Arginine methylationof the p30 C/EBPα oncoprotein regulates progenitor proliferationand myeloid differentiation

The transcription factor CCAAT enhancer binding protein alpha (C/EBPα) is a master regulator of myelopoiesis. CEBPA encodes a long (p42) and a truncated (p30) protein isoform from a single mRNA. Mutations that abnormally enhance expression of p30 are associated with acute myelogenous leukemia (AML). We show by mutational analysis that three highly conserved arginine residues (R140,147,154) located at the p30 C/EBPα N-terminus, previously found to be methylated, are involved in myeloid lineage commitment, progenitor proliferation, and differentiation. The conservative amino acid substitution with lysine that retains the amino acid side chain charge enhanced progenitor proliferation, while a non-conservative substitution with uncharged side chains (alanine or leucine) impaired proliferation and enhanced granulopoietic differentiation. Analysis of protein-protein interactions (PPI) suggested that arginine methylation of p30 C/EBPα differentially determines its capacity to interact with SWI/SNF and MLL complexes. Pharmacological targeting of p30 C/EBPα arginine methylation may have clinical relevance in myeloproliferative and inflammatory diseases, in neutropenia, and in leukemic stem cells. To investigate the role of arginine methylation in celll differentiation and proliferation, the three arginine residues of-interest were replaced by alanine (A), leucine (L) or lysine (K). Mouse CebpaΔ/ΔCebpbΔ/Δ v-Abl transformed pre-B cells ( termed dKO-B cells) were retrovirally infected with virus carrying pMSCV-IRES-EGFP-based p30 constructs (including WT or A, L, K mutated constructs). After virus infection, EGFP+ cells were sorted at day 4 p.i. and subjected to bulk RNA-sequencing. Quadruplicate samples harvested in different batches were processed altogether (RNA extraction, library preparation and sequencing).