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. 2022 Mar 25;30(4):567–574. doi: 10.1038/s41417-022-00450-9

Fig. 5. SUMOylation inhibition decreased IRF4 level through downregulating DOT1L and H3K79me2 at IRF4 promoter region.

Fig. 5

A TAK-981 synergizes with Len at decreasing IRF4 mRNA level in MM1S and MMR10R cell lines determined by quantitative PCR (qPCR). B Western blot showing decreased DOT1L level upon TAK-981 treatment in MM1S and MMR10R (Top) and H929 and KMS11 (Bottom) cell lines. Cells were treated as described in (Fig. 4). C TAK-981 treatment decreases DOT1L mRNA levels in both MM1S and MMR10R cell lines and Len has no effect on DOT1L level in MMR10R cell line measured by qPCR. D TAK-981 treatment decreased the occupancy of DOT1L and H3K79me2 on the IRF4 promoter region as measured by ChIP assay. ChIP was performed using an anti-DOT1L and H3K79me2 antibody in MM1S and MMR10R treated with TAK-981 (0.1 µM) or Len 2.5 µM for 48 h. The occupancy was normalized to DNA input and calculated relative to IgG control. E Overexpression of DOT1L compensated the decrease of IRF4 mRNA level caused by TAK-981 treatment. MM1S cells were transduced with plasmid MSCB-hDot1Lwt expressing DOT1L(DOT1L) or empty vector (EV) by electroporation then treated with TAK-981(0.1 µM) for 48 h. IRF4 mRNA level was determined by qPCR. Data presented as mean ± SD. ns, not significant; ***p < 0.001. F Overexpression of DOT1L compensated the decrease of IRF4 protein level caused by TAK-981 treatment. Western blot presenting IRF4 and DOT1L protein level in same treatment of (E). GAPDH, loading control. Quantified protein level was labeled below each blot. G UBA2 level correlates with DOT1L expression in patient specimens. Analysis of cohort (GSE2658) of 559 MM patients. Patients with high SAE2 (UBA2; UBA2high group) showed higher DOT1L level than patients with low SAE2 (UBA2; UBA2low group). Data were analyzed using unpaired Student t tests: Data presented as mean ± SD. ****p < 0.0001.