The Theoretical And Experimental Study Of Nucleosome Positioning On The Sequences Containing GAA Triplet Repeats And R5Y5 Motif

All eukaryotes package their genomes into a repeating nucleoprotein complex known as the nucleosome. The nucleosome positioning at a given base pair is the probability that a nucleosome starts at that base pair. Nucleosome positioning regulates many biology processes occurred in chromatin level, such as DNA replication, DNA repair, DNA recombination, alternative splicing and so on. For example, nucleosome affect the access of DNA-binding proteins to their template, as linker DNA between nucleosomes is far more accessible than the DNA in contact with the histones. The genomic positions of nucleosome can also influence the ability of other proteins to bind to the post-translational modifications on histones. So, the nucleosome is not just a static building block of chromatin structure, and the nucleosome properties, including positioning, turnover and histone variations and modifications, play essential roles in gene regulation by affecting the transcriptional competence of various chromatin regions. Nucleosome positioning along the genome might be affected primarily by the preferences of DNA sequence and external factors, such as chromatin remodeling, DNA methylation, histone variants, post-translational modifications, and polymerase Ⅱ binding. The preference of DNA sequence is one of important factors to determine the nucleosome positioning in the genome.About 40 human genetic diseases are associated with trinucleotide repeats in specific site of genome, such as GAA and CAG triplet repeats. Increasing evidence has shown that chromatin structure may be responsible for the human genetic diseases associated with triplet repeats. Several studies suggested that the expansion of GAA triplet-repeats in the first intron of the FRDA could cause epigenetic changes, such as H3-K9 hypermethylation and reductions in histone acetylation. The efficiency of nucleosome assembly on pure GAA22 repeat duplex was only half that of the pUC control DNA. However, less information is available on how GAA triplet-repeats affect the local nucleosome positioning in healthy individuals. It is important to consider the behavior of both healthy and disease length repeat tracts at the nucleosome level to better understand the mechanisms of the FXN gene silencing.In the recent 30 years, many efforts were attempted to search the sequence motif with strong nucleosome positioning properties in nucleosome DNA. The N-gram extension, DNA bendability matrix and base pair stacking energy model were employed by EN Trifonov to analyze the nucleosome occupancy data in human, Caenorhabditis elegans and Saccharomyces cerevisiae. All three approaches converge to the same sequence motif RRRRRYYYYY (R5Y5) with 10.4-bp periodicity, a consensus of strong nucleosome motif. In the R5Y5 motif, R is purine and Y is pyrimidine. After the R5Y5 motif was published, the nucleosome motif had drawn great contention. So far, there are no strong experimental evidence to investigate whether and how the R5Y5 motif affects nucleosome positioning.In this paper, based on the effect of preference of DNA sequence on nucleosome positioning, the statistical analysis and chromatin reconstitution assay were employed to investigate the nucleosome positioning on the sequences containing GAA triplet repeats and R5Y5 motif. The main contributions are summarized as follows.Firstly, the levels of nucleosome occupancy around 18 triplet-repeats across the human genome were computed statistically. The results showed that the ability to form nucleosome of triplet-repeats containing AA or TT was weak. Then, we preferred to pay greater attention to nucleosome occupancy on GAA triplet repeats. Surprisingly, A-tracts were frequently adjacent to GAA triplet-repeats in the human genome. The results suggested that the GAA triplet-repeats could induce local nucleosome depletion in vivo and that the A-tract upstream of repeats could further enhance the depletion surrounding GAA triplet-repeats. Nucleosome occupancy on special regions of the genome containing GAA triplet repeats also indicated that GAA triplet-repeats had a weak ability to form nucleosomes in unaffected individuals.Secondly, six histones (H2A, H2B, H3, H4, H2A.Z and H3.3) were expressed and purified from E. coli BL21 cells containing pET-histone expression plasmids. Three histone octamers, which were canonical histone octamer, histone octamer containing variant H2A.Z and histone octamer containing variant H3.3, were reconstituted for assembling the nucleosome and chromatin structure in vitro.Thirdly, chromatin reconstitution assay demonstrated that GAA triplet repeats can cause local nucleosome depletion. In this work, three recombinant plasmids containing GAA7, GAA27 and GAA42 repeats were constructed, and other four recombinant plasmids containing CAG7, CAG27, CAG44 and 601-sequence were also constructed as control sample. Chromatins were assembled on plasmids-containing GAA and CAG triplet-repeats and the 601 sequence. The successful formation and alignment of nucleosomes were examined by micrococcal nuclease digestion, electron microscopy and sucrose density gradient centrifugation. The results showed that chromatin arrays on the GAA triplet-repeat-containing template adopted a looser and more open structure than that on the 601-containing template. The analytical ultracentrifugation in sedimentation velocity experiments further demonstrated that GAA triplet-repeat inserts destabilized the ability of recombinant plasmids to assemble nucleosomes.Lastly, nucleosome reconstitution assay demonstrated that the sequences contain R5Y5 motif have strong affinities to histones in vitro. Based on 10.5-bp periodicity of TA and R5Y5 sequence motif on nucleosome DNA, six sequences with different affinity to histones were designed to assembly the nucleosome structure. The results showed that both R5Y5 motif and TA periodicity extremely significantly affect the canonical nucleosome formation. The influence of TA periodicity on canonical nucleosome assembly was more remarkable than that of R5Y5 motif. To assemble nucleosome contain histone variant H2A.Z, the sequences with TA periodicity had significantly higher affinity to histone octamer, but we did not determine the significant difference of the ability of R5Y5 to form nucleosome. While the nucleosome containing histone variant H3.3 was reconstituted, TA periodicity was also a strong nucleosome positioning signals, and the significant difference of the ability of R5Y5 to form nucleosome was also not determined. But there was not significant statistically difference between sequences only with R5Y5 motif and that only with TA periodicity to form nucleosome containing histone variant H3.3.