## 4 Discussion  
In _S. cerevisiae_, nucleosome positioning is crucial for regulating gene expression [32]. It has been observed that \(-1\) nucleosomes in long nucleosome-depleted regions (NDRs) remain stable during transcription, whereas those in short NDRs may disassemble or shift upstream. This suggests a nuanced interaction between NDR length and transcriptional regulation [43]. Genes with long NDRs allow transcription factors to bind directly to NDR sequences without displacing the \(-1\) nucleosome, resulting in higher gene expression. In contrast, genes with short NDRs required energy to move the \(-1\) nucleosome upstream, leading to lower gene expression (Figure 8). Our experimental data support this view, although not all the genes conformed to this pattern. Future studies should delve deeper into the epigenetic mechanisms through which NDRs influence gene expression. Figure 7: Comparative analysis of GFP expression levels. (**A**) The relative mRNA expression levels of the GFP gene under the regulation of L-NDR and NDR\(-\) were determined by RT-qPCR. (**B**) The GFP expression levels under the regulation of L-NDR and NDR\(-\) were determined by an ELISA kit. (**C**) The expression of GFP protein under the regulation of L-NDR and NDR\(-\) was detected by Western blotting using anti-GFP antibodies. (**D**) The relative mRNA expression levels of the GFP gene under the regulation of S-NDR and NDR+ were determined by RT-qPCR. (**E**) The GFP expression levels under the regulation of S-NDR and NDR+ were determined by an ELISA kit. (**F**) The expression of GFP protein under the regulation of S-NDR and NDR+ was detected by Western blotting using anti-GFP antibodies. In (**A,B,D,E**), the mean \(\pm\) SEM were determined from each paired NDR with three replications. A paired t-test was down (two-tailed). * \(p\) < 0.05, **_p_ < 0.01, ***_p_ < 0.001, ***_p_ < 0.001, ***_p_ < 0.0001. RT-qPCR, reverse transcription quantitative polymerase chain reaction. Currently, the main purpose of the transgenic design is to increase the level of exogenous gene expression based on the one-dimensional sequence information of the gene transcription start region, but research on how to regulate exogenous gene expression levels is still immature. We proposed a method that may regulate the expression of exogenous genes from the perspective of the chromosome structure of \(\pm 1\) nucleosome and the NDR in the transcriptional regulatory region. It was found that the length of NDR sequences between the \(-1\) nucleosome and the \(+1\) nucleosome in the transcriptional start region of _S. cerevisiae_ genes was closely related to gene expression levels. Based on the conclusion, we took the \(-1\) nucleosome, the NDR sequence, and the \(+1\) nucleosome as the transcriptional regulatory sequences to explore the effect of NDR length on the expression levels of GFP. For the transcriptional regulatory sequences of long NDRs (\(>\)150 bp), it was found that the relative mRNA expression levels and protein expression levels of GFP were significantly higher than that of short NDRs (\(<\)150 bp). When the lengths of long NDRs were shortened by 80 bp to a length less than 150 bp, the relative mRNA expression levels and protein expression levels of GFP were significantly decreased. Conversely, when the length of short NDRs is lengthened by 80 bp to a length greater than 150 bp, the relative mRNA expression levels and protein expression levels of GFP were significantly increased. Those results indicated that the length of the NDR sequence is a key factor in regulating the expression of exogenous genes, and the expression level of exogenous genes can be increased by increasing the length of the NDR sequence. Conversely, shortening the length of the NDR sequence can decrease the expression levels of exogenous genes. As mentioned above, this may be related to the effect of NDR length on transcription factor binding. Long NDR is conducive to transcription factor binding, and the gene expression level is high.