Intranuclear relocalization of matrix binding sites during T cell activation detected by amplified fluorescence in situ hybridization

We describe a method for analyzing the nuclear localization of specific DNA sequences, with special emphasis on their binding status to the nuclear ma trix, depending on the developmental stage of the cells. This method employ s high-resolution fluorescence in situ hybridization procedures. For our st udies, it was important to examine the nuclear localization of a particular gene locus. Previously, however, it was not possible to detect a single-co py genomic sequence using a DNA probe less than several kilobases in size. We describe here a signal amplification technique based on tyramide which m akes such a task possible. Using this method, we monitored single-copy loci using a short, 509-bp DNA sequence that binds in vivo to the T cell factor SATB1 within T cell nuclei, high-salt-extracted nuclei (histone-depleted n uclei generating "halos" with distended chromatin loops), and the nuclear m atrix, before and after T cell activation. We found that these loci were an chored onto the nuclear matrix, creating new bases of chromatin loops, only after T cell activation. This experimental strategy, therefore, enabled us to detect the changes in higher order chromatin structure upon activation and study gene regulation at a new dimension: the loop domain structure. Th e methods shown here can be widely applied to explore other functions invol ving chromatin, including recombination and replication. (C) 1999 Academic Press.