DYNAMIC CHANGES IN THE HIGHER-LEVEL CHROMATIN ORGANIZATION OF SPECIFIC SEQUENCES REVEALED BY IN-SITU HYBRIDIZATION TO NUCLEAR HALOS

A novel approach to study the higher level packaging of specific DNA s equences has been developed by coupling high-resolution fluorescence h ybridization with biochemical fractionation to remove histones and dis tend DNA loops to form morphologically reproducible nuclear ''halos.'' Results demonstrate consistent differences in the organization of spe cific sequences, and further suggest a relationship to functional acti vity. Pulse-incorporated bromodeoxyuridine representing nascent replic ating DNA localized with the base of the chromatin loops in discrete c lustered patterns characteristic of intact cells, whereas at increasin g chase times, the replicated DNA was consistently found further out o n the extended region of the halo. Fluorescence hybridization to uniqu e loci for four transcriptionally inactive sequences produced long str ings of signal extending out onto the DNA halo or ''loop,'' whereas fo ur transcriptionally active sequences remained tightly condensed as si ngle spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single sp ots of fluorescence signal. Interestingly, two transcriptionally activ e, tandemly repeated gene clusters exhibited strikingly different pack aging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the stru ctural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of c hromatin packaging involving both stable and transient structural asso ciations, and provide precedent for an approach whereby different bioc hemical fractionation methods may be used to unravel various aspects o f the complex higher-level organization of the genome.