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Identifying the Molecular Mechanisms by which Disruptor of Telomere silencing 1‐Like (DOT1L), a Histone 3, Lysine 79 (H3K79) Methyltransferase, Regulates Mammalian Hematopoiesis
Author(s) -
Malcom Carrie,
PiaseckaSrader Joanna,
Alvarez Nehemiah,
Slawson Chad,
Fields Patrick
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.583.1
Subject(s) - histone h3 , biology , methyltransferase , histone methyltransferase , microbiology and biotechnology , gene silencing , embryonic stem cell , genetics , epigenetics , methylation , gene
D isruptor o f T elomere silencing 1 ‐ L ike (DOT1L), is a histone 3, lysine 79 (H3K79) methyltransferase. The enzyme has been implicated in multiple processes, including activation of transcription, regulation of the cell cycle, leukemogenesis, and mouse embryonic development. Previous studies in our lab found that Dot1L deficiency results in an erythropoietic defect, leading to lethal anemia at around mid‐gestation (Feng et al., 2010). The precise molecular mechanism(s) by which DOT1L regulates embryonic hematopoiesis has not yet been elucidated and is the overall objective of this study. Specifically, we sought to determine whether the methyltransferase activity of DOT1L is essential for hematopoiesis. DOT1L is a large protein (1540aa) and involved in several, diverse processes, but only its methyltransferase activity has been documented. Additional functional domains of the protein might be responsible for its diverse activities. We developed a murine embryonic stem cell (mESC)‐based culture system in which to examine the role of DOT1L in hematopoiesis, in vitro . Using the Cas9/CRISPR system, we created two different mutations in Dot1L in cultured mESCs: a Dot1L knockout and a Dot1L point mutant. Knockout mESCs contain a large, out‐of‐frame deletion in both alleles of Dot1L , while the point mutant contains a single amino acid change in the methyltransferase domain of Dot1L , thereby eliminating its methyltransferase activity, but preserving the rest of the protein. We then induced these Dot1L knockout and point mutant mESC clones, along with wildtype mESCs, to undergo hematopoiesis in culture. Our data showed that this protocol leads to similar erythropoietic defects as those observed in vivo . Dot1L knockout and point mutant mESCs are capable of forming the same types of hematopoietic colonies as wildtype mESCs (both erythroid and myeloid), but there is a decrease in colony size and number compared to wildtype. These results suggest that the methyltransferase activity of DOT1L plays a predominant role in the activity of the protein as a whole, and is entirely responsible for its function in facilitating early, murine hematopoiesis. Support or Funding Information R01 DK091277, Fields (PI), 04/01/12‐03/31/17NIH/NIDDK Role of the Histone Methyltransferase DOT1L in Embryonic Erythropoiesis