Downregulation of Protein Arginine Methyltransferase – 4 (PRMT4) Promotes MiR-223 Expression and Myeloid Differentiation.

Abstract 3632

Protein arginine methyltransferase 4 is a Type I member of PRMT family, that catalyses the addition of a methyl-group to arginine residues of a wide range of proteins, including histones, transcription factors, and RNA binding proteins. PRMT4 has been shown to regulate gene expression through its interaction with various transcription factors and via methylation of numerous substrates. Although PRMT4 has been reported to play an important role in T cell development, lung development and adipocyte differentiation in mouse models, the function of PRMT4 during hematopoiesis has not been studied.

To investigate the function of PRMT4 in the hematopoietic system, we utilized human CD34+ haematopoietic stem/progenitor cells (HSPCs). We observed that PRMT4 protein level is markedly downregulated during the myeloid differentiation of CD34+ cells without a significant change in the mRNA level. We then utilized a loss of function approach, using short hairpin RNAs, and found that knockdown of PRMT4 leads to an acceleration of myeloid differentiation, with a concomitant loss of the clonogenic potential of the cells. Interestingly, knocking down PRMT4 results in upregulation of miR-223, a myeloid specific microRNA. We also found that, during the myeloid differentiation of CD34+ cells, miR-223 expression steadily increased. Using a microRNA target prediction program, we identified a binding site for miR-223 in the 3′-UTR region of PRMT4 and found that when we over-expressed miR-223 in CD34+ cells, PRMT4 protein expression decreased. To determine the importance of PRMT4 downregulation in myeloid differentiation, we expressed the PRMT4-ORF (that should not be regulated by microRNAs) in CD34+ cells. The forced expression of PRMT4, that lacks the 3′-UTR region, leads to a block in myeloid differentiation and the inability of cells to up-regulate miR-223 during differentiation. Taken together, these data indicate a regulatory loop between PRMT4 and miR-223 that controls the differentiation of CD34+ toward the myeloid lineage.

To examine how PRMT4 regulates transcription of miR-223, we examined the miR-223 locus and found a RUNX1 binding site in the promoter of pri-miR-223. We discovered that PRMT4 interacts with RUNX1 and methylates RUNX1 at a specific arginine residue. This results in the recruitment of several novel interacting partners, which appear to control the expression of miR-223. Thus our results indicate that PRMT4 regulate the transcription of miR-223 transcription via its effects on RUNX1. Our study demonstrates a novel function of PRMT4 in myeloid differentiation, through regulation of RUNX1 function and miR-223 expression.