## 3 Results and Discussion
### Permutation Test and Analysis of Z-DNA Overlap with Enhancer Elements  
To evaluate the statistical significance of overlaps between predicted Z-DNA regions and enhancer-related elements (TE, SE, and SE-E) in six cell lines, permutation tests were conducted as described in Section 2.3 of the Materials and Methods. We compared the actual overlaps between Z-DNA sequences and enhancer annotations against overlaps expected by random genomic distribution. Specifically, predicted Z-DNA regions were intersected with SE-E, TE, SE, and background regions, and their overlap frequencies were evaluated against intersections obtained using 1000 randomly generated genomic coordinate sets, matched by length and excluding centromeric and telomeric regions (see details in Materials and Methods, Section 2.3). Figure 2 presents the results of the permutation test. This test was based on calculating match scores between Z-DNA coordinates and regulatory elements for both their actual predictions and randomly generated ones. Lower \(p\)-values indicate a lower probability of the coordinate overlaps occurring by chance. Background regions consistently demonstrated high \(p\)-values (median = 0.994, interquartile range: 0.950-0.998), indicating that intersections within these genomic segments occur predominantly due to random chance. In contrast, super-enhancer enhancers and typical enhancers showed significantly lower median \(p\)-values (SE-E median range: 0.006-0.035; TE median range: 0.0006-0.0035), clearly signifying a statistically significant enrichment of Z-DNA loci in these regulatory elements. Notably, super-enhancer regions, despite containing spacer elements characterized by lower regulatory activity, also exhibited substantial enrichment (median \(p\)-value range: 0.014-0.066), markedly distinct from the background. Figure 1: Heat maps of Z-DNA distribution across enhancer elements in the HCT116 cell line: Comparison of original (**Top Row**) and Z-DNA-intersected (**Bottom Row**) datasets across typical enhancers, super-enhancer enhancers, super-enhancers, and background regions (BG) with chromosomal binning (25 bins per chromosome, hg38 assembly). These robust statistical differences suggest that the observed intersection between Z-DNA regions and enhancer elements is likely not coincidental and indicates a potential functional relationship. It is known that Z-DNA serves as a crucial tool in the regulation of gene transcription. Studies have shown that conserved Z-DNA flipons in promoter regions of humans and mice are associated with increased transcription reinitiation rates, indicating higher levels of mRNA synthesis in such genes. Additionally, these regions exhibit an enrichment of transcription factors and histone marks of active chromatin [31]. This observation is further supported by the elevated occurrence of Z-DNA motifs in the promoters of birds with shortened ontogenetic periods. Genes with such promoters are involved in key regulatory pathways related to growth and development [54]. Enhancers and super-enhancers are functionally similar to promoters, as all these sequences act as cis-regulatory elements. It has been demonstrated that both enhancers and promoters exhibit a significantly increased presence of Z-DNA-forming regions--by 3- and 6.7-fold, respectively--suggesting a regulatory role for Z-DNA [40]. Thus, the permutation test conducted here corroborates previous findings on the enrichment of Z-DNA motifs in cis-regulatory elements. Given the established role of Z-DNA in promoters and enhancers, we hypothesize that super-enhancers with a higher density of Z-DNA-forming sequences may also be associated with more intense transcription of their target genes. Furthermore, the previously described ability of Z-DNA to maintain a transcriptionally active state in promoters due to its structural properties may also apply to super-enhancers. That is, in super-enhancers, Z-DNA could serve both as a transcription factor (TF)-binding element and as a structural component.