The Regulation of Transcription Factors Binding Site Occupancies by Their Concentrations

The precise control of transcription factor (TF) concentrations is crucial for While TF concentration gradients are well-characterized in model organisms like Drosophila and Caenorhabditis elegans, their quantitative relationship with genomic occupancy in mammals is less understood. Recent studies have highlighted the significance of TF dosage, with aberrant levels linked to disorders such as Pierre-Robin Syndrome (PRS) and cancer. Aberrant TF levels are linked to disorders such as Pierre-Robin Syndrome (PRS) and cancer. Nonetheless, endogenous TF levels naturally fluctuate within cell populations, influencing cell fate decisions without compromising cellular identity. In mES cells, fluctuations in OCT4 and SOX2 levels influence cell fate, while variations in NANOG concentration regulate the timing of pluripotency exit. In this study, we quantified changes in pluripotent TF binding site occupancy as a function of physiological TF concentration variations. Using cell sorting, ChIP-seq, ATAC-seq, and motif enrichment analysis, we identified distinct concentration-dependent binding patterns. We found that NANOG binds regions enriched in OCT4-SOX2 motifs independently of its concentration but expands to additional pluripotency-associated regions at higher levels. SOX2 binding occupancy does not scale with its concentration; instead, its occupancy shifts to regions enriched with differentiation-associated motifs at higher levels. OCT4 displays an intermediate pattern, with binding occupancy initially increasing like NANOG but redistributing among binding sites beyond a threshold, similar to SOX2. These concentration-dependent binding dynamics reflect the role of each TF in balancing pluripotency and differentiation.

Our results suggest that a simple mass-action models may not fully capture TF binding dynamics in mES cells. This study reveals distinct concentration-dependent mechanisms through which OCT4, SOX2, and NANOG regulate pluripotency and differentiation. Finally, our observations suggest that gradient-based regulation of cell fate decisions during embryonic development may be a conserved mechanism across model organisms.