## 4 Discussion
In contrast, short NDR reduces the binding efficiency of transcription factors, resulting in decreased gene expression levels. In eukaryotes, the regulation of gene expression is a complex process that is affected by a variety of factors, including the TATA box, DNA methylation, histone modification, and transcription factor binding. It has been demonstrated that the presence of nucleosomes impedes the formation of TATA frames and preinitiation complexes, rendering it  
Figure 8: Diagram of the mechanism of gene expression regulated by NDR sequence. (**A**) For NDR \(>\) 150 bp, the length of the NDR is sufficient. TF can bind directly to the NDR sequence. The \(-1\) nucleosome does not need to move and consume additional energy. Expression levels of relevant genes are high. (**B**) For NDR \(<\) 150 bp, the length of the NDR is short. TF cannot directly bind to the NDR sequence. The \(-1\) nucleosome must move upstream or disassemble to extend the NDR sequence. This process requires more energy, which reduces binding efficiency. Expression levels of relevant genes are low. \(-1\)N represents \(-1\) nucleosome, \(+1\)N represents \(+1\) nucleosome, and NDR is the nucleosome-depleted region between the \(-1\) nucleosome and \(+1\) nucleosome. essentially impossible for TATA-binding proteins and the RNA polymerase II transcription machinery to bind to nucleosome DNA. Consequently, NDRs are generated in poly(dA:dT) bundles that bind to the core promoter and initiate transcription [36; 44]. NDRs are essential functional elements for initiating transcription and replication, providing entry points on chromatin for protein complexes involved in RNA transcription and DNA replication [45; 46; 47]. Furthermore, histone modifications are of great importance in the regulation of chromatin-associated processes. These modifications include methylation, acetylation and ubiquitination of lysine residues, as well as phosphorylation of serine residues. They are essential for a number of functions, including transcription and DNA repair. Among these modifications, acetylation occurs predominantly on nucleosomes flanking the NDR [48; 49; 50]. It has been demonstrated that ATP-dependent nucleosome remodeling complexes can localize \(+1\) and \(-1\) nucleosomes through the NDR [47]. In conclusion, gene expression regulation involves a multitude of molecular mechanisms, and a comprehensive understanding of these mechanisms is of paramount importance. Finally, it should be stressed that, in this study, we mainly focused on the effects of the \(-1\) nucleosome, \(+1\) nucleosome, and NDR length on exogenous gene expression. One study has shown that when large genomic regions from a foreign yeast species are introduced into _S. cerevisiae_, the distance between nucleosomes is characteristic of _S. cerevisiae_, and when foreign DNA is introduced into _S. cerevisiae_, nucleosome-depleted regions may also occur in coding regions [51]. Therefore, we thought that the transcription system would still form the structure of the \(-1\) and \(+1\) nucleosomes when it is integrated into the chromosome of _S. cerevisiae_. By experimental analysis, we ensured that the transgene design is consistent with the expectation, which means that the transcription system including NDR really preserve the nucleosome occupancy signatures in _S. cerevisiae_. Meanwhile, it is important to note that this was only a preliminary study investigating the relationship between NDR length and the expression of exogenous genes, and there are some limitations. In the future, more precise design is needed to explore the regulation of exogenous gene expression. For example, first, more appropriate transcriptional regulatory sequences in _S. cerevisiae_ genes should be selected. The translation start site should be located at the 3'-end or downstream of the \(+1\) nucleosome sequences in the selected transcriptional regulatory sequences. Then, the part of the coding sequence of the _S. cerevisiae_ gene is replaced by the coding sequence of the exogenous gene.