Recombinant HMG-I was also found to stabilize the CCAAT box DNA binding activity of recombinant NF-Y, as well as the native NF-Y complex, in vitro. Together, these results suggest a functional HMG-I(Y) protein binding site has been identified in the NF-Y complex and mapped to the conserved DBD and AT-hook regions of NF-YA and HMG-I(Y), respectively. This protein-protein interaction site may function to modulate NF-Y activity through stabilization of NF-Y binding to its CCAAT box DNA-binding site.
Currie
R A
RA
Laboratory of Gene Regulation, The Picower Institute for Medical Research, Manhasset, New York 11030, USA. racurrie@picower.edu
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D003094:Collagen / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D025741:HMGA1a Protein; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D014157:Transcription Factors / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009363:Neoplasm Proteins; D014157:Transcription Factors; D025741:HMGA1a Protein; D003094:Collagen; D004247:DNA