Figure 5. DOT1L-ubiquitin and DOT1L-acidic patch interfaces are both required for DOT1L-mediated H3K79me2 and cell proliferation in MLL-rearranged acute leukemia cells.

(A) Western blots for the indicated proteins in MV4; 11 cells, for parental lines (left) or those prerescued with wild-type (WT) murine Dot1L (right; this exogenous Dot1L cDNA is resistant to the sgRNA used for targeting human DOT1L [DOT1L sg]), after mock treatment (−) or doxycycline-induced depletion of endogenous DOT1L by CRISPR/Cas9 (+) for 2, 4, or 6 days.

(B) Rescue of the endogenous DOT1L depletion-related H3K79me2 loss by exogenous WT or mutant Dot1L, as indicated. Lanes 1–2 show effect of DOT1L loss in the absence of exogenous Dot1L.

(C) Relative cell proliferation following depletion of endogenous DOT1L in MV4; 11 cells, which were either parental lines (no rescue) or those prerescued with exogenous WT or mutant Dot1L, as indicated. Relative proliferation plotted on y axis after normalizing to the cell number of the culture under the doxycycline-treated condition against cell number without doxycycline treatment and then normalized to day 0 (n = 3 biological replicates; presented as mean ± SE).

See also Figure S5.