Fig 1: K20R mutation induces hyperplasia in the mouse spleen.(A) Hematoxylin and eosin (H&E) histological staining of liver and spleen sections from the K20r and Ezh2 wild-type mice (8-month-old). Scale bar: 200 μm. (B) The anti-Ki67 and anti-GFI1B immunostaining of spleen sections from the K20r and Ezh2 wild-type mice (8-month-old). Scale bar: 50 mm. (C) Left: protein lysates were extracted from the spleen of the K20r and Ezh2 wild-type mice and blotted with indicated antibodies. Right: band intensities were quantified and normalized to that of histone H3 control. Significance was indicated as a two-tailed, unpaired, t-test. Values are expressed as the mean ± SEM. *p<0.05. (D) RNA levels of indicated hematopoietic regulatory genes were measured by quantitative RT-PCR (RT-qPCR) from the spleens of the Ezh2 wild-type and K20r mice (4-month-old). The mRNA levels were measured in triplicate by RT-qPCR. (E) Protein lysates were extracted from the spleens of the Ezh2 wild-type and K20r mice and detected with anti-EZH2, SUZ12, EED, and GFI1B antibodies, using the actin antibody as a control. The protein band intensity values are means ± SEM (n=3). Significance is indicated as a two-tailed, unpaired, t-test. *p<0.05.**p<0.01. (F) Model: Left panel: The lysine residue 20 (K20) of EZH2 is methylated by SET7 methyltransferase and the level of methylated K20 is reversibly removed by KDM1A demethylase. L3MBTL3 preferentially binds to the methylated K20 in EZH2 to recruit the CRL4DCAF5 ubiquitin E3 ligase complex to target the methylated EZH2 for ubiquitin-dependent proteolysis. Right panel: The K20 methylation is negatively regulated by the phosphorylation of serine 21 (S21) by the PI3K-activated AKT. Conversely, the S21 phosphorylation is mutually exclusive to the methylation of K20, resulting in the methylation-phosphorylation switch to control the activity and proteolysis of EZH2 for H3K27 trimethylation. Figure 8—source data 1.Original photos and bots for Figure 8. Figure 8—source data 2.Original table sources for quantification of Figure 8 plots.