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.