Experimental Study Of The DNA Sequence-Dependent Nucleosome Positioning

The repeating unit of chromatin is the nucleosome, which is formed by wrapping~145–147bp of DNA around a histone octamer core.The nucleosome positioning refers to the histoneoctamer precise location in the DNA double helix. DNA sequences and trans-acting factor aretwo main factors affecting nucleosome positioning. Studies on nucleosome assembly andpositioning mechanism have a very important role for understanding of many biologicalprocesses, including transcription factor binding and transcriptional regulation mechanism.So far，it has been found that more than40kinds of human genetic diseases of thenervous system are associated with DNA repeats sequences’ expansion or deletion. However,over the past years, the study of genetic expansion diseases mainly focused on the abnormalsecondary structure formation and genetic instability, and there was very little understand of thenucleosome positioning and it’s implication with the chromatin structure. We construct threerecombinant plasmids containing repeat sequence associated with three diseases respectivelyand plasmid pUC601as control. The histone and plasmids were used to assemble chromatinstructure in vitro, and then analyzed by agarose gel electrophoresis after micrococcal nucleasedigestion. In vitro, the plasmid containing (GAA)42repeats preferentially excludednucleosomes than pUC601. The recombinant plasmids’ ability to form chromatin structure waschanged because of the insert of the different repeats sequence fragment. Therefore, thecharacteristics of three repeat sequence’s nucleosome positioning were preliminarilyinvestigated in the plasmid level, and the results lay the foundation for understanding of themechanism of such diseases.DNA sequence，one of the important components of nucleosome, plays a key role innucleosome forming process. Currently, some theoretical model of the nucleosome positioningis constructed based on the DNA sequence motifs such as (RRRRRYYYYY)n. The motifbased on the statistical rules is conversial, and lacks the experimental evidence. Based ontheoretical model mentioned above, we designed DNA sequences, which nucleosome formationability is different. After a salt dilution method was employed to assembly nucleosome, theaffinity was analyzed by SDS-PAGE. The results showed that the nucleosome assembly abilityof DNA sequence is consistent with that derived from theoretical prediction.