CCAAT-binding factor NF-Y and RFX are required for in vivo assembly of a nucleoprotein complex that spans 250 base pairs: the invariant chain promoter as a model.
The events that lead to promoter accessibility within chromatin are not completely understood. The invariant chain (Ii) promoter was used as a model to determine the contribution of different DNA-binding factors in establishing occupancy of a complex promoter. Gamma interferon induction of the Ii promoter requires the cooperation of multiple cis elements including distal S, X, and Y/CCAAT elements along with proximal GC and Y/CCAAT elements. The heteromeric transcription factor NF-Y binds to both Y/CCAAT elements. Genomic footprinting was used to analyze in vivo protein-DNA contacts for integrated Ii promoters bearing mutations in each element. The results reveal a hierarchy of transcription factor loading with NF-Y binding to the distal Y/CCAAT element being required for establishing protein-DNA interactions over the entire 250 bp analyzed. Mutation of the X box disrupts binding primarily at the adjacent Y/CCAAT element along with a lesser effect on GC box binding. Importantly, this finding is verified with a cell line which lacks a functional X-box-binding factor, RFX, providing physiological validity for the strategy described here. Mutation of both the S element and the GC box results in either no or little effect on transcription factor binding. However, mutation of the proximal Y/CCAAT element disrupts binding to the adjacent GC box and partially reduces binding in the distal S/X/Y domain. The crucial role for NF-Y in establishing promoter occupancy may be related to its histone fold motif, the essential component for assembling nucleosome-like structures.
Linhoff
M W
MW
Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 27599, USA.
Wright
K L
KL
Ting
J P
JP
D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics*; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005802:Genes, MHC Class II / Q000235:genetics; D005909:Glioblastoma; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000944:Antigens, Differentiation, B-Lymphocyte; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D000949:Histocompatibility Antigens Class II; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; C045084:invariant chain; D004247:DNA