Dissecting the impact of transcription factor dose on cell reprogramming heterogeneity using scTF-seq

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Cellular reprogramming often results in diverse cell fates, with underlying mechanisms poorly understood.
• Understanding transcription factor (TF) function is crucial for controlling cell fate and reprogramming outcomes.

Purpose of the Study:

• To develop and apply a novel single-cell technique, scTF-seq, for dissecting TF function and dose-dependent effects in reprogramming.
• To create a comprehensive gain-of-function atlas for mouse TFs and analyze their impact on cell fate decisions.

Main Methods:

• Developed single-cell transcription factor sequencing (scTF-seq), a method for barcoded, doxycycline-inducible TF overexpression and transcriptomic analysis.
• Applied scTF-seq to mouse embryonic multipotent stromal cells to generate a TF gain-of-function atlas.
• Analyzed TF dose-dependent transcriptomic changes and cell state transitions at single-cell resolution.

Main Results:

• Generated a gain-of-function atlas for 384 mouse TFs, identifying key regulators of lineage specification and cell cycle control.
• Uncovered how TF dose shapes reprogramming heterogeneity, revealing dose-dependent and stochastic cell state transitions.
• Classified TFs by capacity and dose sensitivity, and demonstrated context-dependent TF interactions (synergistic to antagonistic) based on relative TF dose.

Conclusions:

• scTF-seq enables high-resolution dissection of TF function, dose, and cell fate control during reprogramming.
• Provides a framework for understanding and predicting reprogramming outcomes, crucial for gene regulation research.
• Advances cell engineering strategies by offering insights into precise control of cell fate determination.