Computational Molecular Biology 2025, Vol.15, No.2, 91-101 http://bioscipublisher.com/index.php/cmb 94 4 Based on the Key Regulatory Factors Revealed by the Integrated Analysis 4.1 Identification of key transcription factors Through the integrated analysis of single-cell RNA-seq and ATAC-seq, a direct objective was to identify the key transcription factors that drive cell fate determination (Hamrud et al., 2025). Traditional methods often infer the importance of known factors based on their expression changes. However, integrative analysis can more powerfully determine whether a certain factor is "critical" because it can simultaneously consider the upstream epigenetic state and downstream gene activation of the factor (Zhang et al., 2023). Specifically, in the integration of data, we can identify candidate key factors from two perspectives: First, if the gene of a certain transcription factor is significantly upregulated during the differentiation process and an open enhancer appears in the chromatin near its locus at the same stage, it indicates that the activation of this factor is permitted by epigenetic regulation and can be suspected as a phylogenetic determinant. For instance, studies on the hematopoietic differentiation of human fetal liver have found that the transcription factor HLF is highly expressed in the early HSC/MPP stage, and its promoter region is specifically open in HSC cells but gradually closes in downstream differentiated cells. This suggests that HLF may play a key role in maintaining the status of stem cells. Further functional experiments also demonstrated that the self-renewal ability of stem cells decreased after HLF knockout. Therefore, the clues initially provided by the integrated analysis were thus verified. 4.2 Cis-regulating element and enhancer interaction Cis-regulatory elements, including gene promoters, enhancers, silencers, etc., are DNA sequences that determine when and where genes are expressed. Enhancers are particularly important as they can move away from the gene itself and enhance transcription by contacting the promoter through DNA loops. Single-cell ATAC-seq can capture the opening of enhancers on a genome-wide scale, while scRNA-seq tells us the expression of the corresponding genes. Integrating the two can establish enhancer-gene associations and evaluate their significance in cell fate determination (Figure 1) (Finkbeiner et al., 2022). In integrative analysis, researchers often select open regions close to a certain gene as candidate enhancers based on chromatin accessibility data, and then examine the association between these open regions and gene expression. For instance, Pervolarakis et al. conducted a combined analysis of scRNA and scATAC on mammary epithelial cells and discovered that there were specific open enhancers near some genes related to the maintenance of mammary epithelial cell identity (such as those responsible for lumen/basal surface characteristics). Moreover, the openness of these enhancers is positively correlated with the expression levels of the corresponding genes in different cells. This suggests that these enhancers are likely to directly regulate the corresponding genes, thereby affecting the cell phenotype (Xu et al., 2021). 4.3 Analysis of gene-regulatory networks By combining the integrated information of key transcription factors and cis-regulatory elements, we can reconstruct the gene regulatory network for cell fate determination. In this kind of network, nodes include transcription factor genes and other regulatory genes, while edges represent regulatory relationships. For instance, transcription factor A regulates the expression of gene B through enhancer X. Integrating single-cell data can help us build more refined and reliable networks. One example is the previously mentioned study on the development of interneurons in primates. By integrating scRNA and scATAC, the authors constructed a gene regulatory network during development: they paired enhancers that were significantly open at different stages with genes that were significantly expressed, and inferred a number of important factor regulatory relationships. For instance, it was found that the DLX family of transcription factors not only have upregulated expression themselves, but also regulate the enhancer opening of a series of downstream neuronal functional genes near their binding sites, thereby establishing the "DLX regulatory module", which is crucial for the fate of interneurons (Hamrud et al., 2025). At the same time, they also identified some regulatory edges that only exist in primates. For instance, the enhancer of the TH gene has been opened to be associated with the FOXP2 factor, which might imply that the unique FOXP2 regulatory circuit in humans has intervened in the differentiation of certain types of interneurons.
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