Immunophenotypic characterization of these mice was performed by conducting confocal analysis of brain sections, stained with antibodies specific for the pan-OL lineage marker OLIG2, for OPC-specific surface receptor (e.g., platelet-derived growth factor receptor- (PDGFR)) and for differentiation markers (e.g., CC1 or MBP). methylation of histone H4R3, as critical for developmental myelination. PRMT5 pharmacological inhibition, CRISPR/cas9 targeting, or genetic ablation decrease p53-dependent survival and impair differentiation without affecting proliferation. Conditional ablation of in progenitors results in hypomyelination, reduced survival and differentiation. Decreased histone H4R3 symmetric methylation is followed by increased nuclear acetylation of H4K5, and is rescued by pharmacological inhibition of histone acetyltransferases. Data obtained using purified histones further validate the results obtained in mice and in cultured Fosphenytoin disodium oligodendrocyte progenitors. Together, these results identify PRMT5 as critical for oligodendrocyte differentiation and developmental myelination by modulating the cross-talk between histone arginine methylation and lysine acetylation. Introduction Brain function is highly specialized and dependent on the integrated action of several cell types. Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS) and are responsible for ensuring axonal conduction and neuronal support1. Their number is tightly regulated and dependent on differentiation, survival, and proliferation of oligodendrocyte progenitor cells (OPCs). Therefore, understanding the basic processes regulating OL cell number is key for the advancement in neurobiology. We and others have previously contributed to elucidating the molecular mechanisms governing proliferation and differentiation of OPC2C4. Among the latter, we reported decreased acetylation of lysine residues on histone tails as an essential event for the differentiation of OPCs into OL2,5C9. Based on these and additional studies10,11, we proposed a mechanism of developmental myelination driven by de-repression of inhibitory molecules9,12. Besides modifications of lysine residues, repressive modifications of nucleosomal histones include the symmetric dimethylation of arginines (-NG, -NG-dimethyl arginine), which is mediated by class-II protein arginine methyltransferases (PRMTs) such as PRMT513,14 and PRMT915,16. PRMT5 is expressed in the brain and enriched in the OL lineage17C19. Its activity is thought to negatively regulate gene expression due to methylation of multiple arginine residues on nucleosomal histone tails20C22. PRMT5 is also expressed at high levels in proneural gliomas, which are transcriptionally related Rabbit Polyclonal to ATG4D to OPCs23,24, and arise from their transformation25,26. PRMT5 levels positively correlate with malignancy and negatively correlate with glioma patients survival27,28, therefore justifying the efforts to identify specific pharmacological inhibitors as potential therapeutic targets27,29C33. Despite several studies highlighting the importance of Fosphenytoin disodium PRMT5 in malignancies, the physiological role of this enzyme in the OL lineage remains poorly understood. Previous studies in neural stem cells underlined the importance of PRMT5 in the regulation of pre-mRNA splicing34. Another study in a glial cell line suggested this enzyme could affect OL differentiation by affecting transcription, although the mechanistic aspects were Fosphenytoin disodium not elucidated19. Based on this cumulative evidence, we reasoned that a thorough characterization of PRMT5 in the OL lineage is timely and may shed some light on a better understanding of the regulation of OL cell number in the brain. In this study, we adopted several strategies to address this key question including: a detailed characterization of mice with cell-lineage-specific ablation of in immature oligodendrocyte progenitors or in oligodendrocytes, the use of CRISPR/Cas9 and pharmacological inhibitors to interfere with PRMT5 function in primary OPC cultures, transcriptomic analyses, and biochemical assays using synthetic proteins and modified histone peptides. Because the study of symmetric arginine methylation relies on the high quality of reagents, in this study we extensively characterized the specificity of all the commercially available antibodies to study this modification and selected those with the highest level of discriminatory power from other modifications (including asymmetric methylation at the same residue). Overall, this comprehensive study identifies PRMT5 as a key regulator of the number of myelinating cells in the CNS, by modulating survival of differentiating progenitors and orchestrating a tight coordination between symmetric histone arginine methylation and decreased histone lysine acetylation at the transition between growth arrest and differentiation. Results PRMT5 expression and activity in the oligodendrocyte lineage To characterize the expression pattern of in OL lineage cells, we measured its transcript levels in RNA samples obtained from cultured primary oligodendrocyte progenitors (OPCs) kept.