Figure 5.

DOT1L’s enzymatic activity is essential in hematopoiesis, but its AF9-binding domain is dispensable. (A) Experimental approach. BM harvested from 5-FU-treated Mx-Cre⁺Dot1l^f/f B6-CD45.2 donors was co-transduced ex vivo with MSCV-based constructs expressing NUP98-HOXA10HD IRES-mCherry and eGFP alone vs. eGFP + DOT1L-WT, DOT1L-D10, DOT1L I867A or DOT1L-RCR. After ex vivo expansion, these cells were transplanted into irradiated B6-CD45.1 recipients. After hematopoietic reconstitution, poly(I:C) was administered to inactivate endogenous Dot1l, followed by analysis of chimerism and Dot1l excision; (B) Representative examples of flow cytometric data. Numbers show the percentage of mCherry⁺eGFP⁺ cells among blood myeloid cells; (C) Flow cytometric analysis of blood CD45.2⁺ donor-derived CD11b⁺Gr1⁺ myeloid cells for mCherry and eGFP expression, before (blue) and 16 wks after (red) poly(I:C) (5 i.p. doses). Data from individual mice are shown. Increased representation of cells expressing DOT1L-WT, DOT1L-D10, and DOT1L-I867A, but not eGFP or DOT1L-RCR was seen after poly(I:C); (D) Flow cytometric analysis of blood CD45.2⁺ donor-derived B220⁺CD19⁺ myeloid cells for mCherry and eGFP expression, before (blue) and 16 wks after (red) poly(I:C) (5 i.p. doses). Data from individual mice are shown. Increased representation of cells expressing DOT1L-WT, DOT1L-D10, and DOT1L-I867A, but not eGFP or DOT1L-RCR was seen after poly(I:C); (E) LT-HSCs after poly(I:C) administration at the termination of the study. DOT1L-WT, DOT1L-D10 and DOT1L-I867A recipients have LT-HSCs that are comprised mostly of eGFP+ donor cells; (F) PCR analysis of Dot1l excision in sort-purified blood CD45.2⁺mCherry⁺eGFP⁺ myeloid cells 20 weeks after poly(I:C) administration. (G) DOT1L-WT, DOT1L-D10 and DOT1L-I867A recipients had ongoing myelopoiesis with fully excised Dot1l, while eGFP and DOT1L-RCR recipients had selected cells escaping Dot1l inactivation. (ns p > 0.05, ** p ≤ 0.01, **** p ≤ 0.0001).