Figure 2.
Loss of RUNX1 R233/R237 methylation in RUNX1 R233K/R237K double-mutant Runx1KTAMK/KTAMKmice expands a phenotypic HSC pool with less quiescence. (A) A schema showing loss of RUNX1 methylation in RUNX1 R233K/R237K double-mutant Runx1KTAMK/KTAMK mice. Two arginine-to-lysine (RTAMR-to-KTAMK) mutations (red) preclude RUNX1 methylation by PRMT1. (B) Increase of phenotypic HSCs in Runx1KTAMK/KTAMK mice. The left panel shows representative FACS plots of Lin–Sca1+cKit+ (LSK) cells in Runx1+/+ (upper), Runx1+/KTAMK (middle), and Runx1KTAMK/KTAMK (lower) mice. The LT-HSC subset is shown in red. Numbers shown are mean percentages of total live bone marrow cells. The right graphs show mean frequencies of CD34–Flt3– LSK cells (upper), CD34+Flt3– LSK cells, and CD34+Flt3+ LSK cells (lower) in Runx1+/+ (blue), Runx1+/KTAMK (light green), and Runx1KTAMK/KTAMK (red) mice (n = 7-13 mice; 8-16 weeks old). (C) Phenotypic analysis of HSCs and MPPs using SLAM markers. The graph shows mean frequencies of HSCs and MPPs in Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) mice (n = 4 mice; 8 weeks old). (D) Loss of quiescence in Runx1KTAMK/KTAMK LT-HSCs. The upper panel shows representative FACS plots of CD34–Flt3– LSK cells in Runx1+/+ (left) and Runx1KTAMK/KTAMK (right) mice. The G0 subset is shown in red. The lower graph shows mean frequencies of each cell cycle subset among LT-HSCs from Runx1+/+ (blue), Runx1+/KTAMK (light green), and Runx1KTAMK/KTAMK (red) mice (n = 4-6 mice; 7-15 weeks old). (E) Increase of colony-forming units in total bone marrow cells from Runx1+/KTAMK (light green) and Runx1KTAMK/KTAMK (red) mice, compared with Runx1+/+ mice (blue) (n = 12 from 4 mice per genotype; 9 to 11 weeks old; from 4 independent experiments). (F) Competitive reconstitution assay in which Runx1+/+ (blue) or Runx1KTAMK/KTAMK (red) bone marrow cells were transplanted into irradiated recipient mice along with competitor cells. Lines show the frequency of donor-derived cells in peripheral blood after the first BMT at the indicated time intervals (n = 9 to 10 mice from 3 independent experiments). Donor cells were from mice 9 to 12 weeks old. Data on B cells and T cells are shown in supplemental Figure 1C. NS, not significant.

Loss of RUNX1 R233/R237 methylation in RUNX1 R233K/R237K double-mutant Runx1KTAMK/KTAMKmice expands a phenotypic HSC pool with less quiescence. (A) A schema showing loss of RUNX1 methylation in RUNX1 R233K/R237K double-mutant Runx1KTAMK/KTAMK mice. Two arginine-to-lysine (RTAMR-to-KTAMK) mutations (red) preclude RUNX1 methylation by PRMT1. (B) Increase of phenotypic HSCs in Runx1KTAMK/KTAMK mice. The left panel shows representative FACS plots of Lin–Sca1+cKit+ (LSK) cells in Runx1+/+ (upper), Runx1+/KTAMK (middle), and Runx1KTAMK/KTAMK (lower) mice. The LT-HSC subset is shown in red. Numbers shown are mean percentages of total live bone marrow cells. The right graphs show mean frequencies of CD34–Flt3– LSK cells (upper), CD34+Flt3– LSK cells, and CD34+Flt3+ LSK cells (lower) in Runx1+/+ (blue), Runx1+/KTAMK (light green), and Runx1KTAMK/KTAMK (red) mice (n = 7-13 mice; 8-16 weeks old). (C) Phenotypic analysis of HSCs and MPPs using SLAM markers. The graph shows mean frequencies of HSCs and MPPs in Runx1+/+ (blue) and Runx1KTAMK/KTAMK (red) mice (n = 4 mice; 8 weeks old). (D) Loss of quiescence in Runx1KTAMK/KTAMK LT-HSCs. The upper panel shows representative FACS plots of CD34–Flt3– LSK cells in Runx1+/+ (left) and Runx1KTAMK/KTAMK (right) mice. The G0 subset is shown in red. The lower graph shows mean frequencies of each cell cycle subset among LT-HSCs from Runx1+/+ (blue), Runx1+/KTAMK (light green), and Runx1KTAMK/KTAMK (red) mice (n = 4-6 mice; 7-15 weeks old). (E) Increase of colony-forming units in total bone marrow cells from Runx1+/KTAMK (light green) and Runx1KTAMK/KTAMK (red) mice, compared with Runx1+/+ mice (blue) (n = 12 from 4 mice per genotype; 9 to 11 weeks old; from 4 independent experiments). (F) Competitive reconstitution assay in which Runx1+/+ (blue) or Runx1KTAMK/KTAMK (red) bone marrow cells were transplanted into irradiated recipient mice along with competitor cells. Lines show the frequency of donor-derived cells in peripheral blood after the first BMT at the indicated time intervals (n = 9 to 10 mice from 3 independent experiments). Donor cells were from mice 9 to 12 weeks old. Data on B cells and T cells are shown in supplemental Figure 1C. NS, not significant.

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