The nuclear factor-Y (NF-Y), a trimeric, CCAAT-binding transcriptional activator with histone-like subunits, was until recently considered a prototypical promoter transcription factor. However, recent in vivo chromatin immunoprecipitation assays associated with microarray methodologies (chromatin immunoprecipitation on chip experiments) have indicated that a large portion of target sites (40%-50%) are located outside of core promoters. We applied the tethered particle motion technique to the major histocompatibility complex class II enhancer-promoter region to characterize i), the progressive compaction of DNA due to increasing concentrations of NF-Y, ii), the role of specific subunits and domains of NF-Y in the process, and iii), the interplay between NF-Y and the regulatory factor-X, which cooperatively binds to the X-box adjacent to the CCAAT box. Our study shows that NF-Y has histone-like activity, since it binds DNA nonspecifically with high affinity to compact it. This activity, which depends on the presence of all trimer subunits and of their glutamine-rich domains, seems to be attenuated by the transcriptional cofactor regulatory factor-X. Most importantly NF-Y-induced DNA compaction may facilitate promoter-enhancer interactions, which are known to be critical for expression regulation.