Effects Of Nucleosome Distribution On Different Steps Of Eukaryotic Gene Expression

The process of eukaryotes gene expression is precise and collaborative, and may affect by many kinds of factors. Some reports demonstrated that chromatin remodeling complex not only involved in the control of initiation of transcription in eukaryotes but also involved in every step of gene expression. At the same time, increasingly evidence suggests that nucleosome, as the basic unit of the nuclear chromatin, not only protects DNA not to combine with some proteins, it may also have some other functions.We have found that nucleosome positioning is regular in previous analysis. The nucleosome positioning in main function site of the gene, especially in translation start site and 5’or 3’splicing site, is specific. At the same time, we also found that the nucleosome positioning in different function site of the gene and the distribution of PoⅢ, the specific DNA methylation modification is consistent. This suggests that nucleosome positioning involved in mark and identify the different function of gene sites to regulate eukaryotes gene expression. To prove this hypothesis, we changed the translation start site of LacZ reporter gene to changed nucleosome positioning in order to analyze the relationship between nucleosome positioning and gene expression. The results show that, if we change the typical nucleosome positioning around the translation start site of LacZ reporter gene, that will affect cell recognized the translation start sites. Then, we insert different introns which have different GC dinucleotide content in different positions of LacZ reporter gene to explore the relationship between the nucleosome positioning around 5’and3’splicing sites and gene expression. The results show that, insert different introns which have different GC dinucleotide content in different positions of LacZ reporter gene indeed affect the expression of LacZ reporter gene. At the same time, the result indicate that as the position of intron move downstream, the LacZ gene expression first raised, then slowly drops. We believe that this trend may due to the change of nucleosome positioning of LacZ gene which is because insert introns. The nucleosome positioning around the translation start site is phased. We suspect that insert intron around the translation start site may make this phase more stable, so gene expression level slightly increased. However, with the position of introns moving downstream, the nucleosome positioning become more and more fuzzy, but insert intron may make the nucleosome positioning around this area more stable, and this may lead to the decrease of gene expression. We also compare the intron splicing results between different LacZ reporter genes which insert different introns which have different GC dinucleotide content. The result showes that the constructs which insert intron at 477bp from ATG cannot completely splice and these results also confirm this hypothesis. Next we plan insert intron at 2765bp from ATG of LacZ gene downstream to further prove our hypothesis. At the same time, we also want to change different promoter to verify above experimental results. Because the above experiments are base on a plasmid, we also prepared to choose some of above LacZ reporter gene constructs insert into yeast genome in order to find the expression difference between plasmid and genome to further explore the relationship between nucleosome positioning and gene expression.At the same time, we also plan to use bioinformatics means to compare the published CHIP-Seq and RNA-Seq experimental results of yeast BY4741 strains. Then derive a formula to explain the relationship between nucleosome positioning and gene expression in order to further confirm our experimental results. We believe that, the research about nucleosome positioning will help us to enhance our understanding of the chromatin structure and function.