Homodimer-mediated phosphorylation of C/EBPa-p42 S16 modulates acute myeloid leukaemia differentiation through liquid-liquid phase separation [ChIP-seq]

CCAAT/enhancer binding protein a (C/EBPa) regulates myeloid differentiation, and its dysregulation contributes to acute myeloid leukaemia (AML) progress. Clarifying its functional implementation mechanism is of great significance for its further clinical application. Here, we show that C/EBPa regulates AML cell differentiation through liquid-liquid phase separation (LLPS), which can be disrupted by C/EBPa-p30. Considering that C/EBPa-p30 inhibits the functions of C/EBPa through the LZ region, a small peptide TAT-LZ that could instantaneously interfere with the homodimerization of C/EBPa-p42 was constructed, and dynamic inhibition of C/EBPa phase separation was observed, demonstrating the importance of C/EBPa-p42 homodimers for its LLPS. Mechanistically, homodimerization of C/EBPa-p42 mediated its phosphorylation at the novel phosphorylation site S16, which promoted LLPS and subsequent AML cell differentiation. Finally, decreasing the endogenous C/EBPa-p30/C/EBPa-p42 ratio rescued the phase separation of C/EBPa in AML cells, which provided a new insight for the treatment of the AML. Overall design: Here, we show that C/EBPa promotes AML cell differentiation through LLPS, which can be disrupted by its dominant negative mutant C/EBPa-p30. Mechanistically, homodimer-mediated phosphorylation of C/EBPa S16 modulates the LLPS and thus governs AML differentiation. At last, we decreased the endogenous C/EBPa-p30/C/EBPa ratio to rescue the phase separation of C/EBPa in AML cells and significantly enhance drug efficacy. Our study clearly depicts a model in which LLPS is dependent on homodimers-mediated phosphorylation, which lays a theoretical foundation for understanding the regulatory mechanism of LLPS by an endogenous dominant negative mutant.