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

The transcription factor CCAAT enhancer binding protein alpha (C/EBPa) 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/EBPa 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/EBPa differentially determines its capacity to interact with SWI/SNF and MLL complexes. Pharmacological targeting of p30 C/EBPa arginine methylation may have clinical relevance in myeloproliferative and inflammatory diseases, in neutropenia, and in leukemic stem cells. Overall design: 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 Cebpa?/?Cebpb?/? 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).

CCAAT增强子结合蛋白α（CCAAT enhancer binding protein alpha, C/EBPa）是髓系生成的主调控因子。CEBPA基因可通过单一mRNA编码两种蛋白异构体：全长型（p42）与截短型（p30）。异常增强p30表达的突变与急性髓系白血病（acute myelogenous leukemia, AML）相关。本研究通过突变分析证实，位于p30 C/EBPa N端的3个高度保守精氨酸残基（R140、147、154）——此前已被发现存在甲基化修饰——参与髓系谱系定向、祖细胞增殖与分化过程。将该位点精氨酸替换为赖氨酸的保守氨基酸置换（保留氨基酸侧链电荷）可增强祖细胞增殖，而替换为不带电荷侧链的丙氨酸或亮氨酸的非保守置换则会损害增殖并促进粒细胞生成分化。蛋白质相互作用（protein-protein interactions, PPI）分析表明，p30 C/EBPa的精氨酸甲基化可差异化调控其与SWI/SNF及MLL复合物的结合能力。靶向p30 C/EBPa精氨酸甲基化的药理学手段，或在骨髓增殖性疾病、炎症性疾病、中性粒细胞减少症以及白血病干细胞相关研究中具有临床应用价值。实验设计：为探究精氨酸甲基化在细胞分化与增殖中的作用，本研究将3个目标精氨酸残基分别替换为丙氨酸（A）、亮氨酸（L）或赖氨酸（K）。将小鼠Cebpa⁻/⁻Cebpb⁻/⁻ v-Abl转化的前B细胞（命名为dKO-B细胞）用携带基于pMSCV-IRES-EGFP的p30构建体（包括野生型及A、L、K突变型构建体）的逆转录病毒感染。病毒感染后，于感染后第4天分选EGFP阳性细胞并进行批量RNA测序。不同批次收获的四份重复样本统一进行后续处理，包括RNA提取、文库制备及测序。