Overlapping Requirements for Tet2 and Tet3 in Normal Development and Hematopoietic Stem Cell Emergence

Abstract

Summary The Tet family of methylcytosine dioxygenases (Tet1, Tet2 and Tet3) convert 5-methylcytosine to 5-hydroxymethylcytosine. To date, functional overlap among Tet family members has not been examined systematically in the context of embryonic development. To clarify the potential for overlap among Tet enzymes during development, we mutated the zebrafish orthologs of Tet1, Tet2 and Tet3, and examined single, double and triple mutant genotypes. Here, we identify Tet2 and Tet3 as the major 5-methylcytosine dioxygenases in the zebrafish embryo and uncover a combined requirement for Tet2 and Tet3 in hematopoietic stem cell (HSC) emergence. We demonstrate that Notch signaling in the hemogenic endothelium is regulated by Tet2/3 prior to HSC emergence and show that restoring expression of the downstream gata2b/scl/runx1 transcriptional network can rescue HSCs in tet2/3 double mutant larvae. Our results reveal essential, overlapping functions for tet genes during embryonic development and uncover a requirement for 5hmC in regulating HSC production.

Active DNA demethylation at enhancers during the vertebrate phylotypic period

“…The triple morphants were severely affected, with the majority displaying embryonic lethality, while the embryos that survived gastrulation (23%) displayed short and blended axes, impaired head structures, small eyes and reduced pigmentation (Supplementary Fig. 17a), comparable to the defects recently described in a tet1 - tet2 - tet3 zebrafish knockout 57 . We next performed WGBS (for details, see Supplementary Table 1) on morphant embryos ( n = 50) and compared the 5mC profiles to their counterparts in wildtype embryos.…”

RUNX1 regulates site specificity of DNA demethylation by recruitment of DNA demethylation machineries in hematopoietic cells

“…58 Furthermore, both TET2 and TET3 regulate GlcNAcylation by interacting with O-GlcNAc transferase. 59 Our data indicate that both TET2 and TET3 enhance RUNX1-mediated DNA demethylation, and each effect is highly overlapped, supporting the functional overlap between TET2 and TET3.…”

DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes

“…RNAi mediated silencing of TET2 in cord blood progenitor CD34+ cells lowers 5hmCG levels and skews differentiation toward granulocytes and away from lymphoid and erythroid lineages (Pronier et al, 2011). When both TET2 and TET3 are mutated in zebrafish, there are reductions in the number of hematopoietic stem cells emerging during embryonic development (Li et al, 2015). Furthermore, TET2 mutations are implicated in the development of T-cell lymphomas, myeloproliferation, and myeloid malignancies, where bone marrow precursor cells are affected (Moran-Crusio et al, 2011; Pronier et al, 2011; Muto et al, 2014).…”