Similar to p21, H3R2 dimethylation was enriched at the p27 promoter. However, there was no significant change of H3R2me2a at the TSS of the p57 promoter. These results indicate a direct role for PRMT6 in repression of p21 and p27 promoter activity. The effect of PRMT6 overexpression on CIP/KIP inhibitor expression was further investigated in the U2OS cell system. As expected, high PRMT6 level correlated with a Haloperidol hydrochloride global rise of the H3R2me2a mark. Analysis of the CIP/KIP inhibitor level revealed a detectable downregulation of p21 and p27 but not of p57. In order to confirm that these effects rely on the active enzyme the effects of a catalytically inactive mutant of PRMT6 were investigated. The glutamic acid at position 164 is a critical residue of the double E loop, a motif required for the enzymatic activity of PRMTs. Expression of PRMT6- E164Q did not alter H3R2me2a levels nor suppressed p21 or p27 expression. Interestingly, we noticed higher levels of p27 compared to mock GS 143 condition, which may indicate a selective dominant-negative effect of the PRMT6 mutant. Taken together, these results indicate that PRMT6 acts to reduce the expression of p21. Here, we report a role for PRMT6 in cell cycle regulation and show that PRMT6 negatively regulates p21 protein expression in three human cell lines tested. Expression of PRMT6 leads to increased levels of H3R2me2a on the p21 and p27 promoters in U2OS cells, leading to reduced mRNA and protein expression of these genes. Furthermore, knockdown of PRMT6 and increased p21 and p27 levels coincide with an accumulation of cells in G2. This is in accordance with the finding that overexpression of p21 results in CDK1 inhibition and G2 arrest in U2OS cells. Overexpression of PRMT6 leads to a weak but detectable downregulation of p21 and p27 expression. Possibly, PRMT6 action requires assistance by co-regulators to fully exert its repressive function. To date only a few interactors of PRMT6 have been described in the literature, the HIV proteins Tat and Ref, and the human DNA polymerase beta. However, it has been shown before that PRMT6 regulates transcription through recruitment to target promoters. Since PRMT6 lacks a DNA binding domain, we postulate the existence of additional binding partners of PRMT6 for efficient promoter targeting. At present such PRMT6-interacting proteins remain to be identified and this may involve transient interactions as PRMT6 has been shown to sediment as a monomer in glycerol gradients. Moreover, it is not clear whether the cell cycle effects under PRMT6 knockdown conditions solely rely on the activation of p21 and p27. Interestingly, another arginine methyltransferase, CARM1/ PRMT4, regulates the half-life of the p21 transcript through methylation of the mRNA binding protein HuD. The authors suggest that methylated HuD maintains PC12 cells in a proliferative state by committing p21 mRNA to its decay system. Loss of CARM1/PRMT4 leads to increased p21 protein level, whereas p27 expression is not affected.