Rescue of erythroid differentiation and β-globin transcription by expression of rat PRMT1 in PRMT1 KD cells. (A) Detection of exogenous shRNA-resistant Flag-tagged rat PRMT1 expressed in stably transfected PRMT1 KD MEL cell clones by Western blotting using anti-Flag and anti-PRMT1 antibodies. (B) Stable MEL clones transfected with control, shPRMT1, or shRNA vectors plus PRMT1 rescue-construct were incubated with 1.5% DMSO, and total RNA was extracted at days 0, 3, and 5. The β^maj-globin mRNA expression was analyzed by qRT-PCR and normalized to β-actin (top), and hemoglobinization was assayed by benzidine staining at day 5 (bottom). (C) ChIP assays of PRMT1 at the HS2 enhancer and β^maj-promoter in MEL cell lines harboring the vector control, shPRMT1, and shPRMT1 plus PRMT1 rescue-construct. (D) ChIP assays of dimethyl H4R3 and H3K9/K14ac at the HS2 enhancer and β^maj-promoter in MEL cell lines harboring the vector control, shPRMT1, and shPRMT1 plus PRMT1 rescue-construct. Significant differences by Student t test: *P < .05, **P < .01. (E) A simplified model illustrating the role of asymmetric dimethyl H4R3, introduced by PRMT1, in transcription regulation. PRMT1 is initially recruited to specific gene loci by sequence-specific transcription factors including USF1. Subsequently, H4R3 dimethylation of neighboring nucleosomes serves as a signal for the recruitment of HATs and the transcription machinery. Shown are the mean ± SDM of 3 independent experiments.