Chromatin Structure: Nucleosome Formation and Positioning
Doctoral thesis, 1999
In all eukaryotic cells the DNA is complexed with a group of highly basic histone proteins forming nucleosomes and higher order chromatin structures. In the nucleosome the DNA is tightly wrapped, almost two turns, around an octamer of histone proteins. The octamer in turn is built as a tripartite assembly of a (H3/H4)2-tetramer flanked by two (H2A/H2B)-dimers. The tripartite nature exhibits an interesting feature upon nucleosome formation. The nucleosomal wrapping about the tetramer complex is stabilised energetically four-fold by the addition of the dimers. In addition, the histone octamer is designed to bind virtually all sequences that exist in the genome, but intrinsic properties of the DNA, such as curvature and flexibility, modulate the association of a particular stretch with the histone octamer. The protruding histone terminal tails also contribute to nucleosome stability. The tails help folding on the mononucleosomal level of intrinsically static DNA, but not for flexible DNA. On the chromatin level the tails mediate folding interactions to approximately 800cal/mol which is in the order of magnitude for sequence dependent variations of nucleosome positioning. In addition, we have also found that acetylation of the histone tails facilitate nucleosome formation. Using an in vitro selection strategy, sequences from the mouse genome were identified that exhibited a high affinity for histone octamers. These were categorised by their sequence context and were found to fall into three major groups; highly flexible due to abundance of TG/CA dinucleotides, intrinsically curved arising from phased A-tracts and last, a group of sequences with no obvious characteristics. One minor group of (TATAAACGCC)-repeat sequences were found to have the highest affinity in nucleosome formation from genomic material, which is about 350-fold higher than average nucleosomal DNA, largely dependent on their 25-fold higher flexibility relative to average DNA. In addition, extensive TGGA-repeats were identified by a selection study for nucleosome refractory sequences from synthetic DNA. These repeats were found to be abundant constituents of the mouse genome and predominately located to five discrete locations on mouse metaphase chromosomes. Hybridisation to human chromosomes revealed that these repeats are located in telomeric positions.