Identifying the Molecular Mechanisms By Which Disruptor of Telomere Silencing 1-like (DOT1L), a Histone 3, Lysine 79 (H3K79) Methyltransferase, Regulates Mammalian Hematopoiesis

Disruptor of Telomere silencing 1-Like (DOT1L), is a histone 3, lysine 79 (H3K79) methyltransferase that has been implicated in multiple processes, including activation of transcription, regulation of the cell cycle, leukemogenesis, and mouse embryonic development. In previous studies, we found that Dot1L deficiency results in an erythropoietic defect, leading to lethal anemia at around mid-gestation (Feng et al., 2010, Blood). The precise molecular mechanism(s) by which DOT1L regulates embryonic hematopoiesis has not yet been elucidated and is the overall objective of this study. DOT1L is a large protein (1540aa), and it is involved in several, diverse processes. However, its only documented activity has been as an intrinsic, histone methyltransferase. Additional functional domains of the protein might be responsible for its diverse activities, including murine hematopoiesis. We sought to determine whether the methyltransferase activity of DOT1L is essential for hematopoiesis. To test this hypothesis, we developed a Dot1L methyltransferase mutant (Dot1L MM) mouse line. Using the Cas9/CRISPR system, we created a Dot1L point mutation in cultured murine embryonic stem cells (mESC). These mESCs contained a wildtype allele, and the second had a single amino acid change in the methyltransferase domain of Dot1L, thereby eliminating its methyltransferase activity, but preserving the rest of the protein. We injected these mutant mESCs into blastocysts to produce chimeric mice. The chimeras containing the methyltransferase mutation were back crossed onto the C57/BL6 background, producing male and female offspring heterozygous for the mutation. Through intercrosses of the F1 generation, we found that the Dot1L MM mice displayed an embryonic lethality between embryonic days 10.5 and 13.5, similar to the Dot1L knockout mice, as reported in our previous studies. We additionally performed ex vivo blood differentiation assays and extensively self-renewing erythroblast (ESRE) cultures using E10.5 yolk sacs from Dot1L MM and knockout mice. Our data showed that the Dot1L MM and knockout yolk sacs display similar phenotypes. In blood differentiation cultures, Dot1L knockout and MM yolk sac cells form the same types of hematopoietic colonies as in wildtype (both erythroid and myeloid), but there is a decrease in colony size and number compared to the wildtype. In the ESRE cultures, Dot1L knockout and MM yolk sac cells form significantly fewer ESREs and have increased cell death compared to wildtype. Strikingly, the cells in these cultures also exhibit a profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as the viability of hematopoietic progenitors. These results suggest that the methyltransferase activity of DOT1L plays a predominant role in the activity of the protein as a whole, and is responsible for its function in facilitating early murine hematopoiesis. Disclosures No relevant conflicts of interest to declare.

FLT3ITD Expression Confers the Leukemogenic Properties of Growth Factor Independence and Enhanced Self-Renewal to Yolk Sac Progenitors.

Abstract 1576 Congenital leukemias are clinically and biologically distinct from those manifesting in later childhood and in adults. Derivation of the leukemic cell of origin from the primitive hematopoietic elements of the yolk sac compartment rather than definitive hematopoietic precursors may explain the unique features of this class of leukemias. We hypothesized that in the cellular context of the yolk sac precursor, common leukemogenic mutations such as the FLT3 receptor internal tandem duplication (FLT3ITD), induce characteristics allowing expansion or propagation of this developmentally self-limited cell population. Yolk sacs were dissected from pregnant C57BL/6 mice at E9.5, disaggregated and then infected with an MSCV-FLT3ITD-GFP or an MSCV-GFP control virus. To assess growth factor independence, infected yolk sac cells were plated in methylcellulose lacking growth factors. Colony numbers were scored after 7–10 days and demonstrated FLT3ITD was capable of establishing growth factor independence in yolk sac precursors, similar to results obtained using transduced fetal liver and adult bone marrow. To determine whether FLT3ITD expression in yolk sac progenitors enhanced self-renewal, the ability to serially replate was tested. Yolk sac cells were infected as above and plated in methylcellulose containing Il-3, Il-6, Scf and Epo. Colonies were scored and the cells were replated every 7–10 days. In contrast to infected fetal liver or adult bone marrow, FLT3ITD-expressing yolk sac cells were capable of replating beyond the 4th round. Analysis of derived FLT3ITD-expressing yolk sac colonies demonstrated a CD41+CD34+c-Kit+ population with an undifferentiated morphology similar to primary yolk sac progenitors. In summary, aberrant expression of the FLT3ITD mutation in yolk sac cells results in the acquisition of growth factor independence and enhanced self-renewal, characteristics essential for leukemogenesis. Although FLT3ITD expression in fetal liver and bone marrow allows growth factor independence, the distinctive ability to promote enhanced self-renewal as well in yolk sac cells suggests the yolk sac is a uniquely vulnerable target for leukemic initiation during fetal development. Disclosures: No relevant conflicts of interest to declare.