Dnmt3a^−/−HSCs are characterized by increased Dot1l expression and increased H3K79 methylation. (A) RNA-seq signal density tracks of messenger RNA expression of Dot1l in murine Dnmt3a^−/− HSCs purified as Hoechst side population-KSL and CD150⁺. Dnmt3a^−/− HSCs were purified after Mx-Cre–mediated deletion and serial transplantation as have previously reported^9,27  compared with HSCs from WT HSCs from 4-, 12-, and 24-month-old mice (m04_RNA, m12_RNA, and m24_RNA, respectively). (B) Average FPKM (fragments per kilobase of transcript per million mapped reads) value of Dot1L in WT vs Dnmt3a^−/− HSCs (2 independently obtained biological replicates of each cohort represented). mos, months. (C) Dot1l expression determined by qRT-PCR relative to glyceraldehyde-3-phosphate dehydrogenase expression in WT HSCs (12 months of age) compared with 2 biologic replicates of Dnmt3a^−/− HSCs purified after Mx-Cre–mediated deletion and serial transplantation (calculated by 2^-ΔΔCt equation). Assay performed in triplicate. Error bars represent standard deviation. (D) ChIP-seq of H3K79me2 of Dnmt3a^fl/fl-Rosa26-Cre-ER^T2 and Dnmt3a^wt/wt-Rosa26-Cre-ER^T2 HSCs isolated from primarily transplanted mice after tamoxifen-induced deletion. Average normalized signal density of H3K79me2 at transcription start sites, protein coding start sites, undermethylated regions (UMR) and DNA methylation canyons in Dnmt3a^−/− HSCs (red) and WT HSCs (black). (D) Representative DNA methylation canyon that expands with Dnmt3a deletion (DNA methylation, red; canyon extend, gray) and associated H3K79me2 in WT HSCs (blue) and Dnmt3a^−/− HSCs (dark red). (E) Average normalized H3K79me2 signal at DNA methylation canyons that expand with Dnmt3a deletion (Dnmt3a^−/− expand, red; WT expand, gold), canyons that do not change with Dnmt3a deletion (Dnmt3a^−/− no change, green; WT no change, teal), and canyons that contract with Dnmt3a deletion (Dnmt3a^−/− contract, blue; WT contract, purple). P value was determined using unpaired 2-way Student t test. ***P < .001.