CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.
NF-Y binds a CCAAT motif found in many eukaryotic polymerase II-dependent promoters. In the HLA-DRA promoter it has been demonstrated that stereo-specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild-type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF-Y binding. Interaction between NF-Y and X box binding proteins was demonstrated by reciprocal co-immunoprecipitation in the absence of DNA and co-affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein-DNA interactions and suggest that NF-Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element.
Wright
K L
KL
Lineberger Comprehensive Cancer Center, Department of Microbiology-Immunology, University of North Carolina at Chapel Hill 27599.
Vilen
B J
BJ
Itoh-Lindstrom
Y
Y
Moore
T L
TL
Li
G
G
Criscitiello
M
M
Cogswell
P
P
Clarke
J B
JB
Ting
J P
JP
D017920:Antiporters; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics; D000076988:Sulfate Transporters; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017920:Antiporters; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D004268:DNA-Binding Proteins; D000949:Histocompatibility Antigens Class II; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011506:Proteins; C081011:SLC26A3 protein, human; D000076988:Sulfate Transporters; D014157:Transcription Factors