Computational Molecular Biology 2025, Vol.15, No.2, 91-101 http://bioscipublisher.com/index.php/cmb 93 developed methods such as Drop-seq and 10x Genomics Chromium encapsulated single cells with microdroplets and added microbeads with cell barcodes, introducing UMI (Unique Molecular Identifier) to quantify mRNA copy numbers. Large-scale parallel sequencing and PCR bias correction have been achieved (Babcock and Weir, 2023). The current mainstream RNA-SEQ protocols typically rely on 3 'end tag sequencing, which can efficiently sequence thousands of cells and obtain their gene expression matrices (Aryamanesh, 2022). 2.2 Application in cell typing and tumor research A typical application of RNA-SEq is to identify cell types and states in complex tissues. For instance, Travaglini et al. constructed a single-cell transcriptional map of the human lung and identified new subtypes of alveolar cells, etc., expanding the types of human lung cells from over 40 previously to nearly 60 (Rehman et al., 2022). For instance, the application of scRNA-seq in tumor tissues can classify tumor cells into different subpopulations based on gene expression, and simultaneously analyze multiple components such as immune cells and fibroblasts in the tumor microenvironment, providing a basis for studying tumor heterogeneity and immune escape (Saddala et al., 2023). Especially in highly heterogeneous cancers such as breast cancer, scRNA-seq reveals that significant intercellular differences still exist under traditional molecular typing, and drug resistation-related tumor subclones can be discovered (Tian et al., 2018). Therefore, scRNA-seq has been widely applied in numerous fields such as oncology, development, reproduction, and neurology for cell classification, marker identification, pathway enrichment, and pseudo-temporal trajectory analysis, etc. (Khan et al., 2023). 3 Transcriptional and Epigenetic Regulatory Mechanisms of Cell Fate Determination 3.1 Regulation of transcription factors and gene expression Transcription factors (TF) are proteins that directly bind to DNA to regulate gene transcription and play a core role in cell fate determination (Kumar and Sharma, 2021). The reason why different cell types have distinct functions and morphologies is largely due to the fact that their specific combinations of transcription factors drive different gene expression programs. For example, during the process of muscle cell differentiation, the expression of transcription factors such as myogenic regulatory factors (such as MyoD) can induce the activation of muscle-specific genes, thereby pushing precursor cells towards the fate of muscle cells (Gurdon et al., 2020). Similarly, in neuronal differentiation, the initiation of basic helical-ring-helix transcription factors such as Neurogenin can induce stem cells to transform into neuronal lineages. These examples demonstrate the classic model where a single "master regulator" transcription factor can determine cell fate. However, in a broader context, the determination of cell fate is often the result of the synergistic action of multiple transcription factors (Larcombe et al., 2022). For example, the core transcription factor network that maintains pluripotency of embryonic stem cells includes OCT4, SOX2, NANOG, etc. They mutually regulate and jointly maintain the fate of stem cells. When any one of them is disturbed, differentiation will be triggered (Balsalobre and Drouin, 2022). 3.2 The role of chromatin accessibility in fate determination Chromatin accessibility refers to the degree of openness of chromatin structure, that is, whether regulatory proteins such as transcription factors can conveniently access DNA sequences (Fan and Huang, 2021). In the process of cell fate determination, the dynamic change of chromatin accessibility is an important prerequisite for gene regulation (Kim et al., 2022). Developmental biology research indicates that there are extensive chromatin remodeling events in the early stage of embryonic development, gradually confining the genome of pluripotent stem cells to specific lineages of epigenetic states. 3.3 Interactive regulation of epigenetics and transcription A typical example is the formation and function of super-enhancers. These regions are usually occupied by core transcription factors determined by cell identity and recruit large-scale coactivators and chromatin regulatory proteins (Balsalobre and Drouin, 2022). For instance, integrating the data of scRNA-seq and scATAC-seq can reveal that in certain cells, the gene of a key transcription factor simultaneously shows high expression and its promoter/enhancer is highly open, and the binding sites of the factor itself are enriched, suggesting the synergy of its self-regulation and chromatin remodeling (Fan and Huang, 2021).
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