Phospho-regulation of ASCL1-mediated chromatin opening during cellular reprogramming.

Area of Science:

• Cell Biology
• Developmental Biology
• Neuroscience

Background:

• The proneural transcription factor ASCL1 is crucial for neurogenesis and neuronal reprogramming.
• However, its reprogramming efficiency varies across cell types, indicating unknown competence factors and barriers.
• Understanding ASCL1 regulation is key to overcoming these barriers for directed differentiation.

Purpose of the Study:

• To investigate how ASCL1 levels and phosphorylation impact its activity during mouse embryonic stem cell differentiation.
• To identify mechanisms regulating ASCL1's competence in directing neuronal reprogramming.
• To explore strategies for enhancing ASCL1-mediated cell reprogramming.

Main Methods:

• Manipulating ASCL1 phosphorylation levels in mouse embryonic stem cells.
• Assessing reprogramming efficiency in mesodermal, neuroectodermal, and pluripotent cells.
• Utilizing RNA-sequencing (RNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on neuroectoderm.
• Analyzing the impact of phosphomutant and phosphomimetic ASCL1 variants on protein stability and function.

Main Results:

• Inhibition of ASCL1 phosphorylation significantly enhanced reprogramming of mesodermal and neuroectodermal cells.
• Pluripotent cells remained resistant to ASCL1-driven neuronal differentiation.
• Un(der)phosphorylated ASCL1 increased chromatin accessibility near neuronal genes and boosted their expression.
• Protein stability had a minor role; amino acid charge changes did not fully explain the enhanced activity of certain ASCL1 mutants.

Conclusions:

• ASCL1 phosphorylation state critically modulates its proneural activity and reprogramming capacity.
• Targeting ASCL1 phosphorylation can overcome barriers to directed neuronal differentiation.
• These findings provide insights for optimizing reprogramming protocols for regenerative medicine and cancer therapies.