Deletion of Dnmt1 prevents development of MLL-AF9-induced leukemia. (A) Relative expression of Dnmt1, Dnmt3a, and Dnmt3b in LSK (n = 5), GMP (n = 4), and L-GMP (n = 5) cells. (A.H.U.) Array hybridization units. (B) Kaplan-Meier survival curve of mice transplanted with MLL-AF9-transduced Dnmt1^Δ/Δ (n = 6) or control Dnmt1^fl/fl (n = 9) cells.

Haploinsufficiency of Dnmt1 and DNA methylation inhibitors impair the self-renewal and proliferation of L-GMPs. (A) Blast CFUs per 1000 Dnmt1^Δ/+ L-GMPs (n = 5) compared with control Dnmt1^+/+ (n = 3) or Dnmt1^fl/+ (n = 2) after 7 d of culture in methylcellulose. (B) Serial CFU assay of Dnmt1^Δ/+ L-GMPs (white bars; n = 6) compared with control Dnmt1^+/+ (black bars; n = 6) or Dnmt1^fl/+ (gray bars; n = 6). Bars represent average number of CFUs formed from individual replated colonies. Ratios indicate the number of colonies giving rise to secondary or tertiary colonies over total. (C) Growth curve of L-GMPs (dotted line) and GMPs (solid line) treated with 0.1 μM decitabine or 10 μM zebularine for 72 h.

Haploinsufficiency of Dnmt1 results in reduced bivalent chromatin marks in L-GMPs. (A,B) Enrichment of histone marks H3K4me3 (A) and H3K27me3 (B) at the Ccnd2, Tgfbi, Dusp6, Timp2, and Klf4 loci in Dnmt1^Δ/+ and control Dnmt1^fl/+ L-GMPs. (C) Ratio of H3K4me3:H3K27me3 in Dnmt1^Δ/+ and control L-GMPs. (D) In LSK cells, transcription is initiated by Pol II at promoters lacking DNA methylation and marked by H3K4me3. DOT1L-mediated H3K79me2 is distributed broadly across promoter and ORF regions. (E) In L-GMPs, bivalent chromatin domains are established by the addition of H3K27me3. DNA methylation acts to enforce transcriptional repression of these loci. (F) Upon haploinsufficiency of Dnmt1, these loci lose DNA methylation, have reduced bivalent chromatin marks, and gain H3K79me2. This allows Pol II binding, deposition of H3K9ac, and active transcription.

Haploinsufficiency of Dnmt1 delays progression of AML but does not alter normal hematopoiesis. (A) Kaplan-Meier survival curve of mice transplanted with MLL-AF9-transduced Dnmt1^fl/+ Mx-Cre⁺ (n = 5) or control Dnmt1^fl/fl (n = 8) cells. The arrow denotes the start of the polyI–polyC injections. (B) Western blot demonstrating DNMT1 protein levels in Dnmt1^fl/+ Mx-Cre⁺ or control leukemias. (C) WBC counts obtained from Dnmt1^fl/+ Mx-Cre⁺ or control mice (n ≥ 3), collected before and up to 12 wk after polyI–polyC injection. (D) Total number of LSK and GMP cells in Dnmt1^Δ/+ (n = 3) or control (n = 3) mice.

Dnmt1 haploinsufficiency results in derepression of genes normally silenced and marked by bivalent chromatin domains in L-GMPs. (A) Venn diagram demonstrating the overlap between 1317 genes repressed in L-GMPs (orange circle) (Krivtsov et al. 2006) and 680 genes derepressed in Dnmt1^Δ/+ L-GMPs (blue circle). The 155-gene overlap represents our “derepressed in Dnmt1^Δ/+ L-GMPs” signature. (B) Graphical representation of genes marked by H3K27me3 and H3K79me2 in LSK cells. Our 155-gene signature is highlighted (red circles) compared with genome-wide (gray circles). (C) Graphical representation of genes marked by H3K27me3 and H3K79me2 in L-GMPs. (D) Graphical representation of genes marked by H3K27me3 and H3K4me3 in L-GMPs.