DISSECTING REGULATORY MECHANISMS UNDERLYING CELLULAR DIFFERENTIATION PROCESSES

Abstract

Cellular differentiation Cellular differentiation is a core biological phenomenon that allows unspecialized cells to develop specific phenotypic and functional identities, which is essential in the development, tissue homeostasis, and disease pathogenesis. The regulatory mechanisms that control differentiation are key to further development of regenerative medicine and targeted therapeutic strategies. This paper sought to separate the regulatory network complexes that govern the process of cellular differentiation integrating transcriptional, epigenetic, and signaling pathway analyses. In this regard, we have used both high-throughput and targeted methods, such as RNA sequencing (RNA-seq) to enumerate changes in global gene expression as well as quantitative real-time PCR (qRT-PCR) to validate highly regulated genes and CRISPR-mediated gene modulation to study functional roles of selected regulatory factors. The identification of critical regulators and their interactions was performed with bioinformatics analyses, such as pathway enrichment and gene network modeling. We found that there are unique gene expression signatures that are linked to various stages of differentiation, indicating the ability of transcription factors and epigenetic modifiers to be coordinately regulated. Important signaling pathways, such as Wnt and Notch, were identified to be important in the regulation of lineage-specific gene expression. This was validated functionally by showing that perturbation of the chosen regulatory genes had a tremendous effect on differentiation efficiency and cellular phenotype. To sum up, the current research offers detailed information about the multi-layered regulatory processes governing differentiation of cells, which can be used as a source of therapeutic intervention and furthers our knowledge about cell fate determination.