In Vitro Experimental Study Of Theoretical Model Of Nucleosome Positioning On Saccharomyces Cerevisiae

As the basic unit of eukaryotic chromatin, the dynamic positioning of nucleosome ongenome can affect gene expression of eukaryotes. It was reported that the DNA sequenceis the main factor influenced nucleosome positioning. Besides, nucleosome positioning arealso affected by chromatin remodeling, histone variants, histone modification, alternativesplicing, etc.Special theoretical model predicting nucleosome positioning of model organisms is theimportant method to study nucleosome characteristics on eukaryotes genome and itsfunction on gene expression. The heterogeneous distribution and dynamic positioning ofnucleosome on eukaryotes genome is involved with gene expression. According to thecalculation results using PCSF(position correlation scoring function) model with1bp step,147bp window in the genome, the sequences with top, medium and lower abilities to formnucleosome were selected from each of Chromosome1,3, and14of Saccharomycescerevisiae, respectively. Then the validity of this theoretical model has been verifiedthrough experiments in vitro, which are listed as follows:1. The recombinant plasmids were reconstructed. The DNA sequences were amplified onthe genome template which was extracted by grinding with glass beads. The sequencesamplified were digested with restriction enzyme and inserted into multiple clone site ofpUC19. The sequences, which were used to assembly nucleosomes, were abundantlyamplified with biotin and Cy3fluorescence labeling primers and purified.2. The H2A, H2B, H3and H4proteins were expressed and purified, then constituted toform octamer. And protein standard curve was profiled. The correlation coefficient is0.9824. The concentration and purity of proteins were tested and prepared to assemblenucleosome.3. Nucleosomes were assembled using the gradient salt dialysis method in vitro. Gibbsfree energy in the reaction was calculated by biotin labeling assay, which was employed tocompare the affinities of the target sequences to octamer. The result indicated that the abilities to assembly nucleosome of five sequences are consistent with that in thetheoretical prediction in chromosome1、3、14of Saccharomyces cerevisiae respectively.The Keqaverage value of each group of top, medium, and lower sequences is consistentwith the results in the theoretical model absolutely. In a word, the theoretical model can beapplied to predict the nucleosome occupancy in Saccharomyces cerevisiae genomeefficiently.4. Cy3labeling assay indicated that the affinities to octamer of seven of the ninesequences were in accordance with that in the theoretical model.5. The affinities to octamer containing H2A.Z of six of the nine sequences were similarto the canonical octamer at the mass ratio0.8, while that of7sequences were similar at theratio1.0. However, there apparently exists nucleosome sliding on the all sequencestemplates in the reconstituted H2A.Z variant-containing nucleosome assay.