## 3 Results
### Impacts of Nucleosome Assembly and Disassembly on Native Gene Expression in S. cerevisiae  
In order to investigate the impact of \(-1\) nucleosomes on gene duplication, we utilized the \(-1\) nucleosome sequences derived from _YBL025W_, _YBL032W_, _YBL071W-A_, _YAR008W_, _YAR027W_ and _YAL030W_ genes along with commercially available nucleosome assembly kits for in vitro nucleosome assembly. Subsequently, isothermal amplification PCR was conducted, employing both naked DNA and nucleosome-assembled DNA sequences as templates. The results indicated that, compared with naked DNA, the amplification products significantly decreased in the presence of nucleosomes (Figure 3A). This reduction was attributed to nucleosome DNA being wrapped by proteins, which prevented primer binding. Consequently, only a minor fraction of unencapsulated DNA fragments served as templates during isothermal amplification, thereby diminishing the amplification yield. These observations indicate that the formation of the \(-1\) nucleosome impedes DNA replication, thereby decreasing the amplification efficiency.  
We further analyzed the expression levels of L-NDR genes (_YBL025W_, _YBL032W_, and _YBL071W-A_) and S-NDR genes (_YAR008W_, _YAR027W_, and _YAL030W_) in _S. cerevisiae_ using RT-qPCR. For each gene, both high- and low-expression strains were examined to determine the effect of \(-1\) nucleosomes on downstream native gene expression (Figure 3B). Absolute quantitative PCR was performed using the \(-1\) nucleosome DNA sequence as a template to evaluate the effects of \(-1\) nucleosome disintegration or sliding on the DNA abundance. The results revealed no statistically significant difference in the amplification of the \(-1\) nucleosome DNA sequence between the two groups of L-NDR genes. However, there was a significant reduction in \(-1\) nucleosome DNA amplific  
Figure 3: Effects of \(-1\) nucleosome on downstream gene expression. (**A**) Examination of \(-1\) nucleosome assembly impact on DNA replication in vitro via isothermal amplification PCR. Lanes 1–6: PCR templates are \(-1\) nucleosome DNA sequences without \(-1\) nucleosomes. Lanes 7–12: PCR templates include \(-1\) nucleosome DNA sequences with \(-1\) nucleosomes. (**B**) Measurement of mRNA relative expression levels for genes _YBL071W-A_, _YBL032W_, _YBL025W_, _YAL030W_, _YAR008W_, and _YAR027W_ in _S. cerevisiae_ using RT-qPCR. (**C**) Absolute qPCR detection of \(-1\) nucleosome DNA copy number, using _TUB1_ as a reference gene. In panels (**B,C**), “high” indicates elevated expression levels of the genes _YBL071W-A_, _YBL032W_, _YBL025W_, _YAL030W_, _YAR008W_, and _YAR027W_ during a specific period, while “low” denotes reduced expression levels of the same genes. qPCR: quantitative polymerase chain reaction.  
groups (Figure 3C). These results suggest that during transcription, the \(-1\) nucleosome of S-NDR genes at the high expression stage may disintegrate or slide, increasing the NDR sequence length and available physical space for transcription factor binding, thereby enhancing transcription efficiency. Conversely, for L-NDR genes, the position of the \(-1\) nucleosome and NDR sequence length likely remained unchanged, resulting in a minimal impact on their transcription levels.