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 no t completely understood. The invariant chain (Ii) promoter was used as a model to determine the contribution of different DNA-binding factor s in establishing occupancy of a complex promoter. Gamma interferon in duction of the Ii promoter requires the cooperation of multiple cis el ements including distal S, X, and Y/CCAAT elements along with proximal GC and Y/CCAAT elements. The heteromeric transcription factor NF-Y bi nds to both Y/CCAAT elements, Genomic footprinting was used to analyze in vivo protein-DNA contacts for integrated Ii promoters bearing muta tions 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 t he GC box results in either no or little effect on transcription facto r binding. However, mutation of the proximal Y/CCAAT element disrupts binding to the adjacent GC box and partially reduces binding in the di stal S/X/Y domain. The crucial role for NF-Y in establishing promoter occupancy may be related to its histone fold motif, the essential comp onent for assembling nucleosome-like structures.